![[Help]](/netaicon1/PTO/help.gif)
![[Home]](/netaicon1/PTO/home.gif)
![[Boolean Search]](/netaicon1/PTO/boolean.gif)
![[Manual]](/netaicon1/PTO/manual.gif)
![[Number Search]](/netaicon1/PTO/number.gif)
![[PTDLs]](/netaicon1/PTO/ptdl.gif)
![[CURR_LIST]](/netaicon1/PTO/hitlist.gif)
![[Shopping Cart]](/netaicon1/PTO/cart.gif)
![[Order Copy]](/netaicon1/PTO/order.gif)
![[Image]](/netaicon1/PTO/image.gif)
| United States Patent Application |
20080233175
|
| Kind Code
|
A1
|
|
Steer; Brian
; et al.
|
September 25, 2008
|
Glucanases, Nucleic Acids Encoding Them And Methods For Making And Using
Them
Abstract
The invention relates to polypeptides having glucanase, e.g.,
endoglucanase, mannanase, xylanase activity or a combination of these
activities, and polynucleotides encoding them. In one aspect, the
glucanase activity is an endoglucanase activity (e.g.,
endo-1,4-beta-D-glucan 4-glucano hydrolase activity) and comprises
hydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulose
derivatives (e.g., carboxy methyl cellulose and hydroxy ethyl cellulose)
lichenin, beta-1,4 bonds in mixed beta-1,3 glucans, such as cereal
beta-D-glucans or xyloglucans and other plant material containing
cellulosic parts. In addition, methods of designing new enzymes and
methods of use thereof are also provided. In alternative aspects, the new
glucanases e.g., endoglucanases, mannanases, xylanases have increased
activity and stability at increased pH and temperature.
| Inventors: |
Steer; Brian; (San Diego, CA)
; Callen; Walter; (San Diego, CA)
; Healey; Shaun; (Carlsbad, CA)
; Pulliam; Derrick; (Durham, NC)
|
| Correspondence Address:
|
VERENIUM C/O MOFO S.D.
12531 HIGH BLUFF DRIVE, SUITE 100
SAN DIEGO
CA
92130-2040
US
|
| Family ID:
|
33564024
|
| Appl. No.:
|
10/560957
|
| Filed:
|
July 2, 2004 |
| PCT Filed:
|
July 2, 2004 |
| PCT NO:
|
PCT/US04/21492 |
| 371 Date:
|
April 3, 2007 |
Related U.S. Patent Documents
| | | | |
|---|
|
| Application Number | Filing Date | Patent Number |
|---|
| | 60484725 | Jul 2, 2003 | |
|
|
| Current U.S. Class: |
424/439 |
| Current CPC Class: |
C12N 9/2405 20130101; A23L 29/10 20160801; C12Y 302/01004 20130101; G01N 2333/924 20130101; G01N 2500/00 20130101; Y02E 50/17 20130101; D21C 1/00 20130101; C12P 7/10 20130101; C12N 11/16 20130101; C12N 11/14 20130101; C12N 11/02 20130101; G01N 33/573 20130101; A23C 19/04 20130101; A23C 9/1322 20130101; G01N 33/53 20130101; A23L 2/52 20130101; A23K 40/10 20160501; A23K 20/189 20160501; A23K 10/14 20160501; A23K 50/10 20160501; A23L 33/18 20160801; Y02E 50/16 20130101; C12N 9/2437 20130101 |
| Class at Publication: |
424/439 |
| International Class: |
A61K 47/00 20060101 A61K047/00 |
Claims
1: An isolated, synthetic or recombinant nucleic acid comprising (a) a
nucleic acid sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or more or complete (100%) sequence identity to SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,
SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ
ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID
NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID
NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID
NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID
NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID
NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID
NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID
NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ
ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,
SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID
NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ
ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,
SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:199, SEQ ID
NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ
ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179,
SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID
NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ
ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207,
SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID
NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ
ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235,
SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID
NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ
ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263,
SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID
NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ
ID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291,
SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID
NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ
ID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319,
SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID
NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:337, SEQ
ID NO:339, SEQ ID NO:341, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347,
SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:353, SEQ ID NO:355, SEQ ID
NO:357, SEQ ID NO:359, SEQ ID NO:361, SEQ ID NO:363, SEQ ID NO:365, SEQ
ID NO:367, SEQ ID NO:369, SEQ ID NO:371, SEQ ID NO:373, SEQ ID NO:375,
SEQ ID NO:377, SEQ ID NO:379, SEQ ID NO:381, SEQ ID NO:383, SEQ ID
NO:385, SEQ ID NO:387, SEQ ID NO:389, SEQ ID NO:391, SEQ ID NO:393, SEQ
ID NO:395, SEQ ID NO:397, SEQ ID NO:399, SEQ ID NO:401, SEQ ID NO:403,
SEQ ID NO:405, SEQ ID NO:407, SEQ ID NO:409, SEQ ID NO:411, SEQ ID
NO:413, SEQ ID NO:415, SEQ ID NO:417, SEQ ID NO:419, SEQ ID NO:421, SEQ
ID NO:423, SEQ ID NO:425, SEQ ID NO:427, SEQ ID NO:429, SEQ ID NO:431,
SEQ ID NO:433, SEQ ID NO:435, SEQ ID NO:437, SEQ ID NO:439, SEQ ID
NO:441, SEQ ID NO:443, SEQ ID NO:445, SEQ ID NO:447, SEQ ID NO:449, SEQ
ID NO:451, SEQ ID NO:453, SEQ ID NO:455, SEQ ID NO:457, SEQ ID NO:459,
SEQ ID NO:461, SEQ ID NO:463, SEQ ID NO:465, SEQ ID NO:467, SEQ ID
NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ ID NO:475, SEQ ID NO:477, SEQ
ID NO:479, SEQ ID NO:481, SEQ ID NO:483, SEQ ID NO:485, SEQ ID NO:487,
SEQ ID NO:489, SEQ ID NO:491, SEQ ID NO:493, SEQ ID NO:495, SEQ ID
NO:497, SEQ ID NO:499, SEQ ID NO:501, SEQ ID NO:503, SEQ ID NO:505, SEQ
ID NO:507, SEQ ID NO:509, SEQ ID NO:511, SEQ ID NO:513, SEQ ID NO:515 or
SEQ ID NO:517, over a region of at least about 20, 30, 40, 50, 60, 75 or
100, or 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, 1050, 1100, 1150 or more residues, or over the
full length of the gene or transcript, wherein the nucleic acid encodes
at least one polypeptide having: (i) a glucanase, mannanase or xylanase
activity, or (ii) immunogenic activity and can generate an antibody that
specifically binds to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ
ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID
NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID
NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID
NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID
NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID
NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID
NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID
NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ
ID NO:10, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ
ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128,
SEQ ID NO:130, SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID
NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID NO:144; NO:146, SEQ ID
NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ
ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166,
SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID
NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ
ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194,
SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID
NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ
ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222,
SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID
NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ
ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250,
SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID
NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ
ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278,
SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ ID
NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ
ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:306,
SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID
NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ
ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334,
SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340, SEQ ID NO:342, SEQ ID
NO:344, SEQ ID NO:346, SEQ ID NO:348, SEQ ID NO:350, SEQ ID NO:352, SEQ
ID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362,
SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368, SEQ ID NO:370, SEQ ID
NO:372, SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378, SEQ ID NO:380, SEQ
ID NO:382, SEQ ID NO:384, SEQ ID NO:386, SEQ ID NO:388, SEQ ID NO:390,
SEQ ID NO:392, SEQ ID NO:394, SEQ ID NO:396, SEQ ID NO:398, SEQ ID
NO:400, SEQ ID NO:402, SEQ ID NO:404, SEQ ID NO:406, SEQ ID NO:408, SEQ
ID NO:410, SEQ ID NO:412, SEQ ID NO:414, SEQ ID NO:416, SEQ ID NO:418,
SEQ ID NO:420, SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, SEQ ID
NO:428, SEQ ID NO:430, SEQ ID NO:432, SEQ ID NO:434, SEQ ID NO:436, SEQ
ID NO:438, SEQ ID NO:440, SEQ ID NO:442, SEQ ID NO:444, SEQ ID NO:446,
SEQ ID NO:448, SEQ ID NO:450, SEQ ID NO:452, SEQ ID NO:454, SEQ ID
NO:456, SEQ ID NO:458, SEQ ID NO:460, SEQ ID NO:462, SEQ ID NO:464, SEQ
ID NO:466, SEQ ID NO:468, SEQ ID NO:470, SEQ ID NO:472, SEQ ID NO:474,
SEQ ID NO:476, SEQ ID NO:478, SEQ ID NO:480, SEQ ID NO:482, SEQ ID
NO:484, SEQ ID NO:486, SEQ ID NO:488, SEQ ID NO:490, SEQ ID NO:492, SEQ
ID NO:494, SEQ ID NO:496, SEQ ID NO:498, SEQ ID NO:500, SEQ ID NO:502,
SEQ ID NO:504, SEQ ID NO:506, SEQ ID NO:508, SEQ ID NO:510, SEQ ID
NO:512, SEQ ID NO:514, SEQ ID NO:516 or SEQ ID NO:518; (b) the nucleic
acid sequence of (a), wherein the sequence identities are determined by
analysis with a sequence comparison algorithm or by a visual inspections
(c) the nucleic acid sequence of (b), wherein the sequence comparison
algorithm comprises a BLASTN program using as defaults a wordlength (W)
of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both
strands, and all other options are set to default; (d) a nucleic acid
sequence encoding a polypeptide having a sequence as set forth in SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID
NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID
NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID
NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID
NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID
NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID
NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:10, SEQ ID NO:112, SEQ ID
NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ
ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132;
SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID
NO:142; SEQ ID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID
NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ
ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170,
SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID
NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ
ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198,
SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID
NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ
ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226,
SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID
NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ
ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254,
SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID
NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ
ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282,
SEQ ID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID
NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ
ID NO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310,
SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID
NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ
ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:338,
SEQ ID NO:340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID
NO:348, SEQ ID NO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ
ID NO:358, SEQ ID NO:360, SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:366,
SEQ ID NO:368, SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:374, SEQ ID
NO:376, SEQ ID NO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ ID NO:384, SEQ
ID NO:386, SEQ ID NO:388, SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394,
SEQ ID NO:396, SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402, SEQ ID
NO:404, SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, SEQ ID NO:412, SEQ
ID NO:414, SEQ ID NO:416, SEQ ID NO:418, SEQ ID NO:420, SEQ ID NO:422,
SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, SEQ ID
NO:432, SEQ ID NO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ
ID NO:442, SEQ ID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQ ID NO:450,
SEQ ID NO:452, SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458, SEQ ID
NO:460, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ
ID NO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ ID NO:476, SEQ ID NO:478,
SEQ ID NO:480, SEQ ID NO:482, SEQ ID NO:484, SEQ ID NO:486, SEQ ID
NO:488, SEQ ID NO:490, SEQ ID NO:492, SEQ ID NO:494, SEQ ID NO:496, SEQ
ID NO:498, SEQ ID NO:500, SEQ ID NO:502, SEQ ID NO:504, SEQ ID NO:506,
SEQ ID NO:508, SEQ ID NO:510, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:516
or SEQ ID NO:518; (e) a nucleic acid sequence that hybridizes under
stringent conditions to a nucleic acid comprising SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,
SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ
ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID
NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID
NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID
NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID
NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID
NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID
NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID
NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ
ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,
SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID
NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ
ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,
SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:199, SEQ ID
NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ
ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179,
SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID
NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ
ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207,
SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID
NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ
ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235,
SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID
NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ
ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263,
SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID
NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ
ID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291,
SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID
NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ
ID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319,
SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID
NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:337, SEQ
ID NO:339, SEQ ID NO:341, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347,
SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:353, SEQ ID NO:355, SEQ ID
NO:357, SEQ ID NO:359, SEQ ID NO:361, SEQ ID NO:363, SEQ ID NO:365, SEQ
ID NO:367, SEQ ID NO:369, SEQ ID NO:371, SEQ ID NO:373, SEQ ID NO:375,
SEQ ID NO:377, SEQ ID NO:379, SEQ ID NO:381, SEQ ID NO:383, SEQ ID
NO:385, SEQ ID NO:387, SEQ ID NO:389, SEQ ID NO:391, SEQ ID NO:393, SEQ
ID NO:395, SEQ ID NO:397, SEQ ID NO:399, SEQ ID NO:401, SEQ ID NO:403,
SEQ ID NO:405, SEQ ID NO:407, SEQ ID NO:409, SEQ ID NO:411, SEQ ID
NO:413, SEQ ID NO:415, SEQ ID NO:417, SEQ ID NO:419, SEQ ID NO:421, SEQ
ID NO:423, SEQ ID NO:425, SEQ ID NO:427, SEQ ID NO:429, SEQ ID NO:431,
SEQ ID NO:433, SEQ ID NO:435, SEQ ID NO:437, SEQ ID NO:439, SEQ ID
NO:441, SEQ ID NO:443, SEQ ID NO:445, SEQ ID NO:447, SEQ ID NO:449, SEQ
ID NO:451, SEQ ID NO:453, SEQ ID NO:455, SEQ ID NO:457, SEQ ID NO:459,
SEQ ID NO:461, SEQ ID NO:463, SEQ ID NO:465, SEQ ID NO:467, SEQ ID
NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ ID NO:475, SEQ ID NO:477, SEQ
ID NO:479, SEQ ID NO:481, SEQ ID NO:483, SEQ ID NO:485, SEQ ID NO:487,
SEQ ID NO:489, SEQ ID NO:491, SEQ ID NO:493, SEQ ID NO:495, SEQ ID
NO:497, SEQ ID NO:499, SEQ ID NO:501, SEQ ID NO:503, SEQ ID NO:505, SEQ
ID NO:507, SEQ ID NO:509, SEQ ID NO:511, SEQ ID NO:513, SEQ ID NO:515 or
SEQ ID NO:517, wherein the nucleic acid encodes a polypeptide having a
glucanase, mannanase or xylanase activity, the nucleic acid is at least
about 20, 30, 40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 600, 700,
800, 900, 1000 or more residues in length or the full length of the gene
or transcript, and the stringent conditions include a wash step
comprising a wash in 0.2.times.SSC at a temperature of about 65.degree.
C. for about 15 minutes; (f) the nucleic acid of (a), (b), (c), (d) or
(f), wherein the glucanase, mannanase or xylanase activity comprises: an
endoglucanase activity; or catalyzing hydrolysis of 1,4-beta-D-glycosidic
linkages or internal .beta.-1,3-glucosidic linkages, or, hydrolyzing a
glucan to produce a smaller molecular weight polysaccharide or oligomer;
or an endoglucanase activity comprising an endo-1,4-beta-endoglucanase
activity; or an endoglucanase activity comprising 1,4-beta-D-glycosidic
linkage activity; or an endoglucanase activity comprising hydrolysis of a
1,4-beta-D-glycosidic linkage in a cellulose, a cellulose derivative, a
lichenin or a cereal; or hydrolyzing a glycosidic linkage in a cellulose
derivative; or hydrolyzing a glycosidic linkage in a carboxy methyl
cellulose or a hydroxy ethyl cellulose; or hydrolyzing a glycosidic
linkage in a cereal; or hydrolyzing a glycosidic linkage in a
beta-D-glucan or a xyloglucan; or hydrolyzing polysaccharides comprising
1,4-.beta.-glycoside-linked D-glucopyranoses; or hydrolyzing a cellulose,
a cellulose derivative or a hemicellulose; or hydrolyzing a cellulose or
a hemicellulose in a wood or paper pulp or a wood or paper product; or
catalyzing hydrolysis of glucan in a feed, a food product or a beverage;
or hydrolyzing a glucan in a feed, food product or beverage; hydrolyzing
a glycosidic linkage or a glucan in a cereal-based animal feed, a wort or
a beer, a dough, a fruit or a vegetable; or hydrolyzing a glucan in a
microbial cell, a fungal cell, a mammalian cell or a plant cell; (g) the
nucleic acid of (a), (b), (c), (d), (e) or (f), wherein the glucanase,
mannanase or xylanase activity is thermostable; or the polypeptide
retains a glucanase, mannanase or xylanase activity under conditions
comprising a temperature range of between about 37.degree. C. to about
95.degree. C., or between about 55.degree. C. to about 85.degree. C., or
between about 70.degree. C. to about 75.degree. C., or between about
70.degree. C. to about 95.degree. C., or between about 90.degree. C. to
about 95.degree. C.; (h) the nucleic acid of (a), (b), (c), (d), (e) or
(f), wherein the glucanase activity is thermotolerant; or the polypeptide
retains a glucanase activity after exposure to a temperature in the range
from greater than 37.degree. C. to about 95.degree. C., from greater than
55.degree. C. to about 85.degree. C., or between about 70.degree. C. to
about 75.degree. C., or from greater than 90.degree. C. to about
95.degree. C.
2-26. (canceled)
27: A nucleic acid probe for identifying a nucleic acid encoding a
polypeptide with a glucanase activity, wherein the probe comprises at
least 10 consecutive bases, or at least about 10 to 50, about 20 to 60,
about 30 to 70, about 40 to 80, about 60 to 100, or about 50 to 150
consecutive bases, of a sequence comprising (a) SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID
NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID
NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID
NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID
NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID
NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID
NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID
NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID
NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ
ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,
SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID
NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ
ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,
SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:199, SEQ ID
NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ
ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179,
SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID
NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ
ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207,
SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID
NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ
ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235,
SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID
NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ
ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263,
SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID
NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ
ID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291,
SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID
NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ
ID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319,
SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID
NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:337, SEQ
ID NO:339, SEQ ID NO:341, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347,
SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:353, SEQ ID NO:355, SEQ ID
NO:357, SEQ ID NO:359, SEQ ID NO:361, SEQ ID NO:363, SEQ ID NO:365, SEQ
ID NO:367, SEQ ID NO:369, SEQ ID NO:371, SEQ ID NO:373, SEQ ID NO:375,
SEQ ID NO:377, SEQ ID NO:379, SEQ ID NO:381, SEQ ID NO:383, SEQ ID
NO:385, SEQ ID NO:387, SEQ ID NO:389, SEQ ID NO:391, SEQ ID NO:393, SEQ
ID NO:395, SEQ ID NO:397, SEQ ID NO:399, SEQ ID NO:401, SEQ ID NO:403,
SEQ ID NO:405, SEQ ID NO:407, SEQ ID NO:409, SEQ ID NO:411, SEQ ID
NO:413, SEQ ID NO:415, SEQ ID NO:417, SEQ ID NO:419, SEQ ID NO:421, SEQ
ID NO:423, SEQ ID NO:425, SEQ ID NO:427, SEQ ID NO:429, SEQ ID NO:431,
SEQ ID NO:433, SEQ ID NO:435, SEQ ID NO:437, SEQ ID NO:439, SEQ ID
NO:441, SEQ ID NO:443, SEQ ID NO:445, SEQ ID NO:447, SEQ ID NO:449, SEQ
ID NO:451, SEQ ID NO:453, SEQ ID NO:455, SEQ ID NO:457, SEQ ID NO:459,
SEQ ID NO:461, SEQ ID NO:463, SEQ ID NO:465, SEQ ID NO:467, SEQ ID
NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ ID NO:475, SEQ ID NO:477, SEQ
ID NO:479, SEQ ID NO:481, SEQ ID NO:483, SEQ ID NO:485, SEQ ID NO:487,
SEQ ID NO:489, SEQ ID NO:491, SEQ ID NO:493, SEQ ID NO:495, SEQ ID
NO:497, SEQ ID NO:499, SEQ ID NO:501, SEQ ID NO:503, SEQ ID NO:505, SEQ
ID NO:507, SEQ ID NO:509, SEQ ID NO:511, SEQ ID NO:513, SEQ ID NO:515 or
SEQ ID NO:517, or (b) the sequence of claim 1, wherein the probe
identifies the nucleic acid by binding or hybridization.
28-30. (canceled)
31: An amplification primer pair for amplifying a nucleic acid encoding a
polypeptide having a glucanase, mannanase or xylanase activity, wherein
the primer pair (a) is capable of amplifying a nucleic acid comprising
the sequence of claim 1, wherein a member of the amplification primer
pair comprises an oligonucleotide comprising at least about 10 to 50
consecutive bases of the sequence, or, about 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive bases
of the sequence; or, (b) comprises a first member having a sequence as
set forth by about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more residues of the sequence
of claim 1, and a second member having a sequence as set forth by about
the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30 or more residues of the complementary strand of
the first member.
32-33. (canceled)
34: A glucanase-encoding, mannanase-encoding or xylanase-encoding nucleic
acid comprising: a nucleic acid generated by amplification of a
polynucleotide using the amplification primer pair of claim 31; (b) the
nucleic acid of (a), wherein the amplification is by polymerase chain
reaction (PCR); (c) the nucleic acid of (b), wherein the nucleic acid is
generated by amplification of a gene library; or (d) the nucleic acid of
(c), wherein the gene library is an environmental library.
35-37. (canceled)
38: An isolated, synthetic or recombinant glucanase, mannanase or
xylanase encoded by the glucanase-encoding, mannanase-encoding or
xylanase-encoding nucleic acid of claim 34.
39: A method of amplifying a nucleic acid encoding a polypeptide having a
glucanase, mannanase or xylanase activity comprising amplification of a
template nucleic acid with an amplification primer sequence pair capable
of amplifying the nucleic acid sequence of claim 1.
40: An expression cassette comprising a nucleic acid comprising the
sequence of claim 1.
41: A vector comprising a nucleic acid comprising the sequence of as set
forth in claim 1.
42: A cloning vehicle comprising (a) a nucleic acid comprising the
sequence of claim 1, wherein the cloning vehicle comprises a viral
vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, a
bacteriophage or an artificial chromosome; (b) the cloning vehicle of
(a), wherein the viral vector comprises an adenovirus vector, a
retroviral vector or an adeno-associated viral vector; (c) the cloning
vehicle of (b), comprising a bacterial artificial chromosome (BAC), a
plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial
chromosome (YAC), or a mammalian artificial chromosome (MAC).
43-44. (canceled)
45: A transformed cell comprising (a) a nucleic acid comprising the
sequence of claim 1; (b) an expression cassette as set forth in claim 40;
or (c) the transformed cell of (a) or (b), wherein the cell is a
bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect
cell or a plant cell.
46-47. (canceled)
48: A transgenic non-human animal comprising (a) the sequence of claim 1;
or the trans genic non-human animal of (a), wherein the animal is a
mouse.
49. (canceled)
50: A transgenic plant comprising (a) the sequence of claim 1; or, the
transgenic plant of (a), wherein the plant is a corn plant, a sorghum
plant, a potato plant, a tomato plant, a wheat plant, an oilseed plant, a
rapeseed plant, a soybean plant, a rice plant, a barley plant, a grass,
or a tobacco plant.
51. (canceled)
52: A transgenic seed comprising (a) the sequence of claim 1; or, the
transgenic seed of (a), wherein the seed is a corn seed, a wheat kernel,
an oilseed, a rapeseed, a soybean seed, a palm kernel, a sunflower seed,
a sesame seed, a rice, a barley, a peanut or a tobacco plant seed.
53. (canceled)
54: An antisense oligonucleotide comprising (a) a nucleic acid sequence
complementary to or capable of hybridizing under stringent conditions to
the sequence of claim 1; or, (b) the antisense oligonucleotide of (a),
wherein the antisense oligonucleotide is between about 10 to 50, about 20
to 60, about 30 to 70, about 40 to 80, or about 60 to 100 bases in
length.
55. (canceled)
56: A method of inhibiting the translation of a glucanase, mannanase or
xylanase message in a cell comprising administering to the cell or
expressing in the cell an antisense oligonucleotide comprising a nucleic
acid sequence complementary to or capable of hybridizing under stringent
conditions to the sequence of claim 1.
57: A double-stranded inhibitory RNA (RNAi) molecule comprising (a) a
subsequence of the sequence of claim 1; or, (b) the sequence of (a),
wherein the RNAi is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or
more duplex nucleotides in length.
58. (canceled)
59: A method of inhibiting the expression of a glucanase in a cell
comprising administering to the cell or expressing in the cell a
double-stranded inhibitory RNA (iRNA), wherein the RNA comprises a
subsequence of the sequence of claim 1.
60: An isolated, synthetic or recombinant polypeptide (i) having an amino
acid sequence at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,
60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or
complete (100%) sequence identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID
NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID
NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID
NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID
NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID
NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID
NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID
NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID
NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ
ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,
SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132; SEQ ID
NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ
ID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID
NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ
ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172,
SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID
NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ
ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200,
SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID
NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ
ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228,
SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID
NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ
ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256,
SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID
NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ
ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284,
SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID
NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ
ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312,
SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID
NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ
ID NO:332, SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340,
SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:348, SEQ ID
NO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQ
ID NO:360, SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368,
SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:374, SEQ ID NO:376, SEQ ID
NO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ ID NO:384, SEQ ID NO:386, SEQ
ID NO:388, SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, SEQ ID NO:396,
SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402, SEQ ID NO:404, SEQ ID
NO:406, SEQ ID NO:408, SEQ ID NO:410, SEQ ID NO:412, SEQ ID NO:414, SEQ
ID NO:416, SEQ ID NO:418, SEQ ID NO:420, SEQ ID NO:422, SEQ ID NO:424,
SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, SEQ ID NO:432, SEQ ID
NO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:442, SEQ
ID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQ ID NO:450, SEQ ID NO:452,
SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458, SEQ ID NO:460, SEQ ID
NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ ID NO:470, SEQ
ID NO:472, SEQ ID NO:474, SEQ ID NO:476, SEQ ID NO:478, SEQ ID NO:480,
SEQ ID NO:482, SEQ ID NO:484, SEQ ID NO:486, SEQ ID NO:488, SEQ ID
NO:490, SEQ ID NO:492, SEQ ID NO:494, SEQ ID NO:496, SEQ ID NO:498, SEQ
ID NO:500, SEQ ID NO:502, SEQ ID NO:504, SEQ ID NO:506, SEQ ID NO:508,
SEQ ID NO:510, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:516 or SEQ ID
NO:518, over a region of at least about 20, 25, 30, 35, 40, 45, 50, 55,
60, 75 or 100, 150, 200, 250, 300 or more residues, or over the full
length of the polypeptide, wherein the polypeptide has: (i) a glucanase,
mannanase or xylanase activity, or (ii) immunogenic activity and can
generate an antibody that specifically binds to SEQ ID NO:2, SEQ ID NO:4,
SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ
ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID
NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID
NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID
NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID
NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID
NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID
NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID
NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID
NO:106, SEQ ID NO:108, SEQ ID NO:10, SEQ ID NO:112, SEQ ID NO:114, SEQ ID
NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ
ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132; SEQ ID NO:134;
SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ ID
NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID
NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ
ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172,
SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID
NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ
ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200,
SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID
NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ
ID NO:220, SEQ ID NO:222. SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228,
SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID
NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ
ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256,
SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID
NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ
ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284,
SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID
NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ
ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312,
SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID
NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ
ID NO:332, SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340,
SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:348, SEQ ID
NO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQ
ID NO:360, SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368,
SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:374, SEQ ID NO:376, SEQ ID
NO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ ID NO:384, SEQ ID NO:386, SEQ
ID NO:388, SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, SEQ ID NO:396,
SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402, SEQ ID NO:404, SEQ ID
NO:406, SEQ ID NO:408, SEQ ID NO:410, SEQ ID NO:412, SEQ ID NO:414, SEQ
ID NO:416, SEQ ID NO:418, SEQ ID NO:420, SEQ ID NO:422, SEQ ID NO:424,
SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, SEQ ID NO:432, SEQ ID
NO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:442, SEQ
ID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQ ID NO:450, SEQ ID NO:452,
SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458, SEQ ID NO:460, SEQ ID
NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ ID NO:470, SEQ
ID NO:472, SEQ ID NO:474, SEQ ID NO:476, SEQ ID NO:478, SEQ ID NO:480,
SEQ ID NO:482, SEQ ID NO:484, SEQ ID NO:486, SEQ ID NO:488, SEQ ID
NO:490, SEQ ID NO:492, SEQ ID NO:494, SEQ ID NO:496, SEQ ID NO:498, SEQ
ID NO:500, SEQ ID NO:502, SEQ ID NO:504, SEQ ID NO:506, SEQ ID NO:508,
SEQ ID NO:510, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:516 or SEQ ID
NO:518; (ii) the amino acid sequence of (i), wherein the sequence
identities are determined by analysis with a sequence comparison
algorithm or by a visual inspection; (iii) the nucleic acid sequence of
(b), wherein the sequence comparison algorithm comprises a BLAST version
2.2.2 algorithm having a filtering setting set to blastall-p blastp-d "nr
pataa"-F F, and all other options are set to default; (iv) an amino acid
sequence encoded by the nucleic acid of claim 1; (v) the amino acid
sequence of (i), (ii), (iii) or (iv) and having at least one conservative
amino acid residue substitution and retaining a glucanase, mannanase or
xylanase activity, wherein a conservative substitution comprises
substituting an amino acid residue by another amino acid of like
characteristics; (vi) the amino acid sequence of (v), wherein the
conservative substitution comprises replacement of an aliphatic amino
acid with another aliphatic amino acid; or, replacement of a Serine with
a Threonine or vice versa; or, replacement of an acidic residue with
another acidic residue; or, replacement of a residue bearing an amide
group with another residue bearing an amide group; or, exchange of a
basic residue with another basic residue; or, replacement of an aromatic
residue with another aromatic residue, or a combination thereof; (vii)
the amino acid sequence of (vi), wherein the aliphatic residue comprises
Alanine, Valine, Leucine, Isoleucine or a synthetic equivalent thereof;
or, the acidic residue comprises Aspartic acid, Glutamic acid or a
synthetic equivalent thereof; or, the residue comprising an amide group
comprises Aspartic acid, Glutamic acid or a synthetic equivalent thereof;
or, the basic residue comprises Lysine, Arginine or a synthetic
equivalent thereof; or, the aromatic residue comprises Phenylalanine,
Tyrosine or a synthetic equivalent thereof; (viii) the amino acid
sequence of (i), (ii), (iii), (iv), (v), (vi) or (vii), wherein the
glucanase, mannanase or xylanase activity comprises: an endoglucanase
activity; or catalyzing hydrolysis of 1,4-beta-D-glycosidic linkages or
internal .beta.-1,3-glucosidic linkages, or, hydrolyzing a glucan to
produce a smaller molecular weight polysaccharide or oligomer; or an
endoglucanase activity comprising an endo-1,4-beta-endoglucanase
activity; or an endoglucanase activity comprising 1,4-beta-D-glycosidic
linkage activity; or an endoglucanase activity comprising hydrolysis of a
1,4-beta-D-glycosidic linkage in a cellulose, a cellulose derivative, a
lichenin or a cereal; or hydrolyzing a glycosidic linkage in a cellulose
derivative; or hydrolyzing a glycosidic linkage in a carboxy methyl
cellulose or a hydroxy ethyl cellulose; or hydrolyzing a glycosidic
linkage in a cereal; or hydrolyzing a glycosidic linkage in a
beta-D-glucan or a xyloglucan; or hydrolyzing polysaccharides comprising
1,4-.beta.-glycoside-linked D-glucopyranoses; or hydrolyzing a cellulose,
a cellulose derivative or a hemicellulose; or hydrolyzing a cellulose or
a hemicellulose in a wood or paper pulp or a wood or paper product; or
catalyzing hydrolysis of glucan in a feed, a food product or a beverage;
or hydrolyzing a glucan in a feed, food product or beverage; hydrolyzing
a glycosidic linkage or a glucan in a cereal-based animal feed, a wort or
a beer, a dough, a fruit or a vegetable; or hydrolyzing a glucan in a
microbial cell, a fungal cell, a mammalian cell or a plant cell; (ix) the
amino acid sequence of (i), (ii), (iii), (iv), (v), (vi), (vii) or
(viii), wherein the glucanase, mannanase or xylanase activity is
thermostable; or the polypeptide retains a glucanase, mannanase or
xylanase activity under conditions comprising a temperature range of
between about 37.degree. C. to about 95.degree. C., or between about
55.degree. C. to about 85.degree. C., or between about 70.degree. C. to
about 75.degree. C., or between about 70.degree. C. to about 95.degree.
C., or between about 90.degree. C. to about 95.degree. C.; (x) the amino
acid sequence of (i), (ii), (iii), (iv), (v), (vi), (vii) or (viii),
wherein the glucanase activity is thermotolerant; or the polypeptide
retains a glucanase activity after exposure to a temperature in the range
from greater than 37.degree. C. to about 95.degree. C., from greater than
55.degree. C. to about 85.degree. C., or between about 70.degree. C. to
about 75.degree. C., or from greater than 90.degree. C. to about
95.degree. C.; (xi) the amino acid sequence of (i), (ii), (iii), (iv),
(v), (vi), (vii), (viii), (ix) or (x), wherein the glucanase activity
comprises a specific activity at about 37.degree. C. in the range from
about 100 to about 1000 units per milligram of protein, from about 500 to
about 750 units per milligram of protein, from about 500 to about 1200
units per milligram of protein, or from about 750 to about 1000 units per
milligram of protein; (xii) the amino acid sequence of (x), wherein the
thermotolerance comprises retention of at least half of the specific
activity of the glucanase at 37.degree. C. after being heated to an
elevated temperature, or, the thermotolerance comprises retention of
specific activity at 37.degree. C. in the range from about 500 to about
1200 units per milligram of protein after being heated to an elevated
temperature; (xiii) the amino acid sequence of any of (i) through (xii),
wherein the polypeptide comprises at least one glycosylation site; or,
wherein the polypeptide comprises at least one N-linked glycosylation
site, or, wherein the polypeptide is glycosylated after being expressed
in a P. pastoris or a S. pombe; (xiv) the amino acid sequence of any of
(i) through (xiii), wherein the polypeptide retains a glucanase activity
under conditions comprising about pH 6.5, pH 6.0, pH 5.5, 5.0, pH 4.5 or
4.0; or, the polypeptide retains a glucanase activity under conditions
comprising about pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10 or pH 10.5;
(xiv) the amino acid sequence of any of (i) through (xiii), wherein the
polypeptide lacks a signal sequence or a prepro sequence; (xv) the amino
acid sequence of any of (i) through (xiv), and having a heterologous
signal sequence or a heterologous prepro sequence, or having a yeast
signal sequence; or (xv) the amino acid sequence of any of (i) through
(xiv), and having: (a) a heterologous sequence, or (b) a heterologous
sequence not naturally associated with a glucanase, mannanase, or
xylanase.
61-90. (canceled)
91: A protein preparation comprising the polypeptide of claim 60, wherein
the protein preparation comprises a liquid, a solid or a gel.
92: A heterodimer comprising: (a) the polypeptide of claim 60 and a
second domain; the heterodimer of (a), wherein the second domain is a
polypeptide and the heterodimer is a fusion protein; or (c) the
heterodimer of (a) or (b), wherein the second domain is an epitope or a
tag.
93-94. (canceled)
95: A homodimer comprising the polypeptide of claim 60.
96: An immobilized polypeptide, wherein the polypeptide comprises: (a)
the sequence of claim 60; or, (b) the immobilized polypeptide of (a),
wherein the polypeptide is immobilized on a cell, a metal, a resin, a
polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a
bead, a gel, a plate, an array or a capillary tube.
97. (canceled)
98: An array comprising: (a) an immobilized polypeptide comprising the
amino acid sequence of claim 60; or (b) an immobilized nucleic acid
comprising the nucleic acid sequence of claim 1; or (c) a combination
thereof.
99. (canceled)
100: An isolated, synthetic or recombinant antibody (a) that specifically
binds to the polypeptide of claim 60; or (b) the antibody of (a), wherein
the antibody is a monoclonal or a polyclonal antibody.
101. (canceled)
102: A hybridoma comprising an antibody that specifically binds to the
polypeptide of claim 60.
103: A method of isolating or identifying a polypeptide with a glucanase,
mannanase or xylanase activity comprising the steps of: (a) providing the
antibody of claim 100; (b) providing a sample comprising polypeptides;
and (c) contacting the sample of step (b) with the antibody of step (a)
under conditions wherein the antibody can specifically bind to the
polypeptide, thereby isolating or identifying a polypeptide having a
glucanase, mannanase or xylanase activity.
104: A method of making an anti-glucanase, anti-mannanase or
anti-xylanase antibody comprising (a) administering to a non-human animal
the nucleic acid of claim 1 in an amount sufficient to generate a humoral
immune response, thereby making an anti-glucanase, anti-mannanase or
anti-xylanase antibody; or, (b) administering to a non-human animal the
polypeptide of claim 60 in an amount sufficient to generate a humoral
immune response, thereby making an anti-glucanase, anti-mannanase or
anti-xylanase antibody.
105. (canceled)
106: A method of producing a recombinant polypeptide comprising the steps
of: (A) (a) providing a nucleic acid operably linked to a promoter,
wherein the nucleic acid comprises the sequence of claim 1; and (b)
expressing the nucleic acid of step (a) under conditions that allow
expression of the polypeptide, thereby producing a recombinant
polypeptide; or (B) the method of (A), further comprising transforming a
host cell with the nucleic acid of step (A) (a) followed by expressing
the nucleic acid of step (A) (a), thereby producing a recombinant
polypeptide in a transformed cell.
107. (canceled)
108: A method for identifying a polypeptide having a glucanase, mannanase
or xylanase activity comprising the following steps: (a) providing the
polypeptide of claim 60; (b) providing a glucanase, mannanase or xylanase
substrate; and (c) contacting the polypeptide with the substrate of step
(b) and detecting a decrease in the amount of substrate or an increase in
the amount of a reaction product, wherein a decrease in the amount of the
substrate or an increase in the amount of the reaction product detects a
polypeptide having a glucanase, mannanase or xylanase activity.
109: A method for identifying a glucanase, mannanase or xylanase
substrate comprising the following steps: (a) providing the polypeptide
of claim 60; (b) providing a test substrate; and (c) contacting the
polypeptide of step (a) with the test substrate of step (b) and detecting
a decrease in the amount of substrate or an increase in the amount of
reaction product, wherein a decrease in the amount of the substrate or an
increase in the amount of a reaction product identifies the test
substrate as a glucanase, mannanase or xylanase substrate.
110: A method of determining whether a test compound specifically binds
to a polypeptide comprising the following steps: (a) expressing a nucleic
acid or a vector comprising the nucleic acid under conditions permissive
for translation of the nucleic acid to a polypeptide, wherein the nucleic
acid has the sequence of claim 1; (b) providing a test compound; (c)
contacting the polypeptide with the test compound; and (d) determining
whether the test compound of step (b) specifically binds to the
polypeptide.
111: A method of determining whether a test compound specifically binds
to a polypeptide comprising the following steps: (a) providing the
polypeptide of claim 60; (b) providing a test compound; (c) contacting
the polypeptide with the test compound; and (d) determining whether the
test compound of step (b) specifically binds to the polypeptide.
112: A method for identifying a modulator of a glucanase, mannanase or
xylanase activity comprising the following steps: (A) (a) providing the
polypeptide of claim 60; (b) providing a test compound; (c) contacting
the polypeptide of step (a) with the test compound of step (b) and
measuring an activity of the glucanase, wherein a change in the
glucanase, mannanase or xylanase activity measured in the presence of the
test compound compared to the activity in the absence of the test
compound provides a determination that the test compound modulates the
glucanase, mannanase or xylanase activity; or (B) the method of (A),
wherein the glucanase, mannanase or xylanase activity is measured by
providing a glucanase substrate and detecting a decrease in the amount of
the substrate or an increase in the amount of a reaction product, or, an
increase in the amount of the substrate or a decrease in the amount of a
reaction product; or (C) the method of (B), wherein a decrease in the
amount of the substrate or an increase in the amount of the reaction
product with the test compound as compared to the amount of substrate or
reaction product without the test compound identifies the test compound
as an activator of a glucanase, mannanase or xylanase activity; or,
wherein an increase in the amount of the substrate or a decrease in the
amount of the reaction product with the test compound as compared to the
amount of substrate or reaction product without the test compound
identifies the test compound as an inhibitor of a glucanase, mannanase or
xylanase activity.
113-115. (canceled)
116: A computer system comprising (A) a processor and a data storage
device wherein said data storage device has stored thereon a polypeptide
sequence or a nucleic acid sequence, wherein the polypeptide sequence
comprises the sequence of claim 60, or a polypeptide encoded by the
nucleic acid of claim 1; (B) the computer system of (A), further
comprising a sequence comparison algorithm and a data storage device
having at least one reference sequence stored thereon; (C) the computer
system of (B), wherein the sequence comparison algorithm comprises a
computer program that indicates polymorphisms; or (D) the computer system
of (A), (B) or (C), further comprising an identifier that identifies one
or more features in said sequence.
117-119. (canceled)
120: A computer readable medium having stored thereon a polypeptide
sequence or a nucleic acid sequence, wherein the polypeptide sequence
comprises the polypeptide of claim 60; or a polypeptide encoded by the
nucleic acid of claim 1.
121: A method for identifying a feature in a sequence comprising the
steps of: (a) reading the sequence using a computer program which
identifies one or more features in a sequence, wherein the sequence
comprises a polypeptide sequence or a nucleic acid sequence, wherein the
polypeptide sequence comprises the polypeptide of claim 60; or a
polypeptide encoded by the nucleic acid of claim 1; and (b) identifying
one or more features in the sequence with the computer program.
122: A method for comparing a first sequence to a second sequence
comprising the steps of: (A) (a) reading the first sequence and the
second sequence through use of a computer program which compares
sequences, wherein the first sequence comprises a polypeptide sequence or
a nucleic acid sequence, wherein the polypeptide sequence comprises the
polypeptide of claim 60; or a polypeptide encoded by the nucleic acid of
claim 1; and (b) determining differences between the first sequence and
the second sequence with the computer program; (B) the method of (A),
wherein the step of determining differences between the first sequence
and the second sequence further comprises the step of identifying
polymorphisms; (C) the method of (B), further comprising an identifier
that identifies one or more features in a sequence; (D) the method of
(A), (B) or (C), comprising reading the first sequence using a computer
program and identifying one or more features in the sequence.
123-125. (canceled)
126: A method for isolating or recovering a nucleic acid encoding a
polypeptide with a glucanase, mannanase or xylanase activity from an
environmental sample comprising the steps of: (A) (a) providing the
amplification primer pair of claim 31; (b) isolating a nucleic acid from
the environmental sample or treating the environmental sample such that
nucleic acid in the sample is accessible for hybridization to the
amplification primer pair; and, (c) combining the nucleic acid of step
(b) with the amplification primer pair of step (a) and amplifying nucleic
acid from the environmental sample, thereby isolating or recovering a
nucleic acid encoding a polypeptide with a glucanase, mannanase or
xylanase activity from an environmental sample; (B) the method of (A),
wherein each member of the amplification primer pair comprises an
oligonucleotide comprising at least about 10 to 50, or about 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more
consecutive bases of, the sequence of claim 1; (C) (a) providing a
polynucleotide probe comprising the sequence of claim 1; (b) isolating a
nucleic acid from the environmental sample or treating the environmental
sample such that nucleic acid in the sample is accessible for
hybridization to a polynucleotide probe of step (a); (c) combining the
isolated nucleic acid or the treated environmental sample of step (b)
with the polynucleotide probe of step (a); and (d) isolating a nucleic
acid that specifically hybridizes with the polynucleotide probe of step
(a), thereby isolating or recovering a nucleic acid encoding a
polypeptide with a glucanase, mannanase or xylanase activity from an
environmental sample; or (D) the method of (A), (B) or (C), wherein the
environmental sample comprises a water sample, a liquid sample, a soil
sample, an air sample or a biological sample, or, wherein the biological
sample is derived from a bacterial cell, a protozoan cell, an insect
cell, a yeast cell, a plant cell, a fungal cell or a mammalian cell.
127-130. (canceled)
131: A method of generating a variant of a nucleic acid encoding a
polypeptide with a glucanase, mannanase or xylanase activity comprising
the steps of: (A) (a) providing a template nucleic acid comprising the
sequence of claim 1; and (b) modifying, deleting or adding one or more
nucleotides in the template sequence, or a combination thereof, to
generate a variant of the template nucleic acid; or (B) the method of
(A), further comprising expressing the variant nucleic acid to generate a
variant glucanase, mannanase or xylanase polypeptide; (C) the method of
claim (A) or (B), wherein the modifications, additions or deletions are
introduced by a method comprising error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis, site-specific
mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis.TM.
(GSSM.TM.), synthetic ligation reassembly (SLR), recombination, recursive
sequence recombination, phosphothioate-modified DNA mutagenesis,
uracil-containing template mutagenesis, gapped duplex mutagenesis, point
mismatch repair mutagenesis, repair-deficient host strain mutagenesis,
chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification mutagenesis,
artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid
multimer creation, or any combination thereof; or (D) the method of (A)
or (B) iteratively repeated until a glucanase, mannanase or xylanase
having an altered or different activity or an altered or different
stability from that of a polypeptide encoded by the template nucleic acid
is produced; (E) the method of (A), (B), (C) or (D), wherein the variant
glucanase, mannanase or xylanase polypeptide is thermotolerant, and
retains some activity after being exposed to an elevated temperature; (F)
the method of (A), (B), (C) or (D), wherein the variant glucanase,
mannanase or xylanase polypeptide has increased glycosylation as compared
to the glucanase, mannanase or xylanase encoded by a template nucleic
acid; (G) the method of (A), (B), (C) or (D), wherein the variant
glucanase, mannanase or xylanase polypeptide has a glucanase activity
under a high temperature, wherein the glucanase, mannanase or xylanase
encoded by the template nucleic acid is not active under the high
temperature; (H) the method of (A), (B), (C) or (D), wherein the method
is iteratively repeated until a glucanase, mannanase or xylanase coding
sequence having an altered codon usage from that of the template nucleic
acid is produced; or (I) the method of (A), (B), (C) or (D), wherein the
method is iteratively repeated until a glucanase, mannanase or xylanase
gene having higher or lower level of message expression or stability from
that of the template nucleic acid is produced.
132-140. (canceled)
141: A method for modifying codons in a nucleic acid encoding a
polypeptide with a glucanase, mannanase or xylanase activity to increase
or decrease its expression in a host cell, the method comprising the
following steps: (A) (a) providing a nucleic acid encoding a polypeptide
with a glucanase, mannanase or xylanase activity comprising a sequence as
set forth in claim 1; and, (b) identifying a non-preferred or a less
preferred codon in the nucleic acid of step (a) and replacing it with a
preferred or neutrally used codon encoding the same amino acid as the
replaced codon, wherein a preferred codon is a codon over-represented in
coding sequences in genes in the host cell and a non-preferred or less
preferred codon is a codon under-represented in coding sequences in genes
in the host cell, thereby modifying the nucleic acid to increase its
expression in a host cells; (B) (a) providing a nucleic acid encoding a
polypeptide with a glucanase activity comprising the sequence of claim 1;
and, (b) identifying a codon in the nucleic acid of step (a) and
replacing it with a different codon encoding the same amino acid as the
replaced codon, thereby modifying codons in a nucleic acid encoding a
glucanase; or, (C) (a) providing a nucleic acid encoding a glucanase
polypeptide comprising the sequence of claim 1; and, (b) identifying a
non-preferred or a less preferred codon in the nucleic acid of step (a)
and replacing it with a preferred or neutrally used codon encoding the
same amino acid as the replaced codon, wherein a preferred codon is a
codon over-represented in coding sequences in genes in the host cell and
a non-preferred or less preferred codon is a codon under-represented in
coding sequences in genes in the host cell, thereby modifying the nucleic
acid to increase its expression in a host cell; (D) (a) providing a
nucleic acid encoding a glucanase polypeptide comprising the sequence of
claim 1; and (b) identifying at least one preferred codon in the nucleic
acid of step (a) and replacing it with a non-preferred or less preferred
codon encoding the same amino acid as the replaced codon, wherein a
preferred codon is a codon over-represented in coding sequences in genes
in a host cell and a non-preferred or less preferred codon is a codon
under-represented in coding sequences in genes in the host cell, thereby
modifying the nucleic acid to decrease its expression in a host cell; or,
(E) the method of (A), (B), (C) or (D), wherein the host cell is a
bacterial cell, a fungal cell, an insect cell, a yeast cell, a plant cell
or a mammalian cell.
142-145. (canceled)
146: A method for producing a library of nucleic acids encoding a
plurality of modified glucanase, mannanase or xylanase active sites or
substrate binding sites, wherein the modified active sites or substrate
binding sites are derived from a first nucleic acid comprising a sequence
encoding a first active site or a first substrate binding site the method
comprising the following steps: (A) (a) providing a first nucleic acid
encoding a first active site or first substrate binding site, wherein the
first nucleic acid sequence comprises a sequence that hybridizes under
stringent conditions to the sequence of claim 1, and the nucleic acid
encodes a glucanase active site or a glucanase substrate binding site;
(b) providing a set of mutagenic oligonucleotides that encode
naturally-occurring amino acid variants at a plurality of targeted codons
in the first nucleic acid; and, (c) using the set of mutagenic
oligonucleotides to generate a set of active site-encoding or substrate
binding site-encoding variant nucleic acids encoding a range of amino
acid variations at each amino acid codon that was mutagenized, thereby
producing a library of nucleic acids encoding a plurality of modified
glucanase active sites or substrate binding sites; (B) the method of (A),
comprising mutagenizing the first nucleic acid of step (a) by a method
comprising an optimized directed evolution system, Gene Site-Saturation
Mutagenesis.TM. (GSSM.TM.), or a synthetic ligation reassembly (SLR); or
(C) the method of (A), comprising mutagenizing the first nucleic acid of
step (a) or variants by a method comprising error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis, site-specific
mutagenesis, gene reassembly, Gene Site-Saturation Mutagenesis.TM.
(GSSM.TM.), synthetic ligation reassembly (SLR), recombination, recursive
sequence recombination, phosphothioate-modified DNA mutagenesis,
uracil-containing template mutagenesis, gapped duplex mutagenesis, point
mismatch repair mutagenesis, repair-deficient host strain mutagenesis,
chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis
restriction-selection mutagenesis, restriction-purification mutagenesis,
artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid
multimer creation, or a combination thereof.
147-161. (canceled)
162: An isolated, synthetic or recombinant signal sequence comprising, or
consisting of, the sequence of claim 60 or a sequence as set forth in
residues 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1
to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1
to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1
to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1 to 43 or 1 to 44, of SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID
NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID
NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID
NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID
NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID
NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID
NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ
ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122,
SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID
NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ
ID NO:142; SEQ ID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID
NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ
ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170,
SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID
NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ
ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198,
SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID
NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ
ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226,
SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID
NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ
ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254,
SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID
NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ
ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282,
SEQ ID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID
NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ
ID NO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310,
SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID
NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ
ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:338,
SEQ ID NO:340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID
NO:348, SEQ ID NO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ
ID NO:358, SEQ ID NO:360, SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:366,
SEQ ID NO:368, SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:374, SEQ ID
NO:376, SEQ ID NO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ ID NO:384, SEQ
ID NO:386, SEQ ID NO:388, SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394,
SEQ ID NO:396, SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402, SEQ ID
NO:404, SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, SEQ ID NO:412, SEQ
ID NO:414, SEQ ID NO:416, SEQ ID NO:418, SEQ ID NO:420, SEQ ID NO:422,
SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, SEQ ID
NO:432, SEQ ID NO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ
ID NO:442, SEQ ID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQ ID NO:450,
SEQ ID NO:452, SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458, SEQ ID
NO:460, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ
ID NO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ ID NO:476, SEQ ID NO:478,
SEQ ID NO:480, SEQ ID NO:482, SEQ ID NO:484, SEQ ID NO:486, SEQ ID
NO:488, SEQ ID NO:490, SEQ ID NO:492, SEQ ID NO:494, SEQ ID NO:496, SEQ
ID NO:498, SEQ ID NO:500, SEQ ID NO:502, SEQ ID NO:504, SEQ ID NO:506,
SEQ ID NO:508, SEQ ID NO:510, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:516
or SEQ ID NO:518; or, comprising, or consisting of a sequence as set
forth in Table 3.
163: A chimeric polypeptide comprising (A) at least a first domain
comprising signal peptide (SP) having the sequence of claim 162, and at
least a second domain comprising a heterologous polypeptide or peptide,
wherein the heterologous polypeptide or peptide is not naturally
associated with the signal peptide (SP); (B) the chimeric polypeptide of
(A), wherein the heterologous polypeptide or peptide is not a glucanase,
a mannanase or a xylanase, or an endoglucanase; or (C) the chimeric
polypeptide of (B), wherein the heterologous polypeptide or peptide is
amino terminal to, carboxy terminal to or on both ends of the signal
peptide (SP) or a glucanase, a mannanase or a xylanase, or an
endoglucanase catalytic domain (CD).
164: An isolated, synthetic or recombinant nucleic acid encoding a
chimeric polypeptide, wherein the chimeric polypeptide comprises at least
a first domain comprising signal peptide (SP) having the sequence of
claim 162 and at least a second domain comprising a heterologous
polypeptide or peptide, wherein the heterologous polypeptide or peptide
is not naturally associated with the signal peptide (SP).
165-172. (canceled)
173: A method for hydrolyzing, breaking up or disrupting a
glucan-comprising composition comprising the following steps: (A) (a)
providing the polypeptide having a glucanase activity of claim 60, or a
polypeptide encoded by the nucleic acid of claim 1; (b) providing a
composition comprising a glucan; and (c) contacting the polypeptide of
step (a) with the composition of step (b) under conditions wherein the
glucanase hydrolyzes, breaks up or disrupts the glucan-comprising
composition; or (B) the method of (A), wherein the composition comprises
a plant cell, a bacterial cell, a yeast cell, an insect cell, or an
animal cell.
174. (canceled)
175: A dough or a bread product comprising the polypeptide of claim 60.
176: A method of dough conditioning comprising contacting a dough or a
bread product with at least one polypeptide of claim 60 under conditions
sufficient for conditioning the dough.
177: A beverage comprising the polypeptide of claim 60.
178: A method of beverage production comprising (A) administration of at
least one polypeptide of claim 60 to a beverage or a beverage precursor
under conditions sufficient for decreasing the viscosity of the beverage;
or (B) the method of (A), wherein the beverage or beverage precursor is a
wort or a beer.
179. (canceled)
180: A food, a feed or a nutritional supplement comprising the
polypeptide of claim 60.
181: A method for utilizing a glucanase, mannanase or xylanase as a
nutritional supplement in an animal diet, the method comprising: (A)
preparing a nutritional supplement containing a glucanase, mannanase or
xylanase enzyme comprising at least thirty contiguous amino acids of a
polypeptide as set forth in claim 60; and administering the nutritional
supplement to an animal to increase utilization of a xylan contained in a
feed or a food ingested by the animal; (B) the method of (A), wherein the
animal is a human; (C) the method of (A), wherein the animal is a
ruminant or a monogastric animal; (D) the method of (A), (B) or (C),
wherein the glucanase, mannanase or xylanase enzyme is prepared by
expression of a polynucleotide encoding the glucanase in an organism
selected from the group consisting of a bacterium, a yeast, a plant, an
insect, a fungus and an animal; or (E) the method of (A), (B), (C) or
(D), wherein the organism is selected from the group consisting of an S.
pombe, S. cerevisiae, Pichia pastoris, E. coli, Streptomyces sp.,
Bacillus sp. and Lactobacillus sp.
182-186. (canceled)
187: An edible enzyme delivery matrix comprising (a) a thermostable
recombinant glucanase, mannanase or xylanase enzyme; or (b) the edible
enzyme delivery matrix of (a) comprising the polypeptide of claim 60.
188. (canceled)
189: A method for delivering a glucanase, mannanase or xylanase
supplement to an animal, the method comprising: (a) preparing an edible
enzyme delivery matrix in the form of pellets comprising a granulate
edible carrier and a thermostable recombinant glucanase, mannanase or
xylanase enzyme, wherein the pellets readily disperse the glucanase,
mannanase or xylanase enzyme contained therein into aqueous media, and
administering the edible enzyme delivery matrix to the animal; (b) the
method of (a), wherein the recombinant glucanase, mannanase or xylanase
enzyme comprises the polypeptide of claim 60; (c) the method of (a) or
(b), wherein the granulate edible carrier comprises a carrier comprising
a grain germ, a grain germ that is spent of oil, a hay, an alfalfa, a
timothy, a soy hull, a sunflower seed meal, a wheat midd or a combination
thereof; (d) the method of (a), (b) or (c), wherein the edible carrier
comprises grain germ that is spent of oil; (e) the method of (a), (b),
(c) or (d), wherein the glucanase, mannanase or xylanase enzyme is
glycosylated to provide thermostability at pelletizing conditions; (f)
the method of (a), (b), (c), (d) or (e), wherein the delivery matrix is
formed by pelletizing a mixture comprising a grain germ and a glucanase,
mannanase or xylanase; (g) the method of (a), (b), (c), (d), (e) or (f),
wherein the pelletizing conditions include application of steam; (h) the
method of (a), (b), (c), (d), (e), (f) or (g), wherein the pelletizing
conditions comprise application of a temperature in excess of about
80.degree. C. for about 5 minutes and the enzyme retains a specific
activity of at least 350 to about 900 units per milligram of enzyme.
190-196. (canceled)
197: An isolated, synthetic or recombinant nucleic acid comprising (a) a
sequence encoding a polypeptide having a glucanase, mannanase or xylanase
activity and a signal sequence, wherein the nucleic acid comprises the
sequence of claim 1; or (b) the nucleic acid of (a), wherein the signal
sequence is derived from another glucanase, mannanase or xylanase or a
non-glucanase, mannanase or xylanase enzyme.
198. (canceled)
199: An isolated, synthetic or recombinant nucleic acid comprising a
sequence encoding a polypeptide having a glucanase, mannanase or xylanase
activity, wherein the sequence does not contain a signal sequence and the
nucleic acid comprises a sequence of claim 1.
200: A cellulose- or cellulose derivative-composition comprising the
polypeptide of claim 60.
201: A wood, wood pulp or wood product comprising the polypeptide of
claim 60.
202: A paper, paper pulp or paper product comprising the polypeptide of
claim 60.
203: A method for reducing lignin in a paper, a wood or wood product
comprising contacting the paper, wood or wood product with the
polypeptide of claim 60.
204: A detergent composition comprising a polypeptide as set forth in
claim 60.
205: A pharmaceutical composition comprising the polypeptide of claim 60.
206: A method for eliminating or protecting animals from a microorganism
comprising (a) a glucan comprising administering the polypeptide of claim
60; (b) the method of (a), wherein the microorganism is a bacterium; or
(c) the method of (b) wherein the bacterium is a Salmonellae.
207-208. (canceled)
209: A fuel comprising the polypeptide of claim 60.
210: A method for making a fuel comprising contacting a fermentable sugar
with the polypeptide of claim 60.
211: A dairy product comprising: (a) the polypeptide of claim 60; or (b)
the dairy product of (a), comprising a milk, an ice cream, a cheese or a
yogurt.
212. (canceled)
213. A method for improving texture and flavor of a dairy product
comprising the following steps: (a) providing the polypeptide of claim
60; (b) providing a dairy product; and (c) contacting the polypeptide of
step (a) and the dairy product of step (b) under conditions wherein the
glucanase can improve the texture or flavor of the dairy product.
214-220. (canceled)
Description
REFERENCE TO SEQUENCE LISTING SUBMITTED ON A COMPACT DISC
[0001] This application includes a compact disc (submitted in
quadruplicate) containing a sequence listing. The entire content of the
sequence listing is herein incorporated by reference. The sequence
listing is identified on the compact disc as follows.
TABLE-US-00001
File Name Date of Creation Size (bytes)
Sequence Listing.txt Jul. 2, 2004 1,562,624
FIELD OF THE INVENTION
[0002] This invention relates generally to enzymes, polynucleotides
encoding the enzymes, the use of such polynucleotides and polypeptides
and more specifically to polypeptides (e.g., enzymes, antibodies) having
a glucanase, e.g., an endoglucanase, activity, e.g., catalyzing
hydrolysis of internal endo-.beta.-1,4- and/or .beta.-1,3-glucanase
linkages. In one aspect the endoglucanase activity (e.g.,
endo-1,4-beta-D-glucan 4-glucano hydrolase activity) comprises hydrolysis
of 1,4- and/or .beta.-1,3-beta-D-glycosidic linkages in cellulose,
cellulose derivatives (e.g., carboxy methyl cellulose and hydroxy ethyl
cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans, such as
cereal beta-D-glucans or xyloglucans and other plant or organic material
containing cellulosic parts. In one aspect, the polypeptides of the
invention have a xylanase, or a mannanase activity.
BACKGROUND
[0003] Endoglucanases (e.g., endo-beta-1,4-glucanases, EC 3.2.1.4;
endo-beta-1,3(1)-glucanases, EC 3.2.1.6; endo-beta-1,3-glucanases, EC
3.2.1.39) hydrolyze internal .beta.-1,4- and/or .beta.1,3-glucosidic
linkages in cellulose and glucan to produce smaller molecular weight
glucose and glucose oligomers. Glucans are polysaccharides formed from
1,4-.beta.- and/or 1,3-glycoside-linked D-glucopyranose. Endoglucanases
are of considerable commercial value, being used in the food industry,
for baking and fruit and vegetable processing, breakdown of agricultural
waste, in the manufacture of animal feed (e.g., chicken feed), in pulp
and paper production, textile manufacture and household and industrial
cleaning agents. Endoglucanases are produced by fungi and bacteria.
[0004] Beta-glucans are major non-starch polysaccharides of cereals. The
glucan content can vary significantly depending on variety and growth
conditions. The physicochemical properties of this polysaccharide are
such that it gives rise to viscous solutions or even gels under oxidative
conditions. In addition glucans have high water-binding capacity. All of
these characteristics present problems for several industries including
brewing, baking, animal nutrition. In brewing applications, the presence
of glucan results in wort filterability and haze formation issues. In
baking applications (especially for cookies and crackers), glucans can
create sticky doughs that are difficult to machine and reduce biscuit
size. In addition, this carbohydrate is implicated in rapid rehydration
of the baked product resulting in loss of crispiness and reduced
shelf-life. For monogastric animal feed applications with cereal diets,
beta-glucan is a contributing factor to viscosity of gut contents and
thereby adversely affects the digestibility of the feed and animal growth
rate. For ruminant animals, these beta-glucans represent substantial
components of fiber intake and more complete digestion of glucans would
facilitate higher feed conversion efficiencies. It is desirable for
animal feed endoglucanases to be active in the animal stomach.
[0005] Endoglucanases are also important for the digestion of cellulose, a
beta-1,4-linked glucan found in all plant material. Cellulose is the most
abundant polysaccharide in nature. Commercial enzymes that digest
cellulose have utility in the pulp and paper industry, in textile
manufacture and in household and industrial cleaning agents.
[0006] The publications discussed herein are provided solely for their
disclosure prior to the filing date of the present application. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior invention.
SUMMARY OF THE INVENTION
[0007] The invention provides isolated, synthetic or recombinant nucleic
acids comprising a nucleic acid sequence having at least about 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,
66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence
identity to an exemplary nucleic acid of the invention, e.g., SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,
SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ
ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID
NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID
NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID
NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID
NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID
NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID
NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID
NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID
NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ
ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121,
SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID
NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ
ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149,
SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID
NO:199, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ
ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177,
SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID
NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ
ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205,
SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID
NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ
ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233,
SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID
NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ
ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261,
SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID
NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ
ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289,
SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID
NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ
ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317,
SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID
NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ
ID NO:337, SEQ ID NO:339, SEQ ID NO:341, SEQ ID NO:343, SEQ ID NO:345,
SEQ ID NO:347, SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:353, SEQ ID
NO:355, SEQ ID NO:357, SEQ ID NO:359, SEQ ID NO:361, SEQ ID NO:363, SEQ
ID NO:365, SEQ ID NO:367, SEQ ID NO:369, SEQ ID NO:371, SEQ ID NO:373,
SEQ ID NO:375, SEQ ID NO:377, SEQ ID NO:379, SEQ ID NO:381, SEQ ID
NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQ ID NO:389, SEQ ID NO:391, SEQ
ID NO:393, SEQ ID NO:395, SEQ ID NO:397, SEQ ID NO:399, SEQ ID NO:401,
SEQ ID NO:403, SEQ ID NO:405, SEQ ID NO:407, SEQ ID NO:409, SEQ ID
NO:411, SEQ ID NO:413, SEQ ID NO:415, SEQ ID NO:417, SEQ ID NO:419, SEQ
ID NO:421, SEQ ID NO:423, SEQ ID NO:425, SEQ ID NO:427, SEQ ID NO:429,
SEQ ID NO:431, SEQ ID NO:433, SEQ ID NO:435, SEQ ID NO:437, SEQ ID
NO:439, SEQ ID NO:441, SEQ ID NO:443, SEQ ID NO:445, SEQ ID NO:447, SEQ
ID NO:449, SEQ ID NO:451, SEQ ID NO:453, SEQ ID NO:455, SEQ ID NO:457,
SEQ ID NO:459, SEQ ID NO:461, SEQ ID NO:463, SEQ ID NO:465, SEQ ID
NO:467, SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ ID NO:475, SEQ
ID NO:477, SEQ ID NO:479, SEQ ID NO:481, SEQ ID NO:483, SEQ ID NO:485,
SEQ ID NO:487, SEQ ID NO:489, SEQ ID NO:491, SEQ ID NO:493, SEQ ID
NO:495, SEQ ID NO:497, SEQ ID NO:499, SEQ ID NO:501, SEQ ID NO:503, SEQ
ID NO:505, SEQ ID NO:507, SEQ ID NO:509, SEQ ID NO:511, SEQ ID NO:513,
SEQ ID NO:515 or SEQ ID NO:517, over a region of at least about 10, 15,
20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,
500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,
1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750,
1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300, 2350, 2400,
2450, 2500, or more residues, encodes at least one polypeptide having a
glucanase, e.g., an endoglucanase, activity, a xylanase, or a mannanase
activity, and the sequence identities are determined by analysis with a
sequence comparison algorithm or by a visual inspection.
[0008] Exemplary nucleic acids of the invention also include isolated,
synthetic or recombinant nucleic acids encoding a polypeptide of the
invention, e.g., a polypeptide having a sequence as set forth in SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ D) NO:10, SEQ ID NO:12,
SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID
NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID
NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID
NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID
NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID
NO:94, SEQ ID NO:6, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID
NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:10, SEQ ID NO:112, SEQ ID
NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ
ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132;
SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID
NO:142; SEQ ID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID
NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ
ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170,
SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID
NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ
ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198,
SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID
NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ
ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226,
SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID
NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ
ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254,
SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID
NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ
ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282,
SEQ ID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID
NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ
ID NO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310,
SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID
NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ
ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:338,
SEQ ID NO:340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID
NO:348, SEQ ID NO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ
ID NO:358, SEQ ID NO:360, SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:366,
SEQ ID NO:368, SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:374, SEQ ID
NO:376, SEQ ID NO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ ID NO:384, SEQ
ID NO:386, SEQ ID NO:388, SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394,
SEQ ID NO:396, SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402, SEQ ID
NO:404, SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, SEQ ID NO:412, SEQ
ID NO:414, SEQ ID NO:416, SEQ ID NO:418, SEQ ID NO:420, SEQ ID NO:422,
SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, SEQ ID
NO:432, SEQ ID NO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ
ID NO:442, SEQ ID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQ ID NO:450,
SEQ ID NO:452, SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458, SEQ ID
NO:460, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ
ID NO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ ID NO:476, SEQ ID NO:478,
SEQ ID NO:480, SEQ ID NO:482, SEQ ID NO:484, SEQ ID NO:486, SEQ ID
NO:488, SEQ ID NO:490, SEQ ID NO:492, SEQ ID NO:494, SEQ ID NO:496, SEQ
ID NO:498, SEQ ID NO:500, SEQ ID NO:502, SEQ ID NO:504, SEQ ID NO:506,
SEQ ID NO:508, SEQ ID NO:510, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:516
or SEQ ID NO:518, and subsequences thereof and variants thereof. In one
aspect, the polypeptide has an glucanase, e.g., endoglucanase activity,
e.g., catalyzing hydrolysis of internal endo-.beta.-1,4- and/or
1,3-glucanase linkages, a xylanase, or a mannanase activity.
[0009] In one aspect, the invention also provides glucanase-encoding
nucleic acids with a common novelty in that they are derived from mixed
cultures. The invention provides glucanase-encoding nucleic acids
isolated from mixed cultures comprising a nucleic acid sequence having at
least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete
(100%) sequence identity to an exemplary nucleic acid of the invention,
e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,
SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID
NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID
NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID
NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID
NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID
NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID
NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID
NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID
NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ
ID NO:11, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ
ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129,
SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID
NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ
ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157,
SEQ ID NO:199, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID
NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ
ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185,
SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID
NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ
ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213,
SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID
NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ
ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241,
SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID
NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ
ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269,
SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID
NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ
ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297,
SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID
NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ
ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325,
SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID
NO:335, SEQ ID NO:337, SEQ ID NO:339, SEQ ID NO:341, SEQ ID NO:343, SEQ
ID NO:345, SEQ ID NO:347, SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:353,
SEQ ID NO:355, SEQ ID NO:357, SEQ ID NO:359, SEQ ID NO:361, SEQ ID
NO:363, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:369, SEQ ID NO:371, SEQ
ID NO:373, SEQ ID NO:375, SEQ ID NO:377, SEQ ID NO:379, SEQ ID NO:381,
SEQ ID NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQ ID NO:389, SEQ ID
NO:391, SEQ ID NO:393, SEQ ID NO:395, SEQ ID NO:397, SEQ ID NO:399, SEQ
ID NO:401, SEQ ID NO:403, SEQ ID NO:405, SEQ ID NO:407, SEQ ID NO:409,
SEQ ID NO:411, SEQ ID NO:413, SEQ ID NO:415, SEQ ID NO:417, SEQ ID
NO:419, SEQ ID NO:421, SEQ ID NO:423, SEQ ID NO:425, SEQ ID NO:427, SEQ
ID NO:429, SEQ ID NO:431, SEQ ID NO:433, SEQ ID NO:435, SEQ ID NO:437,
SEQ ID NO:439, SEQ ID NO:441, SEQ ID NO:443, SEQ ID NO:445, SEQ ID
NO:447, SEQ ID NO:449, SEQ ID NO:451, SEQ ID NO:453, SEQ ID NO:455, SEQ
ID NO:457, SEQ ID NO:459, SEQ ID NO:461, SEQ ID NO:463, SEQ ID NO:465,
SEQ ID NO:467, SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ ID
NO:475, SEQ ID NO:477, SEQ ID NO:479, SEQ ID NO:481, SEQ ID NO:483, SEQ
ID NO:485, SEQ ID NO:487, SEQ ID NO:489, SEQ ID NO:491, SEQ ID NO:493,
SEQ ID NO:495, SEQ ID NO:497, SEQ ID NO:499, SEQ ID NO:501, SEQ ID
NO:503, SEQ ID NO:505, SEQ ID NO:507, SEQ ID NO:509, SEQ ID NO:511, SEQ
ID NO:513, SEQ ID NO:515 or SEQ ID NO:517, over a region of at least
about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 100, 150, 200, 250,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,
1000, 1050, 1100, 1150, or more residues (bases).
[0010] In one aspect, the invention provides glucanase-encoding nucleic
acids, and the polypeptides encoded by them, with a common novelty in
that they are derived from a common source, e.g., an environmental or an
archaeal source, see Table 1.
[0011] In one aspect, the invention also provides glucanase-encoding
nucleic acids, and the polypeptides encoded by them, with a common
novelty in that they are in a common family 3, family 5, family 6, family
8, family 9, family 12 or family 16, as discussed below, see Tables 2A
and 2B.
[0012] In one aspect, the invention also provides glucanase-encoding
nucleic acids with a common novelty in that they are derived from
environmental sources, e.g., mixed environmental sources. In one aspect,
the invention provides glucanase-encoding nucleic acids isolated from
environmental sources, e.g., mixed environmental sources, comprising a
nucleic acid sequence having at least about 10, 15, 20, 25, 30, 35, 40,
45, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,
64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)
sequence identity to an exemplary nucleic acid of the invention over a
region of at least about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,
500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,
1200 or more, residues, wherein the nucleic acid encodes at least one
polypeptide having a glucanase activity, and the sequence identities are
determined by analysis with a sequence comparison algorithm or by a
visual inspection.
[0013] In one aspect, the sequence comparison algorithm is a BLAST version
2.2.2 algorithm where a filtering setting is set to blastall-p blastp-d
"nr pataa"-F F, and all other options are set to default.
[0014] Another aspect of the invention is an isolated, synthetic or
recombinant nucleic acid including at least 10 consecutive bases of a
nucleic acid sequence of the invention, sequences substantially identical
thereto, and the sequences complementary thereto.
[0015] In one aspect, the glucanase activity of the invention comprises an
endoglucanase activity, e.g., endo-1,4 and/or 1,3-beta-D-glucan 4-glucano
hydrolase activity. In one aspect, the endoglucanase activity comprises
catalyzing hydrolysis of 1,4-beta-D-glycosidic linkages. In one aspect,
the glucanase, e.g., endoglucanase, activity comprises an endo-1,4-
and/or 1,3-beta-endoglucanase activity or endo-.beta.-1,4-glucanase
activity. In one aspect, the glucanase activity (e.g.,
endo-1,4-beta-D-glucan 4-glucano hydrolase activity) comprises hydrolysis
of 1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives
(e.g., carboxy methyl cellulose and hydroxy ethyl cellulose) lichenin,
beta-1,4 bonds in mixed beta-1,3 glucans, such as cereal beta-D-glucans
and other plant material containing cellulosic parts.
[0016] In one aspect, the glucanase, xylanase, or mannanase activity
comprises hydrolyzing a glucan or other polysaccharide to produce a
smaller molecular weight polysaccharide or oligomer. In one aspect, the
glucan comprises a beta-glucan, such as a water soluble beta-glucan. The
water soluble beta-glucan can comprise a dough or a bread product.
[0017] In one aspect, the glucanase activity comprises hydrolyzing
polysaccharides comprising 1,4-.beta.-glycoside-linked D-glucopyranoses.
In one aspect, the glucanase activity comprises hydrolyzing cellulose. In
one aspect, the glucanase activity comprises hydrolyzing cellulose in a
wood or paper pulp or a paper product.
[0018] In one aspect, the glucanase, e.g., endoglucanase, activity
comprises catalyzing hydrolysis of glucans in a beverage or a feed (e.g.,
an animal feed, such as a chicken feed) or a food product. The beverage,
feed or food product can comprise a cereal-based animal feed, a wort or a
beer, a fruit or a vegetable. In one aspect, the invention provides a
food, feed (e.g., an animal feed, such as a chicken feed), a liquid,
e.g., a beverage (such as a fruit juice or a beer) or a beverage
precursor (e.g., a wort), comprising a polypeptide of the invention. The
food can be a dough or a bread product. The beverage or a beverage
precursor can be a fruit juice, a beer or a wort. In one aspect, the
invention provides methods for the clarification of a liquid, e.g., a
juice, such as a fruit juice, or a beer, by treating the liquid with an
enzyme of the invention.
[0019] In one aspect, the invention provides methods of dough conditioning
comprising contacting a dough or a bread product with at least one
polypeptide of the invention under conditions sufficient for conditioning
the dough. In one aspect, the invention provides methods of beverage
production comprising administration of at least one polypeptide of the
invention to a beverage or a beverage precursor under conditions
sufficient for decreasing the viscosity of the beverage.
[0020] In one aspect, the glucanase, e.g., endoglucanase, activity
comprises catalyzing hydrolysis of glucans in a cell, e.g., a plant cell
or a microbial cell.
[0021] In one aspect, the isolated, synthetic or recombinant nucleic acid
encodes a polypeptide having a glucanase, e.g., endoglucanase, a
xylanase, or a mannanase activity that is thermostable. The polypeptide
can retain a glucanase, a xylanase, or a mannanase or other activity
under conditions comprising a temperature range of between about
37.degree. C. to about 95.degree. C.; between about 55.degree. C. to
about 85.degree. C., between about 70.degree. C. to about 95.degree. C.,
or, between about 90.degree. C. to about 95.degree. C.
[0022] In another aspect, the isolated, synthetic or recombinant nucleic
acid encodes a polypeptide having a glucanase, e.g., endoglucanase, a
xylanase, or a mannanase activity that is thermotolerant. The polypeptide
can retain a glucanase or other activity after exposure to a temperature
in the range from greater than 37.degree. C. to about 95.degree. C. or
anywhere in the range from greater than 55.degree. C. to about 85.degree.
C. The polypeptide can retain a glucanase or other activity after
exposure to a temperature in the range between about 1.degree. C. to
about 5.degree. C., between about 5.degree. C. to about 15.degree. C.,
between about 15.degree. C. to about 25.degree. C., between about
25.degree. C. to about 37.degree. C., between about 37.degree. C. to
about 95.degree. C., between about 55.degree. C. to about 85.degree. C.,
between about 70.degree. C. to about 75.degree. C., or between about
90.degree. C. to about 95.degree. C., or more. In one aspect, the
polypeptide retains a glucanase or other activity after exposure to a
temperature in the range from greater than 90.degree. C. to about
95.degree. C. at pH 4.5.
[0023] The invention provides isolated, synthetic or recombinant nucleic
acids comprising a sequence that hybridizes under stringent conditions to
a nucleic acid comprising a sequence of the invention, e.g., a sequence
as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ
ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID
NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID
NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID
NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID
NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID
NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID
NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID
NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID
NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID
NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ
ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127,
SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID
NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ
ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155,
SEQ ID NO:157, SEQ ID NO:199, SEQ ID NO:161, SEQ ID NO:163, SEQ ID
NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ
ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183,
SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID
NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ
ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211,
SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID
NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ
ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239,
SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID
NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ
ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267,
SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID
NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ
ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295,
SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ ID
NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ
ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323,
SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID
NO:333, SEQ ID NO:335, SEQ ID NO:337, SEQ ID NO:339, SEQ ID NO:341, SEQ
ID NO:343, SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:349, SEQ ID NO:351,
SEQ ID NO:353, SEQ ID NO:355, SEQ ID NO:357, SEQ ID NO:359, SEQ ID
NO:361, SEQ ID NO:363, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:369, SEQ
ID NO:371, SEQ ID NO:373, SEQ ID NO:375, SEQ ID NO:377, SEQ ID NO:379,
SEQ ID NO:381, SEQ ID NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQ ID
NO:389, SEQ ID NO:391, SEQ ID NO:393, SEQ ID NO:395, SEQ ID NO:397, SEQ
ID NO:399, SEQ ID NO:401, SEQ ID NO:403, SEQ ID NO:405, SEQ ID NO:407,
SEQ ID NO:409, SEQ ID NO:411, SEQ ID NO:413, SEQ ID NO:415, SEQ ID
NO:417, SEQ ID NO:419, SEQ ID NO:421, SEQ ID NO:423, SEQ ID NO:425, SEQ
ID NO:427, SEQ ID NO:429, SEQ ID NO:431, SEQ ID NO:433, SEQ ID NO:435,
SEQ ID NO:437, SEQ ID NO:439, SEQ ID NO:441, SEQ ID NO:443, SEQ ID
NO:445, SEQ ID NO:447, SEQ ID NO:449, SEQ ID NO:451, SEQ ID NO:453, SEQ
ID NO:455, SEQ ID NO:457, SEQ ID NO:459, SEQ ID NO:461, SEQ ID NO:463,
SEQ ID NO:465, SEQ ID NO:467, SEQ ID NO:469, SEQ ID NO:471, SEQ ID
NO:473, SEQ ID NO:475, SEQ ID NO:477, SEQ ID NO:479, SEQ ID NO:481, SEQ
ID NO:483, SEQ ID NO:485, SEQ ID NO:487, SEQ ID NO:489, SEQ ID NO:491,
SEQ ID NO:493, SEQ ID NO:495, SEQ ID NO:497, SEQ ID NO:499, SEQ ID
NO:501, SEQ ID NO:503, SEQ ID NO:505, SEQ ID NO:507, SEQ ID NO:509, SEQ
ID NO:511, SEQ ID NO:513, SEQ ID NO:515 or SEQ ID NO:517, or fragments or
subsequences thereof. In one aspect, the nucleic acid encodes a
polypeptide having a glucanase, e.g., endoglucanase, a xylanase, or a
mannanase activity. The nucleic acid can be at least about 10, 15, 20,
25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200
or more residues in length or the full length of the gene or transcript.
In one aspect, the stringent conditions include a wash step comprising a
wash in 0.2.times.SSC at a temperature of about 65.degree. C. for about
15 minutes.
[0024] The invention provides a nucleic acid probe for identifying a
nucleic acid encoding a polypeptide having a glucanase, e.g.,
endoglucanase, activity, a xylanase, or a mannanase, wherein the probe
comprises at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,
600, 650, 700, 750, 800, 850, 900, 950, 1000 or more, consecutive bases
of a sequence comprising a sequence of the invention, or fragments or
subsequences thereof, wherein the probe identifies the nucleic acid by
binding or hybridization. The probe can comprise an oligonucleotide
comprising at least about 10 to 50, about 20 to 60, about 30 to 70, about
40 to 80, or about 60 to 100 consecutive bases of a sequence comprising a
sequence of the invention, or fragments or subsequences thereof.
[0025] The invention provides a nucleic acid probe for identifying a
nucleic acid encoding a polypeptide having a glucanase, e.g.,
endoglucanase, a xylanase, or a mannanase activity, wherein the probe
comprises a nucleic acid comprising a sequence at least about 10, 15, 20,
30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,
550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more residues having
at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,
61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or
complete (100%) sequence identity to a nucleic acid of the invention,
wherein the sequence identities are determined by analysis with a
sequence comparison algorithm or by visual inspection.
[0026] The probe can comprise an oligonucleotide comprising at least about
10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to
100 consecutive bases of a nucleic acid sequence of the invention, or a
subsequence thereof.
[0027] The invention provides an amplification primer pair for amplifying
a nucleic acid encoding a polypeptide having a glucanase activity,
wherein the primer pair is capable of amplifying a nucleic acid
comprising a sequence of the invention, or fragments or subsequences
thereof. One or each member of the amplification primer sequence pair can
comprise an oligonucleotide comprising at least about 10 to 50
consecutive bases of the sequence, or about 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive bases
of the sequence.
[0028] The invention provides amplification primer pairs, wherein the
primer pair comprises a first member having a sequence as set forth by
about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30 or more residues of a nucleic acid of the
invention, and a second member having a sequence as set forth by about
the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30 or more residues of the complementary strand of
the first member.
[0029] The invention provides glucanase-, e.g., endoglucanase-encoding,
xylanase-encoding, or mannanase-encoding nucleic acids generated by
amplification, e.g., polymerase chain reaction (PCR), using an
amplification primer pair of the invention. The invention provides
glucanases, mannanases, or xylanases generated by amplification, e.g.,
polymerase chain reaction (PCR), using an amplification primer pair of
the invention. The invention provides methods of making glucanases,
mannanases, or xylanases by amplification, e.g., polymerase chain
reaction (PCR), using an amplification primer pair of the invention. In
one aspect, the amplification primer pair amplifies a nucleic acid from a
library, e.g., a gene library, such as an environmental library.
[0030] The invention provides methods of amplifying a nucleic acid
encoding a polypeptide having a glucanase, e.g., endoglucanase, a
mannanase, or a xylanase activity comprising amplification of a template
nucleic acid with an amplification primer sequence pair capable of
amplifying a nucleic acid sequence of the invention, or fragments or
subsequences thereof.
[0031] The invention provides expression cassettes comprising a nucleic
acid of the invention or a subsequence thereof. In one aspect, the
expression cassette can comprise the nucleic acid that is operably linked
to a promoter. The promoter can be a viral, bacterial, mammalian or plant
promoter. In one aspect, the plant promoter can be a potato, rice, corn,
wheat, tobacco or barley promoter. The promoter can be a constitutive
promoter. The constitutive promoter can comprise CaMV35S. In another
aspect, the promoter can be an inducible promoter. In one aspect, the
promoter can be a tissue-specific promoter or an environmentally
regulated or a developmentally regulated promoter. Thus, the promoter can
be, e.g., a seed-specific, a leaf-specific, a root-specific, a
stem-specific or an abscission-induced promoter. In one aspect, the
expression cassette can further comprise a plant or plant virus
expression vector.
[0032] The invention provides cloning vehicles comprising an expression
cassette (e.g., a vector) of the invention or a nucleic acid of the
invention. The cloning vehicle can be a viral vector, a plasmid, a phage,
a phagemid, a cosmid, a fosmid, a bacteriophage or an artificial
chromosome. The viral vector can comprise an adenovirus vector, a
retroviral vector or an adeno-associated viral vector. The cloning
vehicle can comprise a bacterial artificial chromosome (BAC), a plasmid,
a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome
(YAC), or a mammalian artificial chromosome (MAC).
[0033] The invention provides transformed cell comprising a nucleic acid
of the invention or an expression cassette (e.g., a vector) of the
invention, or a cloning vehicle of the invention. In one aspect, the
transformed cell can be a bacterial cell, a mammalian cell, a fungal
cell, a yeast cell, an insect cell or a plant cell. In one aspect, the
plant cell can be a cereal, a potato, wheat, rice, corn, tobacco or
barley cell.
[0034] The invention provides transgenic non-human animals comprising a
nucleic acid of the invention or an expression cassette (e.g., a vector)
of the invention. In one aspect, the animal is a mouse.
[0035] The invention provides transgenic plants comprising a nucleic acid
of the invention or an expression cassette (e.g., a vector) of the
invention. The transgenic plant can be a cereal plant, a corn plant, a
potato plant, a tomato plant, a wheat plant, an oilseed plant, a rapeseed
plant, a soybean plant, a rice plant, a barley plant or a tobacco plant.
[0036] The invention provides transgenic seeds comprising a nucleic acid
of the invention or an expression cassette (e.g., a vector) of the
invention. The transgenic seed can be a cereal plant, a corn seed, a
wheat kernel, an oilseed, a rapeseed, a soybean seed, a palm kernel, a
sunflower seed, a sesame seed, a peanut or a tobacco plant seed.
[0037] The invention provides an antisense oligonucleotide comprising a
nucleic acid sequence complementary to or capable of hybridizing under
stringent conditions to a nucleic acid of the invention. The invention
provides methods of inhibiting the translation of a glucanase, e.g.,
endoglucanase, a mannanase, or a xylanase message in a cell comprising
administering to the cell or expressing in the cell an antisense
oligonucleotide comprising a nucleic acid sequence complementary to or
capable of hybridizing under stringent conditions to a nucleic acid of
the invention. In one aspect, the antisense oligonucleotide is between
about 10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about
60 to 100 bases in length.
[0038] The invention provides methods of inhibiting the translation of a
glucanase, e.g., endoglucanase, a mannanase, or a xylanase message in a
cell comprising administering to the cell or expressing in the cell an
antisense oligonucleotide comprising a nucleic acid sequence
complementary to or capable of hybridizing under stringent conditions to
a nucleic acid of the invention. The invention provides double-stranded
inhibitory RNA (RNAi) molecules comprising a subsequence of a sequence of
the invention. In one aspect, the RNAi is about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25 or more duplex nucleotides in length. The invention
provides methods of inhibiting the expression of a glucanase, e.g.,
endoglucanase, a mannanase, or a xylanase in a cell comprising
administering to the cell or expressing in the cell a double-stranded
inhibitory RNA (iRNA), wherein the RNA comprises a subsequence of a
sequence of the invention.
[0039] The invention provides an isolated, synthetic or recombinant
polypeptide comprising an amino acid sequence having at least about 50%,
51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence
identity to an exemplary polypeptide or peptide of the invention over a
region of at least about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250,
275, 300, 325, 350 or more residues, or over the full length of the
polypeptide, and the sequence identities are determined by analysis with
a sequence comparison algorithm or by a visual inspection. Exemplary
polypeptide or peptide sequences of the invention include SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID
NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID
NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID
NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID
NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID
NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID
NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID
NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ
ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122,
SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID
NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ
ID NO:142; SEQ ID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID
NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ
ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170,
SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID
NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ
ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198,
SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID
NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ
ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226,
SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID
NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ
ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254,
SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID
NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ
ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282,
SEQ ID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID
NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ
ID NO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310,
SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID
NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ
ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:338,
SEQ ID NO:340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID
NO:348, SEQ ID NO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ
ID NO:358, SEQ ID NO:360, SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:366,
SEQ ID NO:368, SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:374, SEQ ID
NO:376, SEQ ID NO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ ID NO:384, SEQ
ID NO:386, SEQ ID NO:388, SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394,
SEQ ID NO:396, SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402, SEQ ID
NO:404, SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, SEQ ID NO:412, SEQ
ID NO:414, SEQ ID NO:416, SEQ ID NO:418, SEQ ID NO:420, SEQ ID NO:422,
SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, SEQ ID
NO:432, SEQ ID NO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ
ID NO:442, SEQ ID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQ ID NO:450,
SEQ ID NO:452, SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458, SEQ ID
NO:460, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ
ID NO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ ID NO:476, SEQ ID NO:478,
SEQ ID NO:480, SEQ ID NO:482, SEQ ID NO:484, SEQ ID NO:486, SEQ ID
NO:488, SEQ ID NO:490, SEQ ID NO:492, SEQ ID NO:494, SEQ ID NO:496, SEQ
ID NO:498, SEQ ID NO:500, SEQ ID NO:502, SEQ ID NO:504, SEQ ID NO:506,
SEQ ID NO:508, SEQ ID NO:510, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:516
or SEQ ID NO:518, and subsequences thereof and variants thereof.
Exemplary polypeptides also include fragments of at least about 10, 15,
20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,
500, 550, 600 or more residues in length, or over the full length of an
enzyme. Exemplary polypeptide or peptide sequences of the invention
include sequence encoded by a nucleic acid of the invention. Exemplary
polypeptide or peptide sequences of the invention include polypeptides or
peptides specifically bound by an antibody of the invention.
[0040] In one aspect, a polypeptide of the invention has at least one
glucanase, e.g., endoglucanase, a mannanase, or a xylanase activity.
[0041] In one aspect, the endoglucanase activity comprises
endo-1,4-beta-D-glucan 4-glucano hydrolase activity. In one aspect, the
endoglucanase activity comprises catalyzing hydrolysis of
1,4-beta-D-glycosidic linkages or 1,3-beta-D-glycosidic linkages. In one
aspect, the endoglucanase activity comprises an
endo-1,4-beta-endoglucanase activity or endo-.beta.-1,4-glucanase
activity, endo-1,3-beta-endoglucanase activity or
endo-.beta.-1,3-glucanase activity. In one aspect, the glucanase activity
(e.g., endo-1,4 and/or 1,3-beta-D-glucan 4-glucano hydrolase activity)
comprises hydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,
cellulose derivatives (e.g., carboxy methyl cellulose and hydroxy ethyl
cellulose) lichenin, beta-1,4- and/or 1,3-bonds in mixed beta-1,3
glucans, such as cereal beta-D-glucans or xyloglucans and other plant
material containing cellulosic parts.
[0042] Another aspect of the invention provides an isolated, synthetic or
recombinant polypeptide or peptide including at least 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more
consecutive bases of a polypeptide or peptide sequence of the invention,
sequences substantially identical thereto, and the sequences
complementary thereto. The peptide can be, e.g., an immunogenic fragment,
a motif (e.g., a binding site), a signal sequence, a prepro sequence or a
catalytic domains (CDs) or active site.
[0043] The invention provides isolated, synthetic or recombinant nucleic
acids comprising a sequence encoding a polypeptide having a glucanase,
e.g., an endoglucanase, a mannanase, or a xylanase activity and a signal
sequence, wherein the nucleic acid comprises a sequence of the invention.
The signal sequence can be derived from another glucanase, mannanase, or
xylanase or a non-glucanase, etc., i.e., a heterologous enzyme. The
invention provides isolated, synthetic or recombinant nucleic acids
comprising a sequence encoding a polypeptide having a glucanase, e.g., an
endoglucanase, a mannanase, or a xylanase activity, wherein the sequence
does not contain a signal sequence and the nucleic acid comprises a
sequence of the invention.
[0044] In one aspect, the glucanase, e.g., endoglucanase, activity
comprises catalyzing hydrolysis of 1,4-beta-D-glycosidic linkages or
1,3-beta-D-glycosidic linkages. In one aspect, the endoglucanase activity
comprises an endo-1,4-beta-endoglucanase activity.
[0045] In one aspect, the endoglucanase activity comprises hydrolyzing a
glucan to produce a smaller molecular weight polysaccharide or oligomer.
In one aspect, the glucan comprises an beta-glucan, such as a water
soluble beta-glucan. The water soluble beta-glucan can comprise a dough
or a bread product.
[0046] In one aspect, the glucanase activity comprises hydrolyzing
polysaccharides comprising 1,4-.beta.-glycoside-linked D-glucopyranoses.
In one aspect, the glucanase activity comprises hydrolyzing cellulose. In
one aspect, the glucanase activity comprises hydrolyzing cellulose in a
wood or paper pulp or a paper product.
[0047] In one aspect, the glucanase, xylanase, or mannanase activity
comprises catalyzing hydrolysis of a glucan or other carbohydrate in a
feed (e.g., an animal feed, such as a chicken feed) or a food product.
The feed or food product can comprise a cereal-based animal feed, a wort
or a beer, a fruit or a vegetable.
[0048] In one aspect, the glucanase, xylanase, or mannanase activity
comprises catalyzing hydrolysis of a glucan or other carbohydrate in a
cell, e.g., a plant cell, a fungal cell, or a microbial (e.g., bacterial)
cell.
[0049] In one aspect, the glucanase, e.g., endoglucanase, mannanase, or
xylanase activity is thermostable. The polypeptide can retain a
glucanase, a mannanase, or a xylanase activity under conditions
comprising a temperature range of between about 1.degree. C. to about
5.degree. C., between about 5.degree. C. to about 15.degree. C., between
about 15.degree. C. to about 25.degree. C., between about 25.degree. C.
to about 37.degree. C., between about 37.degree. C. to about 95.degree.
C., between about 55.degree. C. to about 85.degree. C., between about
70.degree. C. to about 75.degree. C., or between about 90.degree. C. to
about 95.degree. C., or more. In another aspect, the glucanase, e.g.,
endoglucanase, mannanase, or xylanase activity can be thermotolerant. The
polypeptide can retain a glucanase, a mannanase, or a xylanase activity
after exposure to a temperature in the range from greater than 37.degree.
C. to about 95.degree. C., or in the range from greater than 55.degree.
C. to about 85.degree. C. In one aspect, the polypeptide can retain a
glucanase, a mannanase, or a xylanase activity after exposure to a
temperature in the range from greater than 90.degree. C. to about
95.degree. C. at pH 4.5.
[0050] In one aspect, the isolated, synthetic or recombinant polypeptide
can comprise the polypeptide of the invention that lacks a signal
sequence. In one aspect, the isolated, synthetic or recombinant
polypeptide can comprise the polypeptide of the invention comprising a
heterologous signal sequence, such as a heterologous glucanase, or
mannanase, xylanase signal sequence or non-glucanase, mannanase, or
xylanase signal sequence.
[0051] In one aspect, the invention provides chimeric proteins comprising
a first domain comprising a signal sequence of the invention and at least
a second domain. The protein can be a fusion protein. The second domain
can comprise an enzyme. The enzyme can be a glucanase, e.g.,
endoglucanase, a mannanase, or a xylanase.
[0052] The invention provides chimeric polypeptides comprising at least a
first domain comprising signal peptide (SP), a prepro sequence and/or a
catalytic domain (CD) of the invention and at least a second domain
comprising a heterologous polypeptide or peptide, wherein the
heterologous polypeptide or peptide is not naturally associated with the
signal peptide (SP), prepro sequence and/or catalytic domain (CD). In one
aspect, the heterologous polypeptide or peptide is not a glucanase, a
mannanase, or a xylanase. The heterologous polypeptide or peptide can be
amino terminal to, carboxy terminal to or on both ends of the signal
peptide (SP), prepro sequence and/or catalytic domain (CD).
[0053] The invention provides isolated, synthetic or recombinant nucleic
acids encoding a chimeric polypeptide, wherein the chimeric polypeptide
comprises at least a first domain comprising signal peptide (SP), a
prepro domain and/or a catalytic domain (CD) of the invention and at
least a second domain comprising a heterologous polypeptide or peptide,
wherein the heterologous polypeptide or peptide is not naturally
associated with the signal peptide (SP), prepro domain and/or catalytic
domain (CD).
[0054] The invention provides isolated, synthetic or recombinant signal
sequences (e.g., signal peptides) consisting of or comprising a sequence
as set forth in residues 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1
to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1
to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1
to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1 to 43 or 1
to 44, of a polypeptide of the invention, e.g., SEQ ID NO:2, SEQ ID NO:4,
SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ
ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID
NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID
NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID
NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID
NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID
NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID
NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID
NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID
NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ
ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,
SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132; SEQ ID
NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ
ID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID
NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ
ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172,
SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID
NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ
ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200,
SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID
NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ
ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228,
SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID
NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ
ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256,
SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID
NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ
ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284,
SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID
NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ
ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312,
SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID
NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ
ID NO:332, SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340,
SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:348, SEQ ID
NO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQ
ID NO:360, SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368,
SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:374, SEQ ID NO:376, SEQ ID
NO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ ID NO:384, SEQ ID NO:386, SEQ
ID NO:388, SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, SEQ ID NO:396,
SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402, SEQ ID NO:404, SEQ ID
NO:406, SEQ ID NO:408, SEQ ID NO:410, SEQ ID NO:412, SEQ ID NO:414, SEQ
ID NO:416, SEQ ID NO:418, SEQ ID NO:420, SEQ ID NO:422, SEQ ID NO:424,
SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, SEQ ID NO:432, SEQ ID
NO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:442, SEQ
ID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQ ID NO:450, SEQ ID NO:452,
SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458, SEQ ID NO:460, SEQ ID
NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ ID NO:470, SEQ
ID NO:472, SEQ ID NO:474, SEQ ID NO:476, SEQ ID NO:478, SEQ ID NO:480,
SEQ ID NO:482, SEQ ID NO:484, SEQ ID NO:486, SEQ ID NO:488, SEQ ID
NO:490, SEQ ID NO:492, SEQ ID NO:494, SEQ ID NO:496, SEQ ID NO:498, SEQ
ID NO:500, SEQ ID NO:502, SEQ ID NO:504, SEQ ID NO:506, SEQ ID NO:508,
SEQ ID NO:510, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:516, SEQ ID
NO:518. The invention provides isolated, synthetic or recombinant signal
sequences (e.g., signal peptides) consisting of or comprising a sequence
as set forth in Table 3, below.
[0055] In one aspect, the glucanase, e.g., endoglucanase, mannanase, or
xylanase activity comprises a specific activity at about 37.degree. C. in
the range from about 1 to about 1200 units per milligram of protein, or,
about 100 to about 1000 units per milligram of protein. In another
aspect, the glucanase, e.g., endoglucanase, mannanase, or xylanase
activity comprises a specific activity from about 100 to about 1000 units
per milligram of protein, or, from about 500 to about 750 units per
milligram of protein. Alternatively, the glucanase, mannanase, or
xylanase activity comprises a specific activity at 37.degree. C. in the
range from about 1 to about 750 units per milligram of protein, or, from
about 500 to about 1200 units per milligram of protein. In one aspect,
the glucanase, mannanase, or xylanase activity comprises a specific
activity at 37.degree. C. in the range from about 1 to about 500 units
per milligram of protein, or, from about 750 to about 1000 units per
milligram of protein. In another aspect, the glucanase, mannanase, or
xylanase activity comprises a specific activity at 37.degree. C. in the
range from about 1 to about 250 units per milligram of protein.
Alternatively, the glucanase, e.g., endoglucanase, mannanase, or xylanase
activity comprises a specific activity at 37.degree. C. in the range from
about 1 to about 100 units per milligram of protein. In another aspect,
the thermotolerance comprises retention of at least half of the specific
activity of the glucanase, mannanase, or xylanase at 37.degree. C. after
being heated to the elevated temperature. Alternatively, the
thermotolerance can comprise retention of specific activity at 37.degree.
C. in the range from about 1 to about 1200 units per milligram of
protein, or, from about 500 to about 1000 units per milligram of protein,
after being heated to the elevated temperature. In another aspect, the
thermotolerance can comprise retention of specific activity at 37.degree.
C. in the range from about 1 to about 500 units per milligram of protein
after being heated to the elevated temperature.
[0056] The invention provides the isolated, synthetic or recombinant
polypeptide of the invention, wherein the polypeptide comprises at least
one glycosylation site. In one aspect, glycosylation can be an N-linked
glycosylation. In one aspect, the polypeptide can be glycosylated after
being expressed in a P. pastoris or a S. pombe.
[0057] In one aspect, the polypeptide can retain glucanase, e.g.,
endoglucanase, mannanase, or xylanase activity under conditions
comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4. In another
aspect, the polypeptide can retain a glucanase, mannanase, or xylanase
activity under conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5,
pH 9, pH 9.5, pH 10, pH 10.5 or pH 11. In one aspect, the polypeptide can
retain a glucanase, mannanase, or xylanase activity after exposure to
conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4.
In another aspect, the polypeptide can retain a glucanase, mannanase, or
xylanase activity after exposure to conditions comprising about pH 7, pH
7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11.
[0058] The invention provides protein preparations comprising a
polypeptide of the invention, wherein the protein preparation comprises a
liquid, a solid or a gel.
[0059] The invention provides heterodimers comprising a polypeptide of the
invention and a second protein or domain. The second member of the
heterodimer can be a different glycanase, a different enzyme or another
protein. In one aspect, the second domain can be a polypeptide and the
heterodimer can be a fusion protein. In one aspect, the second domain can
be an epitope or a tag. In one aspect, the invention provides homodimers
comprising a polypeptide of the invention.
[0060] The invention provides immobilized polypeptides having glucanase,
e.g., endoglucanase, mannanase, or xylanase activity, wherein the
polypeptide comprises a polypeptide of the invention, a polypeptide
encoded by a nucleic acid of the invention, or a polypeptide comprising a
polypeptide of the invention and a second domain. In one aspect, the
polypeptide can be immobilized on a cell, a metal, a resin, a polymer, a
ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel,
a plate, an array or a capillary tube.
[0061] The invention provides arrays comprising an immobilized nucleic
acid of the invention. The invention provides arrays comprising an
antibody of the invention.
[0062] The invention provides isolated, synthetic or recombinant
antibodies that specifically bind to a polypeptide of the invention or to
a polypeptide encoded by a nucleic acid of the invention. The antibody
can be a monoclonal or a polyclonal antibody. The invention provides
hybridomas comprising an antibody of the invention, e.g., an antibody
that specifically binds to a polypeptide of the invention or to a
polypeptide encoded by a nucleic acid of the invention.
[0063] The invention provides method of isolating or identifying a
polypeptide having glucanase, e.g., endoglucanase, mannanase, or xylanase
activity comprising the steps of: (a) providing an antibody of the
invention; (b) providing a sample comprising polypeptides; and (c)
contacting the sample of step (b) with the antibody of step (a) under
conditions wherein the antibody can specifically bind to the polypeptide,
thereby isolating or identifying a polypeptide having an glucanase,
mannanase, or xylanase activity.
[0064] The invention provides methods of making an anti-glucanase,
mannanase, or xylanase antibody comprising administering to a non-human
animal a nucleic acid of the invention or a polypeptide of the invention
or subsequences thereof in an amount sufficient to generate a humoral
immune response, thereby making an anti-glucanase, mannanase, or xylanase
antibody. The invention provides methods of making an anti-glucanase,
mannanase, or xylanase humoral or cellular immune response comprising
administering to a non-human animal a nucleic acid of the invention or a
polypeptide of the invention or subsequences thereof in an amount
sufficient to generate an immune response.
[0065] The invention provides methods of producing a recombinant
polypeptide comprising the steps of: (a) providing a nucleic acid of the
invention operably linked to a promoter; and (b) expressing the nucleic
acid of step (a) under conditions that allow expression of the
polypeptide, thereby producing a recombinant polypeptide. In one aspect,
the method can further comprise transforming a host cell with the nucleic
acid of step (a) followed by expressing the nucleic acid of step (a),
thereby producing a recombinant polypeptide in a transformed cell.
[0066] The invention provides methods for identifying a polypeptide having
glucanase, e.g., endoglucanase, mannanase, or xylanase activity
comprising the following steps: (a) providing a polypeptide of the
invention; or a polypeptide encoded by a nucleic acid of the invention;
(b) providing glucanase, e.g., endoglucanase, mannanase, or xylanase
substrate; and (c) contacting the polypeptide or a fragment or variant
thereof of step (a) with the substrate of step (b) and detecting a
decrease in the amount of substrate or an increase in the amount of a
reaction product, wherein a decrease in the amount of the substrate or an
increase in the amount of the reaction product detects a polypeptide
having a glucanase, mannanase, or xylanase activity.
[0067] The invention provides methods for identifying glucanase, e.g.,
endoglucanase, mannanase, or xylanase substrate comprising the following
steps: (a) providing a polypeptide of the invention; or a polypeptide
encoded by a nucleic acid of the invention; (b) providing a test
substrate; and (c) contacting the polypeptide of step (a) with the test
substrate of step (b) and detecting a decrease in the amount of substrate
or an increase in the amount of reaction product, wherein a decrease in
the amount of the substrate or an increase in the amount of a reaction
product identifies the test substrate as an glucanase, mannanase, or
xylanase substrate.
[0068] The invention provides methods of determining whether a test
compound specifically binds to a polypeptide comprising the following
steps: (a) expressing a nucleic acid or a vector comprising the nucleic
acid under conditions permissive for translation of the nucleic acid to a
polypeptide, wherein the nucleic acid comprises a nucleic acid of the
invention, or, providing a polypeptide of the invention; (b) providing a
test compound; (c) contacting the polypeptide with the test compound; and
(d) determining whether the test compound of step (b) specifically binds
to the polypeptide.
[0069] The invention provides methods for identifying a modulator of a
glucanase, e.g., endoglucanase, mannanase, or xylanase activity
comprising the following steps: (a) providing a polypeptide of the
invention or a polypeptide encoded by a nucleic acid of the invention;
(b) providing a test compound; (c) contacting the polypeptide of step (a)
with the test compound of step (b) and measuring an activity of the
glucanase, mannanase, or xylanase wherein a change in the glucanase
mannanase, or xylanase activity measured in the presence of the test
compound compared to the activity in the absence of the test compound
provides a determination that the test compound modulates the glucanase,
mannanase, or xylanase activity. In one aspect, the glucanase, mannanase,
or xylanase activity can be measured by providing a glucanase, mannanase,
or xylanase substrate and detecting a decrease in the amount of the
substrate or an increase in the amount of a reaction product, or, an
increase in the amount of the substrate or a decrease in the amount of a
reaction product. A decrease in the amount of the substrate or an
increase in the amount of the reaction product with the test compound as
compared to the amount of substrate or reaction product without the test
compound identifies the test compound as an activator of glucanase,
mannanase, or xylanase activity. An increase in the amount of the
substrate or a decrease in the amount of the reaction product with the
test compound as compared to the amount of substrate or reaction product
without the test compound identifies the test compound as an inhibitor of
glucanase, mannanase, or xylanase activity.
[0070] The invention provides computer systems comprising a processor and
a data storage device wherein said data storage device has stored thereon
a polypeptide sequence or a nucleic acid sequence of the invention (e.g.,
a polypeptide encoded by a nucleic acid of the invention). In one aspect,
the computer system can further comprise a sequence comparison algorithm
and a data storage device having at least one reference sequence stored
thereon. In another aspect, the sequence comparison algorithm comprises a
computer program that indicates polymorphisms. In one aspect, the
computer system can further comprise an identifier that identifies one or
more features in said sequence. The invention provides computer readable
media having stored thereon a polypeptide sequence or a nucleic acid
sequence of the invention. The invention provides methods for identifying
a feature in a sequence comprising the steps of: (a) reading the sequence
using a computer program which identifies one or more features in a
sequence, wherein the sequence comprises a polypeptide sequence or a
nucleic acid sequence of the invention; and (b) identifying one or more
features in the sequence with the computer program. The invention
provides methods for comparing a first sequence to a second sequence
comprising the steps of: (a) reading the first sequence and the second
sequence through use of a computer program which compares sequences,
wherein the first sequence comprises a polypeptide sequence or a nucleic
acid sequence of the invention; and (b) determining differences between
the first sequence and the second sequence with the computer program. The
step of determining differences between the first sequence and the second
sequence can further comprise the step of identifying polymorphisms. In
one aspect, the method can further comprise an identifier that identifies
one or more features in a sequence. In another aspect, the method can
comprise reading the first sequence using a computer program and
identifying one or more features in the sequence.
[0071] The invention provides methods for isolating or recovering a
nucleic acid encoding a polypeptide having a glucanase, mannanase, or
xylanase activity from an environmental sample comprising the steps of:
(a) providing an amplification primer sequence pair for amplifying a
nucleic acid encoding a polypeptide having a glucanase, mannanase, or
xylanase activity, wherein the primer pair is capable of amplifying a
nucleic acid of the invention; (b) isolating a nucleic acid from the
environmental sample or treating the environmental sample such that
nucleic acid in the sample is accessible for hybridization to the
amplification primer pair; and, (c) combining the nucleic acid of step
(b) with the amplification primer pair of step (a) and amplifying nucleic
acid from the environmental sample, thereby isolating or recovering a
nucleic acid encoding a polypeptide having a glucanase, mannanase, or
xylanase activity from an environmental sample. One or each member of the
amplification primer sequence pair can comprise an oligonucleotide
comprising at least about 10 to 50 consecutive bases of a sequence of the
invention. In one aspect, the amplification primer sequence pair is an
amplification pair of the invention.
[0072] The invention provides methods for isolating or recovering a
nucleic acid encoding a polypeptide having a glucanase, mannanase, or
xylanase activity from an environmental sample comprising the steps of:
(a) providing a polynucleotide probe comprising a nucleic acid of the
invention or a subsequence thereof; (b) isolating a nucleic acid from the
environmental sample or treating the environmental sample such that
nucleic acid in the sample is accessible for hybridization to a
polynucleotide probe of step (a); (c) combining the isolated, synthetic
nucleic acid or the treated environmental sample of step (b) with the
polynucleotide probe of step (a); and (d) isolating a nucleic acid that
specifically hybridizes with the polynucleotide probe of step (a),
thereby isolating or recovering a nucleic acid encoding a polypeptide
having a glucanase, mannanase, or xylanase activity from an environmental
sample. The environmental sample can comprise a water sample, a liquid
sample, a soil sample, an air sample or a biological sample. In one
aspect, the biological sample can be derived from a bacterial cell, a
protozoan cell, an insect cell, a yeast cell, a plant cell, a fungal cell
or a mammalian cell.
[0073] The invention provides methods of generating a variant of a nucleic
acid encoding a polypeptide having a glucanase, mannanase, or xylanase
activity comprising the steps of: (a) providing a template nucleic acid
comprising a nucleic acid of the invention; and (b) modifying, deleting
or adding one or more nucleotides in the template sequence, or a
combination thereof, to generate a variant of the template nucleic acid.
In one aspect, the method can further comprise expressing the variant
nucleic acid to generate a variant glucanase, mannanase, or xylanase
polypeptide. The modifications, additions or deletions can be introduced
by a method comprising error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis, site-specific
mutagenesis, gene reassembly, Gene Site Saturation Mutagenesis.TM.
(GSSM.TM.), synthetic ligation reassembly (SLR) or a combination thereof.
In another aspect, the modifications, additions or deletions are
introduced by a method comprising recombination, recursive sequence
recombination, phosphothioate-modified DNA mutagenesis, uracil-containing
template mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification mutagenesis,
artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid
multimer creation and a combination thereof.
[0074] In one aspect, the method can be iteratively repeated until a
glucanase, mannanase, or xylanase having an altered or different activity
or an altered or different stability from that of a polypeptide encoded
by the template nucleic acid is produced. In one aspect, the variant
glucanase, mannanase, or xylanase polypeptide is thermotolerant, and
retains some activity after being exposed to an elevated temperature. In
another aspect, the variant glucanase, mannanase, or xylanase polypeptide
has increased glycosylation as compared to the glucanase, mannanase, or
xylanase encoded by a template nucleic acid. Alternatively, the variant
glucanase, mannanase, or xylanase polypeptide has a glucanase activity
under a high temperature, wherein the glucanase, mannanase, or xylanase
encoded by the template nucleic acid is not active under the high
temperature. In one aspect, the method can be iteratively repeated until
a glucanase, mannanase, or xylanase coding sequence having an altered
codon usage from that of the template nucleic acid is produced. In
another aspect, the method can be iteratively repeated until a glucanase,
mannanase, or xylanase gene having higher or lower level of message
expression or stability from that of the template nucleic acid is
produced.
[0075] The invention provides methods for modifying codons in a nucleic
acid encoding a polypeptide having a glucanase, mannanase, or xylanase
activity to increase its expression in a host cell, the method comprising
the following steps: (a) providing a nucleic acid of the invention
encoding a polypeptide having a glucanase, mannanase, or xylanase
activity; and, (b) identifying a non-preferred or a less preferred codon
in the nucleic acid of step (a) and replacing it with a preferred or
neutrally used codon encoding the same amino acid as the replaced codon,
wherein a preferred codon is a codon over-represented in coding sequences
in genes in the host cell and a non-preferred or less preferred codon is
a codon under-represented in coding sequences in genes in the host cell,
thereby modifying the nucleic acid to increase its expression in a host
cell.
[0076] The invention provides methods for modifying codons in a nucleic
acid encoding a polypeptide having a glucanase, mannanase, or xylanase
activity; the method comprising the following steps: (a) providing a
nucleic acid of the invention; and, (b) identifying a codon in the
nucleic acid of step (a) and replacing it with a different codon encoding
the same amino acid as the replaced codon, thereby modifying codons in a
nucleic acid encoding a glucanase, mannanase, or xylanase.
[0077] The invention provides methods for modifying codons in a nucleic
acid encoding a polypeptide having a glucanase, mannanase, or xylanase
activity to increase its expression in a host cell, the method comprising
the following steps: (a) providing a nucleic acid of the invention
encoding a glucanase, mannanase, or xylanase polypeptide; and, (b)
identifying a non-preferred or a less preferred codon in the nucleic acid
of step (a) and replacing it with a preferred or neutrally used codon
encoding the same amino acid as the replaced codon, wherein a preferred
codon is a codon over-represented in coding sequences in genes in the
host cell and a non-preferred or less preferred codon is a codon
under-represented in coding sequences in genes in the host cell, thereby
modifying the nucleic acid to increase its expression in a host cell.
[0078] The invention provides methods for modifying a codon in a nucleic
acid encoding a polypeptide having a glucanase, mannanase, or xylanase
activity to decrease its expression in a host cell, the method comprising
the following steps: (a) providing a nucleic acid of the invention; and
(b) identifying at least one preferred codon in the nucleic acid of step
(a) and replacing it with a non-preferred or less preferred codon
encoding the same amino acid as the replaced codon, wherein a preferred
codon is a codon over-represented in coding sequences in genes in a host
cell and a non-preferred or less preferred codon is a codon
under-represented in coding sequences in genes in the host cell, thereby
modifying the nucleic acid to decrease its expression in a host cell. In
one aspect, the host cell can be a bacterial cell, a fungal cell, an
insect cell, a yeast cell, a plant cell or a mammalian cell.
[0079] The invention provides methods for producing a library of nucleic
acids encoding a plurality of modified glucanase, mannanase, or xylanase
active sites (catalytic domains (CDs)) or substrate binding sites,
wherein the modified active sites or substrate binding sites are derived
from a first nucleic acid comprising a sequence encoding a first active
site or a first substrate binding site the method comprising the
following steps: (a) providing a first nucleic acid encoding a first
active site or first substrate binding site, wherein the first nucleic
acid sequence comprises a sequence that hybridizes under stringent
conditions to a nucleic acid of the invention, and the nucleic acid
encodes a glucanase, mannanase, or xylanase active site or a glucanase,
mannanase, or xylanase substrate binding site; (b) providing a set of
mutagenic oligonucleotides that encode naturally-occurring amino acid
variants at a plurality of targeted codons in the first nucleic acid;
and, (c) using the set of mutagenic oligonucleotides to generate a set of
active site-encoding or substrate binding site-encoding variant nucleic
acids encoding a range of amino acid variations at each amino acid codon
that was mutagenized, thereby producing a library of nucleic acids
encoding a plurality of modified glucanase, mannanase, or xylanase active
sites or substrate binding sites. In one aspect, the method comprises
mutagenizing the first nucleic acid of step (a) by a method comprising an
optimized directed evolution system, Gene Site-Saturation Mutagenesis.TM.
(GSSM.TM.), synthetic ligation reassembly (SLR), error-prone PCR,
shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis, site-specific
mutagenesis, gene reassembly, synthetic ligation reassembly (SLR) and a
combination thereof. In another aspect, the method comprises mutagenizing
the first nucleic acid of step (a) or variants by a method comprising
recombination, recursive sequence recombination, phosphothioate-modified
DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex
mutagenesis, point mismatch repair mutagenesis, repair-deficient host
strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,
deletion mutagenesis, restriction-selection mutagenesis,
restriction-purification mutagenesis, artificial gene synthesis, ensemble
mutagenesis, chimeric nucleic acid multimer creation and a combination
thereof.
[0080] The invention provides methods for making a small molecule
comprising the following steps: (a) providing a plurality of biosynthetic
enzymes capable of synthesizing or modifying a small molecule, wherein
one of the enzymes comprises a glucanase, mannanase, or xylanase enzyme
encoded by a nucleic acid of the invention; (b) providing a substrate for
at least one of the enzymes of step (a); and (c) reacting the substrate
of step (b) with the enzymes under conditions that facilitate a plurality
of biocatalytic reactions to generate a small molecule by a series of
biocatalytic reactions. The invention provides methods for modifying a
small molecule comprising the following steps: (a) providing a glucanase,
mannanase, or xylanase enzyme, wherein the enzyme comprises a polypeptide
of the invention, or, a polypeptide encoded by a nucleic acid of the
invention, or a subsequence thereof; (b) providing a small molecule; and
(c) reacting the enzyme of step (a) with the small molecule of step (b)
under conditions that facilitate an enzymatic reaction catalyzed by the
glucanase, mannanase, or xylanase enzyme, thereby modifying a small
molecule by a glucanase, mannanase, or xylanase enzymatic reaction. In
one aspect, the method can comprise a plurality of small molecule
substrates for the enzyme of step (a), thereby generating a library of
modified small molecules produced by at least one enzymatic reaction
catalyzed by the glucanase, mannanase, or xylanase enzyme. In one aspect,
the method can comprise a plurality of additional enzymes under
conditions that facilitate a plurality of biocatalytic reactions by the
enzymes to form a library of modified small molecules produced by the
plurality of enzymatic reactions. In another aspect, the method can
further comprise the step of testing the library to determine if a
particular modified small molecule that exhibits a desired activity is
present within the library. The step of testing the library can further
comprise the steps of systematically eliminating all but one of the
biocatalytic reactions used to produce a portion of the plurality of the
modified small molecules within the library by testing the portion of the
modified small molecule for the presence or absence of the particular
modified small molecule with a desired activity, and identifying at least
one specific biocatalytic reaction that produces the particular modified
small molecule of desired activity.
[0081] The invention provides methods for determining a functional
fragment of a glucanase, mannanase, or xylanase enzyme comprising the
steps of: (a) providing a glucanase, mannanase, or xylanase enzyme,
wherein the enzyme comprises a polypeptide of the invention, or a
polypeptide encoded by a nucleic acid of the invention, or a subsequence
thereof; and (b) deleting a plurality of amino acid residues from the
sequence of step (a) and testing the remaining subsequence for a
glucanase, mannanase, or xylanase activity, thereby determining a
functional fragment of a glucanase, mannanase, or xylanase enzyme. In one
aspect, the glucanase, mannanase, or xylanase activity is measured by
providing a glucanase, mannanase, or xylanase substrate and detecting a
decrease in the amount of the substrate or an increase in the amount of a
reaction product.
[0082] The invention provides methods for whole cell engineering of new or
modified phenotypes by using real-time metabolic flux analysis, the
method comprising the following steps: (a) making a modified cell by
modifying the genetic composition of a cell, wherein the genetic
composition is modified by addition to the cell of a nucleic acid of the
invention; (b) culturing the modified cell to generate a plurality of
modified cells; (c) measuring at least one metabolic parameter of the
cell by monitoring the cell culture of step (b) in real time; and, (d)
analyzing the data of step (c) to determine if the measured parameter
differs from a comparable measurement in an unmodified cell under similar
conditions, thereby identifying an engineered phenotype in the cell using
real-time metabolic flux analysis. In one aspect, the genetic composition
of the cell can be modified by a method comprising deletion of a sequence
or modification of a sequence in the cell, or, knocking out the
expression of a gene. In one aspect, the method can further comprise
selecting a cell comprising a newly engineered phenotype. In another
aspect, the method can comprise culturing the selected cell, thereby
generating a new cell strain comprising a newly engineered phenotype.
[0083] The invention provides methods of increasing thermotolerance or
thermostability of a glucanase, mannanase, or xylanase polypeptide, the
method comprising glycosylating a glucanase, mannanase, or xylanase
polypeptide, wherein the polypeptide comprises at least thirty contiguous
amino acids of a polypeptide of the invention; or a polypeptide encoded
by a nucleic acid sequence of the invention, thereby increasing the
thermotolerance or thermostability of the glucanase, mannanase, or
xylanase polypeptide. In one aspect, the glucanase, mannanase, or
xylanase specific activity can be thermostable or thermotolerant at a
temperature in the range from greater than about 37.degree. C. to about
95.degree. C.
[0084] The invention provides methods for overexpressing a recombinant
glucanase, mannanase, or xylanase polypeptide in a cell comprising
expressing a vector comprising a nucleic acid comprising a nucleic acid
of the invention or a nucleic acid sequence of the invention, wherein the
sequence identities are determined by analysis with a sequence comparison
algorithm or by visual inspection, wherein overexpression is effected by
use of a high activity promoter, a dicistronic vector or by gene
amplification of the vector.
[0085] The invention provides methods of making a transgenic plant
comprising the following steps: (a) introducing a heterologous nucleic
acid sequence into the cell, wherein the heterologous nucleic sequence
comprises a nucleic acid sequence of the invention, thereby producing a
transformed plant cell; and (b) producing a transgenic plant from the
transformed cell. In one aspect, the step (a) can further comprise
introducing the heterologous nucleic acid sequence by electroporation or
microinjection of plant cell protoplasts. In another aspect, the step (a)
can further comprise introducing the heterologous nucleic acid sequence
directly to plant tissue by DNA particle bombardment. Alternatively, the
step (a) can further comprise introducing the heterologous nucleic acid
sequence into the plant cell DNA using an Agrobacterium tumefaciens host.
In one aspect, the plant cell can be a potato, corn, rice, wheat,
tobacco, or barley cell.
[0086] The invention provides methods of expressing a heterologous nucleic
acid sequence in a plant cell comprising the following steps: (a)
transforming the plant cell with a heterologous nucleic acid sequence
operably linked to a promoter, wherein the heterologous nucleic sequence
comprises a nucleic acid of the invention; (b) growing the plant under
conditions wherein the heterologous nucleic acids sequence is expressed
in the plant cell. The invention provides methods of expressing a
heterologous nucleic acid sequence in a plant cell comprising the
following steps: (a) transforming the plant cell with a heterologous
nucleic acid sequence operably linked to a promoter, wherein the
heterologous nucleic sequence comprises a sequence of the invention; (b)
growing the plant under conditions wherein the heterologous nucleic acids
sequence is expressed in the plant cell.
[0087] The invention provides methods for hydrolyzing, breaking up or
disrupting a glucan-comprising composition comprising the following
steps: (a) providing a polypeptide of the invention having a glucanase,
mannanase, or xylanase activity, or a polypeptide encoded by a nucleic
acid of the invention; (b) providing a composition comprising a glucan;
and (c) contacting the polypeptide of step (a) with the composition of
step (b) under conditions wherein the glucanase, mannanase, or xylanase
hydrolyzes, breaks up or disrupts the glucan comprising composition. In
one aspect, the composition comprises a plant cell, a bacterial cell, a
yeast cell, an insect cell, or an animal cell. Thus, the composition can
comprise any plant or plant part, any glucan-containing food or feed
(e.g., an animal feed, such as a chicken feed), a waste product and the
like. The invention provides methods for liquefying or removing a
glucan-comprising composition comprising the following steps: (a)
providing a polypeptide of the invention having a glucanase, mannanase,
or xylanase activity, or a polypeptide encoded by a nucleic acid of the
invention; (b) providing a composition comprising a glucan; and (c)
contacting the polypeptide of step (a) with the composition of step (b)
under conditions wherein the glucanase, mannanase, or xylanase removes,
softens or liquefies the glucan-comprising composition.
[0088] The invention provides detergent compositions comprising a
polypeptide of the invention, or a polypeptide encoded by a nucleic acid
of the invention, wherein the polypeptide has a glucanase, e.g.,
endoglucanase, mannanase, or xylanase activity. The glucanase can be a
nonsurface-active glucanase, mannanase, or xylanase or a surface-active
glucanase, mannanase, or xylanase. The glucanase, mannanase, or xylanase
can be formulated in a non-aqueous liquid composition, a cast solid, a
granular form, a particulate form, a compressed tablet, a gel form, a
paste or a slurry form. The invention provides methods for washing an
object comprising the following steps: (a) providing a composition
comprising a polypeptide of the invention having a glucanase, mannanase,
or xylanase activity, or a polypeptide encoded by a nucleic acid of the
invention; (b) providing an object; and (c) contacting the polypeptide of
step (a) and the object of step (b) under conditions wherein the
composition can wash the object.
[0089] The invention provides textiles or fabrics, including, e.g.,
threads, comprising a polypeptide of the invention, or a polypeptide
encoded by a nucleic acid of the invention. In one aspect, the textiles
or fabrics comprise glucan-containing fibers. The invention provides
methods for treating a textile or fabric (e.g., removing a stain from a
composition) comprising the following steps: (a) providing a composition
comprising a polypeptide of the invention having a glucanase e.g.,
endoglucanase, mannanase, or xylanase activity, or a polypeptide encoded
by a nucleic acid of the invention; (b) providing a textile or fabric
comprising a glucan; and (c) contacting the polypeptide of step (a) and
the composition of step (b) under conditions wherein the glucanase,
mannanase, or xylanase can treat the textile or fabric (e.g., remove the
stain). The invention provides methods for improving the finish of a
fabric comprising the following steps: (a) providing a composition
comprising a polypeptide of the invention having a glucanase, mannanase,
or xylanase activity, or a polypeptide encoded by a nucleic acid of the
invention; (b) providing a fabric; and (c) contacting the polypeptide of
step (a) and the fabric of step (b) under conditions wherein the
polypeptide can treat the fabric thereby improving the finish of the
fabric. In one aspect, the fabric is a wool or a silk.
[0090] The invention provides feeds (e.g., an animal feed, such as a
chicken feed) or foods comprising a polypeptide of the invention, or a
polypeptide encoded by a nucleic acid of the invention. The invention
provides methods for hydrolyzing a glucan or other polysaccharide in a
feed or a food prior to consumption by an animal comprising the following
steps: (a) obtaining a feed material comprising a glucanase e.g.,
endoglucanase, mannanase, or xylanase of the invention, or a glucanase,
mannanase, or xylanase encoded by a nucleic acid of the invention; and
(b) adding the polypeptide of step (a) to the feed or food material in an
amount sufficient for a sufficient time period to cause hydrolysis of a
glucan or other polysaccharide and formation of a treated food or feed,
thereby hydrolyzing a glucan or other polysaccharide in the food or the
feed prior to consumption by the animal. In one aspect, the invention
provides methods for hydrolyzing a glucan or other polysaccharide in a
feed or a food after consumption by an animal comprising the following
steps: (a) obtaining a feed material comprising a glucanase, mannanase,
or xylanase of the invention, or a glucanase, mannanase, or xylanase
encoded by a nucleic acid of the invention; (b) adding the polypeptide of
step (a) to the feed or food material; and (c) administering the feed or
food material to the animal, wherein after consumption, the glucanase,
mannanase, or xylanase causes hydrolysis of a glucan or other
polysaccharide in the feed or food in the digestive tract of the animal.
The food or the feed (e.g., an animal feed, such as a chicken feed) can
be, e.g., a cereal, a grain, a corn and the like.
[0091] In another aspect, the invention provides methods for decreasing
the viscosity of a glucans in a composition, e.g., in a food or a feed
(e.g., an animal feed, such as a chicken feed), by treating the
composition with a glucanase of the invention, or, including a glucanase
of the invention in the composition. The food or feed can comprise barley
or wheat, e.g., a food for feed for a high-barley or a high-wheat diet,
such as a poultry diet. In one aspect, the invention provides methods for
minimizing wet droppings by feeding an animal (e.g., a bird, such as a
domestic poultry) a food or a feed treated by or comprising a glucanase,
mannanase, or xylanase of the invention. In one aspect, the invention
provides methods for increasing growth rate and/or feed conversion by
feeding an animal (e.g., a bird, such as a domestic poultry) a food or a
feed treated by or comprising a glucanase, mannanase, or xylanase of the
invention. In one aspect, the invention provides methods for decreasing
excrement by feeding an animal (e.g., a bird, such as a domestic poultry)
a food or a feed treated by or comprising a glucanase, mannanase, or
xylanase of the invention.
[0092] The invention provides food or nutritional supplements for an
animal (e.g., a fowl, such as a chicken) comprising a polypeptide of the
invention, e.g., a polypeptide encoded by the nucleic acid of the
invention. In one aspect, the polypeptide in the food or nutritional
supplement can be glycosylated. The invention provides edible enzyme
delivery matrices comprising a polypeptide of the invention, e.g., a
polypeptide encoded by the nucleic acid of the invention. In one aspect,
the delivery matrix comprises a pellet comprising an enzyme of the
invention, e.g., a pellet comprising a thermotolerant or thermostable
enzyme of the invention). In one aspect, the polypeptide can be
glycosylated (which in one aspect can make the enzyme more thermotolerant
or thermostable). In one aspect, the glucanase e.g., endoglucanase,
mannanase, or xylanase activity is thermotolerant. In another aspect, the
glucanase, mannanase, or xylanase activity is thermostable.
[0093] The invention provides a food, a feed (e.g., an animal feed, such
as a chicken feed) or a nutritional supplement comprising a polypeptide
of the invention. The invention provides methods for utilizing a
glucanase, mannanase, or xylanase as a nutritional supplement in an
animal diet, the method comprising: preparing a nutritional supplement
containing a glucanase, mannanase, or xylanase enzyme comprising at least
thirty contiguous amino acids of a polypeptide of the invention; and
administering the nutritional supplement to an animal to increase
utilization of a glucan or other polysaccharide contained in a feed or a
food ingested by the animal. The animal can be a human, a ruminant or a
monogastric animal. For example, the animal can be a bird, e.g., a
chicken. The glucanase, mannanase, or xylanase enzyme can be prepared by
expression of a polynucleotide encoding the glucanase in an organism such
as a bacterium, a yeast, a plant, an insect, a fungus or an animal.
Exemplary organisms for expressing polypeptides of the invention can be
S. pombe, S. cerevisiae, Pichia sp., e.g., P. pastoris, E. coli,
Streptomyces sp., Bacillus sp. and Lactobacillus sp.
[0094] The invention provides edible enzyme delivery matrix comprising a
thermostable recombinant glucanase, mannanase, or xylanase enzyme, e.g.,
a polypeptide of the invention. The invention provides methods for
delivering a glucanase, mannanase, or xylanase supplement to an animal (a
human, a ruminant, a monogastric animal, a bird, e.g., a chicken), the
method comprising: preparing an edible enzyme delivery matrix in the form
of pellets comprising a granulate edible carrier and a thermostable
isolated, synthetic or recombinant glucanase, mannanase, or xylanase
enzyme, wherein the pellets readily disperse the glucanase, mannanase, or
xylanase enzyme contained therein into aqueous media, and administering
the edible enzyme delivery matrix to the animal. The recombinant
glucanase, mannanase, or xylanase enzyme can comprise a polypeptide of
the invention. The granulate edible carrier can comprise a carrier
selected from the group consisting of a grain germ, a grain germ that is
spent of oil, a hay, an alfalfa, a timothy, a soy hull, a sunflower seed
meal and a wheat midd. The edible carrier can comprise grain germ that is
spent of oil. The glucanase, mannanase, or xylanase enzyme can be
glycosylated to provide thermostability at pelletizing conditions. The
delivery matrix can be formed by pelletizing a mixture comprising a grain
germ and a glucanase, mannanase, or xylanase. The pelletizing conditions
can include application of steam. The pelletizing conditions can comprise
application of a temperature in excess of about 80.degree. C. for about 5
minutes and the enzyme retains a specific activity of at least 350 to
about 900 units per milligram of enzyme.
[0095] The invention provides methods for improving texture and flavor of
a dairy product comprising the following steps: (a) providing a
polypeptide of the invention having a glucanase, mannanase, or xylanase
activity, or a glucanase encoded by a nucleic acid of the invention; (b)
providing a dairy product; and (c) contacting the polypeptide of step (a)
and the dairy product of step (b) under conditions wherein the glucanase,
mannanase, or xylanase can improve the texture or flavor of the dairy
product. In one aspect, the dairy product comprises a cheese or a
yoghurt. The invention provides dairy products comprising a glucanase,
mannanase, or xylanase of the invention, or is encoded by a nucleic acid
of the invention.
[0096] The invention provides methods for improving the extraction of oil
from an oil-rich plant material comprising the following steps: (a)
providing a polypeptide of the invention having a glucanase, mannanase,
or xylanase activity, or a glucanase, mannanase, or xylanase encoded by a
nucleic acid of the invention; (b) providing an oil-rich plant material;
and (c) contacting the polypeptide of step (a) and the oil-rich plant
material. In one aspect, the oil-rich plant material comprises an
oil-rich seed. The oil can be a soybean oil, an olive oil, a rapeseed
(canola) oil or a sunflower oil and the like.
[0097] In one aspect, the invention provides methods using a glucanase,
mannanase, or xylanase of the invention to produce fermentable sugars
that can be converted into fuel ethanol. In one aspect, the invention
provides fuels comprising a polypeptide of the invention having a
glucanase, mannanase, or xylanase activity, or a glucanase encoded by a
nucleic acid of the invention. In one aspect, an enzyme of the invention
is used to catalyze the hydrolysis of celluloses, hemicelluloses and
lignins. The degradation of cellulose may be used for the conversion of
plant biomass into fuels and chemicals. See, e.g., Kohlmann (1996) Adv.
Space Res. 18:251-265; Perez (2002) Int Microbiol. 5:53-63.
[0098] The invention provides methods for preparing a fruit or vegetable
juice, syrup, puree or extract comprising the following steps: (a)
providing a polypeptide of the invention having a glucanase, mannanase,
or xylanase activity, or a glucanase, mannanase, or xylanase encoded by a
nucleic acid of the invention; (b) providing a composition or a liquid
comprising a fruit or vegetable material; and (c) contacting the
polypeptide of step (a) and the composition, thereby preparing the fruit
or vegetable juice, syrup, puree or extract.
[0099] The invention provides papers or paper products or paper pulp
comprising a glucanase, mannanase, or xylanase of the invention, or a
polypeptide encoded by a nucleic acid of the invention. The invention
provides methods for treating a paper or a paper or wood pulp comprising
the following steps: (a) providing a polypeptide of the invention having
a glucanase, mannanase, or xylanase activity, or a glucanase, mannanase,
or xylanase encoded by a nucleic acid of the invention; (b) providing a
composition comprising a paper or a paper or wood pulp; and (c)
contacting the polypeptide of step (a) and the composition of step (b)
under conditions wherein the glucanase, mannanase, or xylanase can treat
the paper or paper or wood pulp. In one aspect, the pharmaceutical
composition acts as a digestive aid or an anti-microbial (e.g., against
Salmonella). In one aspect, the treatment is prophylactic. In one aspect,
the invention provides oral care products comprising a polypeptide of the
invention having a glucanase, mannanase, or xylanase activity, or a
glucanase, mannanase, or xylanase encoded by a nucleic acid of the
invention. The oral care product can comprise a toothpaste, a dental
cream, a gel or a tooth powder, an odontic, a mouth wash, a pre- or post
brushing rinse formulation, a chewing gum, a lozenge or a candy. The
invention provides contact lens cleaning compositions comprising a
polypeptide of the invention having a glucanase, mannanase, or xylanase
activity, or a glucanase, mannanase, or xylanase encoded by a nucleic
acid of the invention.
[0100] In one aspect, the invention provides methods for eliminating or
protecting animals from a microorganism comprising a glucan or other
polysaccharide comprising administering a polypeptide of the invention.
The microorganism can be a bacterium comprising a glucan, e.g.,
Salmonella.
[0101] Another aspect of the invention is a method of making a polypeptide
of the invention. The method includes introducing a nucleic acid encoding
the polypeptide into a host cell, wherein the nucleic acid is operably
linked to a promoter and culturing the host cell under conditions that
allow expression of the nucleic acid. Another aspect of the invention is
a method of making a polypeptide having at least 10 amino acids of a
sequence as set forth in amino acid sequences of the invention. The
method includes introducing a nucleic acid encoding the polypeptide into
a host cell, wherein the nucleic acid is operably linked to a promoter
and culturing the host cell under conditions that allow expression of the
nucleic acid, thereby producing the polypeptide.
[0102] Another aspect of the invention is a method of generating a variant
including obtaining a nucleic acid having a sequence of the invention,
sequences substantially identical thereto, sequences complementary to a
sequence of the invention, fragments comprising at least 30 consecutive
nucleotides of the foregoing sequences and changing one or more
nucleotides in the sequence to another nucleotide, deleting one or more
nucleotides in the sequence, or adding one or more nucleotides to the
sequence.
[0103] Another aspect of the invention is a computer readable medium
having stored thereon a nucleic acid or polypeptide sequence of the
invention. Another aspect of the invention is a computer system including
a processor and a data storage device wherein the data storage device has
stored thereon a nucleic acid or polypeptide sequence of the invention.
Another aspect of the invention is a method for comparing a first
sequence to a reference sequence wherein the first sequence is a nucleic
acid or polypeptide sequence of the invention. The method includes
reading the first sequence and the reference sequence through use of a
computer program that compares sequences; and determining differences
between the first sequence and the reference sequence with the computer
program. Another aspect of the invention is a method for identifying a
feature in a nucleic acid or polypeptide sequence of the invention,
including reading the sequence through the use of a computer program
which identifies features in sequences; and identifying features in the
sequence with the computer program.
[0104] Yet another aspect of the invention is a method of catalyzing the
breakdown of glycan or a derivative thereof, comprising the step of
contacting a sample containing a glucan or other polysaccharide or a
derivative thereof with a polypeptide of the invention under conditions
which facilitate the breakdown of a glucan.
[0105] Another aspect of the invention is an assay for identifying
fragments or variants of a polypeptide of the invention, which retain the
enzymatic function of a polypeptide of the invention. The assay includes
contacting a polypeptide of the invention with a substrate molecule under
conditions which allow the polypeptide fragment or variant to function
and detecting either a decrease in the level of substrate or an increase
in the level of the specific reaction product of the reaction between the
polypeptide and substrate thereby identifying a fragment or variant of
such sequences.
[0106] Another aspect of the invention is a nucleic acid probe of an
oligonucleotide from about 10 to 50 nucleotides in length and having a
segment of at least 10 contiguous nucleotides that is at least 50%
complementary to a nucleic acid target region of a nucleic acid sequence
of the invention; and which hybridizes to the nucleic acid target region
under moderate to highly stringent conditions to form a detectable
target:probe duplex.
[0107] Another aspect of the invention is a polynucleotide probe for
isolation or identification of glucanase, mannanase, or xylanase genes
having a sequence which is the same as, or fully complementary to at
least a nucleic acid sequence of the invention.
[0108] In still another aspect, the invention provides a protein
preparation comprising a polypeptide having an amino acid sequence of the
invention wherein the protein preparation is a liquid. Still another
aspect of the invention provides a protein preparation comprising a
polypeptide having an amino acid sequence of the invention wherein the
polypeptide is a solid.
[0109] Yet another aspect of the invention provides a method for modifying
small molecules, comprising the step of mixing at least one polypeptide
of the invention with at least one small molecule, to produce at least
one modified small molecule via at least one biocatalytic reaction, where
the at least one polypeptide has glucanase, mannanase, or xylanase
activity.
[0110] Another aspect of the invention is a cloning vector of a sequence
that encodes a polypeptide of the invention having a glucanase,
mannanase, or xylanase activity. Another aspect of the invention is a
host cell comprising a sequence that encodes a polypeptide of the
invention. In yet another aspect, the invention provides an expression
vector capable of replicating in a host cell comprising a nucleic acid of
the invention or a nucleic acid encoding a polynucleotide of the
invention.
[0111] In another aspect, the invention provides a method of dough
conditioning comprising contacting dough with at least one polypeptide of
the invention under conditions sufficient for conditioning the dough.
Another aspect of the invention is a method of beverage production
comprising administration of at least one polypeptide of the invention
under conditions sufficient for decreasing the viscosity of wort or beer,
or, increasing the clarity (e.g., clarification) of the beverage.
[0112] The glucanases, e.g., endoglucanases, mannanases, or xylanases of
the invention are used to break down the high molecular weight glucans or
other polysaccharides in animal feed (e.g., a feed for a human, a
ruminant, a monogastric animal, a bird, e.g., a chicken). Adding enzymes
of the invention stimulates growth rates by improving digestibility,
which also improves the quality of the animal litter. Glucanase functions
through the gastrointestinal tract to reduce intestinal viscosity and
increase diffusion of pancreatic enzymes. Additionally, the enzymes of
the invention may be used in the treatment of endosperm cell walls of
feed grains and vegetable proteins. In one aspect of the invention, the
novel enzymes of the invention are administered to an animal in order to
increase the utilization of a glucan or other polysaccharide in the food.
This activity of the enzymes of the invention may be used to break down
insoluble cell wall material, liberating nutrients in the cell walls,
which then become available to the animal. It also changes hemicellulose
to nutritive sugars so that nutrients formerly trapped within the cell
walls are released. Glucanase, mannanase, or xylanase enzymes of the
invention can produce compounds that may be a nutritive source for the
ruminal microflora.
[0113] Another aspect of the invention provides a method for utilizing
glucanase, mannanase, or xylanase as a nutritional supplement in the
diets of animals, comprising preparation of a nutritional supplement
containing a recombinant glucanase enzyme comprising at least thirty
contiguous amino acids of an amino acid sequence of the invention and
administering the nutritional supplement to an animal to increase the
utilization of a glucan or other polysaccharide contained in food
ingested by the animal.
[0114] In another aspect of the invention, a method for delivering a
glucanase, mannanase, or xylanase supplement to an animal is provided,
where the method comprises preparing an edible enzyme delivery matrix in
the form of pellets comprising a granulate edible carrier and a
thermostable recombinant or synthetic glucanase, mannanase, or xylanase
enzyme, wherein the particles readily disperse the glucanase, mannanase,
or xylanase enzyme contained therein into aqueous media, and
administering the edible enzyme delivery matrix to the animal. The
granulate edible carrier may comprise a carrier selected from the group
consisting of grain germ that is spent of oil, hay, alfalfa, timothy, soy
hull, sunflower seed meal and wheat midd. The glucanase, mannanase, or
xylanase enzyme may have an amino acid sequence of the invention.
[0115] In another aspect, the invention provides an isolated, synthetic or
recombinant nucleic acid comprising a sequence of the invention that
encodes a polypeptide having glucanase, mannanase, or xylanase activity,
wherein the sequence contains a signal sequence. The invention also
provides an isolated, synthetic or recombinant nucleic acid comprising a
sequence that encodes a polypeptide of the invention having glucanase,
mannanase, or xylanase activity, and the sequence contains a signal
sequence from another glucanase, mannanase, or xylanase. Additionally,
the invention provides an isolated, synthetic or recombinant nucleic acid
comprising a sequence of the invention that encodes a polypeptide having
glucanase, mannanase, or xylanase activity and the sequence does not
contain a signal sequence.
[0116] The details of one or more embodiments of the invention are set
forth in the accompanying drawings and the description below. Other
features, objects, and advantages of the invention will be apparent from
the description and drawings, and from the claims.
[0117] All publications, patents, patent applications, GenBank sequences
and ATCC deposits, cited herein and the compact disc (submitted in
quadruplicate) containing a sequence listing are hereby expressly
incorporated by reference for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0118] The following drawings are illustrative of aspects of the invention
and are not meant to limit the scope of the invention as encompassed by
the claims.
[0119] FIG. 1 is a block diagram of a computer system.
[0120] FIG. 2 is a flow diagram illustrating one aspect of a process for
comparing a new nucleotide or protein sequence with a database of
sequences in order to determine the homology levels between the new
sequence and the sequences in the database.
[0121] FIG. 3 is a flow diagram illustrating one aspect of a process in a
computer for determining whether two sequences are homologous.
[0122] FIG. 4 is a flow diagram illustrating one aspect of an identifier
process 300 for detecting the presence of a feature in a sequence.
[0123] FIG. 5 is a table summarizing the relative activities of several
exemplary enzymes of the invention under various conditions.
[0124] FIG. 6 is an illustration in graph form of an exemplary set of data
("sample data") that is illustrated as a "standard curve", as discussed
in Example 3.
[0125] FIG. 7 and FIG. 8 illustrate the results of glucanase activity
assays demonstrating improved expression in Pichia pastoris of the
exemplary glucanase of the invention having a sequence as set forth in
SEQ ID NO:464, encoded by a codon-optimized version of SEQ ID NO:5 (i.e.,
the optimized version being SEQ ID NO:463), as discussed in Example 4,
below.
[0126] FIG. 9 illustrates the results of glucanase activity assays showing
the temperature profile of the exemplary glucanase of the invention
encoded by SEQ ID NO:6, as discussed in Example 5, below.
[0127] FIG. 10 illustrates the results of glucanase activity assays
showing the half-life determination of the exemplary glucanase of the
invention encoded by SEQ ID NO:6, as discussed in Example 5, below.
[0128] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION OF THE INVENTION
[0129] The invention provides polypeptides and polynucleotides encoding
them and methods of making and using them. Enzyme activity of the
polypeptides of the invention encompasses polypeptides having a hydrolase
activity, e.g., a glucanase activity, for example, polypeptides capable
of hydrolyzing glycosidic linkages present in a glucan, e.g., catalyzing
hydrolysis of internal .beta.-1,4-glucosidic linkages. Enzyme activity of
the polypeptides of the invention (including antibodies) encompasses
polypeptides having a glucanase, a xylanase, and/or a mannanase activity.
The enzymes of the invention can be used to make and/or process foods,
feeds (e.g., for a human, a ruminant, a monogastric animal, a bird, e.g.,
a chicken), beverages, nutritional supplements, textiles, detergents and
the like. The enzymes of the invention can be used in pharmaceutical
compositions and dietary aids. Glucanases, mannanases, or xylanases of
the invention are useful in food processing, baking, animal feeds or
foods, beverages, detergents, pulp processing and paper processes.
DEFINITIONS
[0130] The term "antibody" includes a peptide or polypeptide derived from,
modeled after or substantially encoded by an immunoglobulin gene or
immunoglobulin genes, or fragments thereof, capable of specifically
binding an antigen or epitope, see, e.g. Fundamental Immunology, Third
Edition, W. E. Paul, ed, Raven Press, N.Y. (1993); Wilson (1994) J.
Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods
25:85-97. The term antibody includes antigen-binding portions, i.e.,
"antigen binding sites," (e.g., fragments, subsequences, complementarity
determining regions (CDRs)) that retain capacity to bind antigen,
including (i) a Fab fragment, a monovalent fragment consisting of the VL,
VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the hinge
region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a
Fv fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),
which consists of a VH domain; and (vi) an isolated complementarity
determining region (CDR). Single chain antibodies are also included by
reference in the term "antibody."
[0131] The terms "array" or "microarray" or "biochip" or "chip" as used
herein is a plurality of target elements, each target element comprising
a defined amount of one or more polypeptides (including antibodies) or
nucleic acids immobilized onto a defined area of a substrate surface, as
discussed in further detail, below.
[0132] As used herein, the terms "computer," "computer program" and
"processor" are used in their broadest general contexts and incorporate
all such devices, as described in detail, below. A "coding sequence of"
or a "sequence encodes" a particular polypeptide or protein, is a nucleic
acid sequence which is transcribed and translated into a polypeptide or
protein when placed under the control of appropriate regulatory
sequences.
[0133] The phrases "nucleic acid" or "nucleic acid sequence" as used
herein refer to an oligonucleotide, nucleotide, polynucleotide, or to a
fragment of any of these, to DNA or RNA of genomic or synthetic origin
which may be single-stranded or double-stranded and may represent a sense
or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or
RNA-like material, natural or synthetic in origin. The phrases "nucleic
acid" or "nucleic acid sequence" includes oligonucleotide, nucleotide,
polynucleotide, or to a fragment of any of these, to DNA or RNA (e.g.,
mRNA, rRNA, tRNA, iRNA) of genomic or synthetic origin which may be
single-stranded or double-stranded and may represent a sense or antisense
strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like
material, natural or synthetic in origin, including, e.g., iRNA,
ribonucleoproteins (e.g., e.g., double stranded iRNAs, e.g., iRNPs). The
term encompasses nucleic acids, i.e., oligonucleotides, containing known
analogues of natural nucleotides. The term also encompasses
nucleic-acid-like structures with synthetic backbones, see e.g., Mata
(1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997)
Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev
6:153-156. "Oligonucleotide" includes either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide strands
which may be chemically synthesized. Such synthetic oligonucleotides have
no 5' phosphate and thus will not ligate to another oligonucleotide
without adding a phosphate with an ATP in the presence of a kinase. A
synthetic oligonucleotide can ligate to a fragment that has not been
dephosphorylated.
[0134] A "coding sequence of" or a "nucleotide sequence encoding" a
particular polypeptide or protein, is a nucleic acid sequence which is
transcribed and translated into a polypeptide or protein when placed
under the control of appropriate regulatory sequences.
[0135] The term "gene" means the segment of DNA involved in producing a
polypeptide chain; it includes regions preceding and following the coding
region (leader and trailer) as well as, where applicable, intervening
sequences (introns) between individual coding segments (exons). "Operably
linked" as used herein refers to a functional relationship between two or
more nucleic acid (e.g., DNA) segments. Typically, it refers to the
functional relationship of transcriptional regulatory sequence to a
transcribed sequence. For example, a promoter is operably linked to a
coding sequence, such as a nucleic acid of the invention, if it
stimulates or modulates the transcription of the coding sequence in an
appropriate host cell or other expression system. Generally, promoter
transcriptional regulatory sequences that are operably linked to a
transcribed sequence are physically contiguous to the transcribed
sequence, i.e., they are cis-acting. However, some transcriptional
regulatory sequences, such as enhancers, need not be physically
contiguous or located in close proximity to the coding sequences whose
transcription they enhance.
[0136] The term "expression cassette" as used herein refers to a
nucleotide sequence which is capable of affecting expression of a
structural gene (i.e., a protein coding sequence, such as a glucanase of
the invention) in a host compatible with such sequences. Expression
cassettes include at least a promoter operably linked with the
polypeptide coding sequence; and, optionally, with other sequences, e.g.,
transcription termination signals. Additional factors necessary or
helpful in effecting expression may also be used, e.g., enhancers. Thus,
expression cassettes also include plasmids, expression vectors,
recombinant viruses, any form of recombinant "naked DNA" vector, and the
like. A "vector" comprises a nucleic acid which can infect, transfect,
transiently or permanently transduce a cell. It will be recognized that a
vector can be a naked nucleic acid, or a nucleic acid complexed with
protein or lipid. The vector optionally comprises viral or bacterial
nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a
viral lipid envelope, etc.). Vectors include, but are not limited to
replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA
may be attached and become replicated. Vectors thus include, but are not
limited to RNA, autonomous self-replicating circular or linear DNA or RNA
(e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.
5,217,879), and include both the expression and non-expression plasmids.
Where a recombinant microorganism or cell culture is described as hosting
an "expression vector" this includes both extra-chromosomal circular and
linear DNA and DNA that has been incorporated into the host
chromosome(s). Where a vector is being maintained by a host cell, the
vector may either be stably replicated by the cells during mitosis as an
autonomous structure, or is incorporated within the host's genome.
[0137] As used herein, the term "promoter" includes all sequences capable
of driving transcription of a coding sequence in a cell, e.g., a plant
cell. Thus, promoters used in the constructs of the invention include
cis-acting transcriptional control elements and regulatory sequences that
are involved in regulating or modulating the timing and/or rate of
transcription of a gene. For example, a promoter can be a cis-acting
transcriptional control element, including an enhancer, a promoter, a
transcription terminator, an origin of replication, a chromosomal
integration sequence, 5' and 3' untranslated regions, or an intronic
sequence, which are involved in transcriptional regulation. These
cis-acting sequences typically interact with proteins or other
biomolecules to carry out (turn on/off, regulate, modulate, etc.)
transcription. "Constitutive" promoters are those that drive expression
continuously under most environmental conditions and states of
development or cell differentiation. "Inducible" or "regulatable"
promoters direct expression of the nucleic acid of the invention under
the influence of environmental conditions or developmental conditions.
Examples of environmental conditions that may affect transcription by
inducible promoters include anaerobic conditions, elevated temperature,
drought, or the presence of light.
[0138] "Tissue-specific" promoters are transcriptional control elements
that are only active in particular cells or tissues or organs, e.g., in
plants or animals. Tissue-specific regulation may be achieved by certain
intrinsic factors which ensure that genes encoding proteins specific to a
given tissue are expressed. Such factors are known to exist in mammals
and plants so as to allow for specific tissues to develop.
[0139] The term "plant" includes whole plants, plant parts (e.g., leaves,
stems, flowers, roots, etc.), plant protoplasts, seeds and plant cells
and progeny of same. The class of plants which can be used in the method
of the invention is generally as broad as the class of higher plants
amenable to transformation techniques, including angiosperms
(monocotyledonous and dicotyledonous plants), as well as gymnosperms. It
includes plants of a variety of ploidy levels, including polyploid,
diploid, haploid and hemizygous states. As used herein, the term
"transgenic plant" includes plants or plant cells into which a
heterologous nucleic acid sequence has been inserted, e.g., the nucleic
acids and various recombinant constructs (e.g., expression cassettes) of
the invention.
[0140] "Plasmids" can be commercially available, publicly available on an
unrestricted basis, or can be constructed from available plasmids in
accord with published procedures. Equivalent plasmids to those described
herein are known in the art and will be apparent to the ordinarily
skilled artisan.
[0141] "Amino acid" or "amino acid sequence" as used herein refer to an
oligopeptide, peptide, polypeptide, or protein sequence, or to a
fragment, portion, or subunit of any of these and to naturally occurring
or synthetic molecules.
[0142] "Amino acid" or "amino acid sequence" include an oligopeptide,
peptide, polypeptide, or protein sequence, or to a fragment, portion, or
subunit of any of these, and to naturally occurring or synthetic
molecules. The term "polypeptide" as used herein, refers to amino acids
joined to each other by peptide bonds or modified peptide bonds, i.e.,
peptide isosteres and may contain modified amino acids other than the 20
gene-encoded amino acids. The polypeptides may be modified by either
natural processes, such as post-translational processing, or by chemical
modification techniques which are well known in the art. Modifications
can occur anywhere in the polypeptide, including the peptide backbone,
the amino acid side-chains and the amino or carboxyl termini. It will be
appreciated that the same type of modification may be present in the same
or varying degrees at several sites in a given polypeptide. Also a given
polypeptide may have many types of modifications. Modifications include
acetylation, acylation, ADP-ribosylation, amidation, covalent attachment
of flavin, covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid or
lipid derivative, covalent attachment of a phosphatidylinositol,
cross-linking cyclization, disulfide bond formation, demethylation,
formation of covalent cross-links, formation of cysteine, formation of
pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI
anchor formation, hydroxylation, iodination, methylation, myristolyation,
oxidation, pegylation, glucan hydrolase processing, phosphorylation,
prenylation, racemization, selenoylation, sulfation and transfer-RNA
mediated addition of amino acids to protein such as arginylation. (See
Creighton, T. E., Proteins--Structure and Molecular Properties 2nd Ed.,
W.H. Freeman and Company, New York (1993); Posttranslational Covalent
Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York,
pp. 1-12 (1983)). The peptides and polypeptides of the invention also
include all "mimetic" and "peptidomimetic" forms, as described in further
detail, below.
[0143] As used herein, the term "isolated" means that the material is
removed from its original environment (e.g., the natural environment if
it is naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not isolated,
but the same polynucleotide or polypeptide, separated from some or all of
the coexisting materials in the natural system, is isolated. Such
polynucleotides could be part of a vector and/or such polynucleotides or
polypeptides could be part of a composition and still be isolated in that
such vector or composition is not part of its natural environment. As
used herein, the term "purified" does not require absolute purity;
rather, it is intended as a relative definition. Individual nucleic acids
obtained from a library have been conventionally purified to
electrophoretic homogeneity. The sequences obtained from these clones
could not be obtained directly either from the library or from total
human DNA. The purified nucleic acids of the invention have been purified
from the remainder of the genomic DNA in the organism by at least
10.sup.4-10.sup.6 fold. However, the term "purified" also includes
nucleic acids which have been purified from the remainder of the genomic
DNA or from other sequences in a library or other environment by at least
one order of magnitude, typically two or three orders and more typically
four or five orders of magnitude.
[0144] As used herein, the term "recombinant" means that the nucleic acid
is adjacent to a "backbone" nucleic acid to which it is not adjacent in
its natural environment. Additionally, to be "enriched" the nucleic acids
will represent 5% or more of the number of nucleic acid inserts in a
population of nucleic acid backbone molecules. Backbone molecules
according to the invention include nucleic acids such as expression
vectors, self-replicating nucleic acids, viruses, integrating nucleic
acids and other vectors or nucleic acids used to maintain or manipulate a
nucleic acid insert of interest. Typically, the enriched nucleic acids
represent 15% or more of the number of nucleic acid inserts in the
population of recombinant backbone molecules. More typically, the
enriched nucleic acids represent 50% or more of the number of nucleic
acid inserts in the population of recombinant backbone molecules. In a
one aspect, the enriched nucleic acids represent 90% or more of the
number of nucleic acid inserts in the population of recombinant backbone
molecules.
[0145] "Recombinant" polypeptides or proteins refer to polypeptides or
proteins produced by recombinant DNA techniques; i.e., produced from
cells transformed by an exogenous DNA construct encoding the desired
polypeptide or protein, "Synthetic" polypeptides or protein are those
prepared by chemical synthesis. Solid-phase chemical peptide synthesis
methods can also be used to synthesize the polypeptide or fragments of
the invention. Such method have been known in the art since the early
1960's (Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (See
also Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2nd
Ed., Pierce Chemical Co., Rockford, Ill., pp. 11-12)) and have recently
been employed in commercially available laboratory peptide design and
synthesis kits (Cambridge Research Biochemicals). Such commercially
available laboratory kits have generally utilized the teachings of H. M.
Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide for
synthesizing peptides upon the tips of a multitude of "rods" or "pins"
all of which are connected to a single plate. When such a system is
utilized, a plate of rods or pins is inverted and inserted into a second
plate of corresponding wells or reservoirs, which contain solutions for
attaching or anchoring an appropriate amino acid to the pin's or rod's
tips. By repeating such a process step, i.e., inverting and inserting the
rod's and pin's tips into appropriate solutions, amino acids are built
into desired peptides. In addition, a number of available FMOC peptide
synthesis systems are available. For example, assembly of a polypeptide
or fragment can be carried out on a solid support using an Applied
Biosystems, Inc. Model 431A automated peptide synthesizer. Such equipment
provides ready access to the peptides of the invention, either by direct
synthesis or by synthesis of a series of fragments that can be coupled
using other known techniques.
[0146] A promoter sequence is "operably linked to" a coding sequence when
RNA polymerase which initiates transcription at the promoter will
transcribe the coding sequence into mRNA.
[0147] "Plasmids" are designated by a lower case "p" preceded and/or
followed by capital letters and/or numbers. The starting plasmids herein
are either commercially available, publicly available on an unrestricted
basis, or can be constructed from available plasmids in accord with
published procedures. In addition, equivalent plasmids to those described
herein are known in the art and will be apparent to the ordinarily
skilled artisan.
[0148] "Digestion" of DNA refers to catalytic cleavage of the DNA with a
restriction enzyme that acts only at certain sequences in the DNA. The
various restriction enzymes used herein are commercially available and
their reaction conditions, cofactors and other requirements were used as
would be known to the ordinarily skilled artisan. For analytical
purposes, typically 1 .mu.g of plasmid or DNA fragment is used with about
2 units of enzyme in about 20 .mu.l of buffer solution. For the purpose
of isolating DNA fragments for plasmid construction, typically 5 to 50
.mu.g of DNA are digested with 20 to 250 units of enzyme in a larger
volume. Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubation times
of about 1 hour at 37.degree. C. are ordinarily used, but may vary in
accordance with the supplier's instructions. After digestion, gel
electrophoresis may be performed to isolate the desired fragment.
[0149] The phrase "substantially identical" in the context of two nucleic
acids or polypeptides, refers to two or more sequences that have, e.g.,
at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,
61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more nucleotide
or amino acid residue (sequence) identity, when compared and aligned for
maximum correspondence, as measured using one of the known sequence
comparison algorithms or by visual inspection. Typically, the substantial
identity exists over a region of at least about 100 residues and most
commonly the sequences are substantially identical over at least about
150-200 residues. In some aspects, the sequences are substantially
identical over the entire length of the coding regions.
[0150] Additionally a "substantially identical" amino acid sequence is a
sequence that differs from a reference sequence by one or more
conservative or non-conservative amino acid substitutions, deletions, or
insertions, particularly when such a substitution occurs at a site that
is not the active site (catalytic domains (CDs)) of the molecule and
provided that the polypeptide essentially retains its functional
properties. A conservative amino acid substitution, for example,
substitutes one amino acid for another of the same class (e.g.,
substitution of one hydrophobic amino acid, such as isoleucine, valine,
leucine, or methionine, for another, or substitution of one polar amino
acid for another, such as substitution of arginine for lysine, glutamic
acid for aspartic acid or glutamine for asparagine). One or more amino
acids can be deleted, for example, from a glucanase polypeptide,
resulting in modification of the structure of the polypeptide, without
significantly altering its biological activity. For example, amino- or
carboxyl-terminal amino acids that are not required for glucanase
biological activity can be removed. Modified polypeptide sequences of the
invention can be assayed for glucanase biological activity by any number
of methods, including contacting the modified polypeptide sequence with a
glucanase substrate and determining whether the modified polypeptide
decreases the amount of specific substrate in the assay or increases the
bioproducts of the enzymatic reaction of a functional glucanase
polypeptide with the substrate.
[0151] "Fragments" as used herein are a portion of a naturally occurring
protein which can exist in at least two different conformations.
Fragments can have the same or substantially the same amino acid sequence
as the naturally occurring protein. "Substantially the same" means that
an amino acid sequence is largely, but not entirely, the same, but
retains at least one functional activity of the sequence to which it is
related. In general two amino acid sequences are "substantially the same"
or "substantially homologous" if they are at least about 85% identical.
Fragments which have different three dimensional structures as the
naturally occurring protein are also included. An example of this, is a
"pro-form" molecule, such as a low activity proprotein that can be
modified by cleavage to produce a mature enzyme with significantly higher
activity.
[0152] "Hybridization" refers to the process by which a nucleic acid
strand joins with a complementary strand through base pairing.
Hybridization reactions can be sensitive and selective so that a
particular sequence of interest can be identified even in samples in
which it is present at low concentrations. Suitably stringent conditions
can be defined by, for example, the concentrations of salt or formamide
in the prehybridization and hybridization solutions, or by the
hybridization temperature and are well known in the art. In particular,
stringency can be increased by reducing the concentration of salt,
increasing the concentration of formamide, or raising the hybridization
temperature. In alternative aspects, nucleic acids of the invention are
defined by their ability to hybridize under various stringency conditions
(e.g., high, medium, and low), as set forth herein.
[0153] For example, hybridization under high stringency conditions could
occur in about 50% formamide at about 37.degree. C. to 42.degree. C.
Hybridization could occur under reduced stringency conditions in about
35% to 25% formamide at about 30.degree. C. to 35.degree. C. In
particular, hybridization could occur under high stringency conditions at
42.degree. C. in 50% formamide, 5.times.SSPE, 0.3% SDS and 200 n/ml
sheared and denatured salmon sperm DNA. Hybridization could occur under
reduced stringency conditions as described above, but in 35% formamide at
a reduced temperature of 35.degree. C. The temperature range
corresponding to a particular level of stringency can be further narrowed
by calculating the purine to pyrimidine ratio of the nucleic acid of
interest and adjusting the temperature accordingly. Variations on the
above ranges and conditions are well known in the art.
[0154] The term "variant" refers to polynucleotides or polypeptides of the
invention modified at one or more base pairs, codons, introns, exons, or
amino acid residues (respectively) yet still retain the biological
activity of a glucanase of the invention. Variants can be produced by any
number of means included methods such as, for example, error-prone PCR,
shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis, site-specific
mutagenesis, gene reassembly, GSSM.TM. and any combination thereof.
[0155] The term "Saturation Mutagenesis" or "Gene Site Saturation
Mutagenesis.sctn." or "GSSM.TM." includes a method that uses degenerate
oligonucleotide primers to introduce point mutations into a
polynucleotide, as described in detail, below.
[0156] The term "optimized directed evolution system" or "optimized
directed evolution" includes a method for reassembling fragments of
related nucleic acid sequences, e.g., related genes, and explained in
detail, below.
[0157] The term "synthetic ligation reassembly" or "SLR" includes a method
of ligating oligonucleotide fragments in a non-stochastic fashion, and
explained in detail, below.
Generating and Manipulating Nucleic Acids
[0158] The invention provides isolated, recombinant and synthetic nucleic
acids (e.g., an exemplary nucleic acid of the invention, including SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,
SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ
ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID
NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID
NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID
NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID
NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID
NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID
NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID
NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID
NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ
ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121,
SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID
NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ
ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149,
SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID
NO:199, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ
ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177,
SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID
NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ
ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205,
SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID
NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ
ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233,
SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID
NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ
ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261,
SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, SEQ ID
NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ
ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQ ID NO:289,
SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297, SEQ ID
NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ
ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQ ID NO:317,
SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325, SEQ ID
NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ
ID NO:337, SEQ ID NO:339, SEQ ID NO:341, SEQ ID NO:343, SEQ ID NO:345,
SEQ ID NO:347, SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:353, SEQ ID
NO:355, SEQ ID NO:357, SEQ ID NO:359, SEQ ID NO:361, SEQ ID NO:363, SEQ
ID NO:365, SEQ ID NO:367, SEQ ID NO:369, SEQ ID NO:371, SEQ ID NO:373,
SEQ ID NO:375, SEQ ID NO:377, SEQ ID NO:379, SEQ ID NO:381, SEQ ID
NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQ ID NO:389, SEQ ID NO:391, SEQ
ID NO:393, SEQ ID NO:395, SEQ ID NO:397, SEQ ID NO:399, SEQ ID NO:401,
SEQ ID NO:403, SEQ ID NO:405, SEQ ID NO:407, SEQ ID NO:409, SEQ ID
NO:411, SEQ ID NO:413, SEQ ID NO:415, SEQ ID NO:417, SEQ ID NO:419, SEQ
ID NO:421, SEQ ID NO:423, SEQ ID NO:425, SEQ ID NO:427, SEQ ID NO:429,
SEQ ID NO:431, SEQ ID NO:433, SEQ ID NO:435, SEQ ID NO:437, SEQ ID
NO:439, SEQ ID NO:441, SEQ ID NO:443, SEQ ID NO:445, SEQ ID NO:447, SEQ
ID NO:449, SEQ ID NO:451, SEQ ID NO:453, SEQ ID NO:455, SEQ ID NO:457,
SEQ ID NO:459, SEQ ID NO:461, SEQ ID NO:463, SEQ ID NO:465, SEQ ID
NO:467, SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ ID NO:475, SEQ
ID NO:477, SEQ ID NO:479, SEQ ID NO:481, SEQ ID NO:483, SEQ ID NO:485,
SEQ ID NO:487, SEQ ID NO:489, SEQ ID NO:491, SEQ ID NO:493, SEQ ID
NO:495, SEQ ID NO:497, SEQ ID NO:499, SEQ ID NO:501, SEQ ID NO:503, SEQ
ID NO:505, SEQ ID NO:507, SEQ ID NO:509, SEQ ID NO:511, SEQ ID NO:513,
SEQ ID NO:515, SEQ ID NO:517, and sequences having a sequence identity to
an exemplary nucleic acid; nucleic acids encoding polypeptides of the
invention, e.g., the exemplary amino acid sequences as set forth in SEQ
ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID
NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID
NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID
NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID
NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID
NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID
NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID
NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID
NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID
NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ
ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120,
SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID
NO:130, SEQ ID NO:132; SEQ ID NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ
ID NO:140; SEQ ID NO:142; SEQ ID NO:144; NO:146, SEQ ID NO:148, SEQ ID
NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ
ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168,
SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID
NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ
ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196,
SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID
NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ
ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224,
SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID
NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ
ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252,
SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID
NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ
ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280,
SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQ ID
NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ
ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308,
SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID
NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ
ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336,
SEQ ID NO:338, SEQ ID NO:340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID
NO:346, SEQ ID NO:348, SEQ ID NO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ
ID NO:356, SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, SEQ ID NO:364,
SEQ ID NO:366, SEQ ID NO:368, SEQ ID NO:370, SEQ ID NO:372, SEQ ID
NO:374, SEQ ID NO:376, SEQ ID NO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ
ID NO:384, SEQ ID NO:386, SEQ ID NO:388, SEQ ID NO:390, SEQ ID NO:392,
SEQ ID NO:394, SEQ ID NO:396, SEQ ID NO:398, SEQ ID NO:400, SEQ ID
NO:402, SEQ ID NO:404, SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, SEQ
ID NO:412, SEQ ID NO:414, SEQ ID NO:416, SEQ ID NO:418, SEQ ID NO:420,
SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQ ID
NO:430, SEQ ID NO:432, SEQ ID NO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ
ID NO:440, SEQ ID NO:442, SEQ ID NO:444, SEQ ID NO:446, SEQ ID NO:448,
SEQ ID NO:450, SEQ ID NO:452, SEQ ID NO:454, SEQ ID NO:456, SEQ ID
NO:458, SEQ ID NO:460, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ
ID NO:468, SEQ ID NO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ ID NO:476,
SEQ ID NO:478, SEQ ID NO:480, SEQ ID NO:482, SEQ ID NO:484, SEQ ID
NO:486, SEQ ID NO:488, SEQ ID NO:490, SEQ ID NO:492, SEQ ID NO:494, SEQ
ID NO:496, SEQ ID NO:498, SEQ ID NO:500, SEQ ID NO:502, SEQ ID NO:504,
SEQ ID NO:506, SEQ ID NO:508, SEQ ID NO:510, SEQ ID NO:512, SEQ ID
NO:514, SEQ ID NO:516, SEQ ID NO:518). The invention also provides
expression cassettes such as expression vectors, comprising nucleic acids
of the invention, which include polynucleotides which encode the
polypeptides of the invention. The invention also includes methods for
discovering new glucanase sequences using the nucleic acids of the
invention. The invention also includes methods for inhibiting the
expression of glucanase genes, transcripts and polypeptides using the
nucleic acids of the invention. Also provided are methods for modifying
the nucleic acids of the invention by, e.g., synthetic ligation
reassembly, optimized directed evolution system and/or saturation
mutagenesis.
[0159] The nucleic acids of the invention can be made, isolated and/or
manipulated by, e.g., cloning and expression of cDNA libraries,
amplification of message or genomic DNA by PCR, and the like.
[0160] For example, the following exemplary sequences of the invention
were initially derived from the following sources, as set forth in Table
1 below:
TABLE-US-00002
TABLE 1
SEQ ID NO: Source
291, 292 Aquifox aeolicus
161, 162 Archaea
175, 176 Archaea
367, 368 Archaea
479, 480 Archaea
495, 496 Archaea
59, 60 Archaea
75, 76 Archaea
109, 110 Bacteria
229, 230 Bacteria
261, 262 Bacteria
263, 264 Bacteria
273, 274 Bacteria
277, 278 Bacteria
287, 288 Bacteria
293, 294 Bacteria
295, 296 Bacteria
331, 332 Bacteria
333, 334 Bacteria
363, 364 Bacteria
365, 366 Bacteria
369, 370 Bacteria
395, 396 Bacteria
397, 398 Bacteria
401, 402 Bacteria
427, 428 Bacteria
433, 434 Bacteria
435, 436 Bacteria
439, 440 Bacteria
447, 448 Bacteria
449, 450 Bacteria
455, 456 Bacteria
483, 484 Bacteria
485, 486 Bacteria
499, 500 Bacteria
5, 6 Bacteria
231, 232 Bacteria
67, 68 Bacteria
517, 518 Bacteria
399, 400 Thermotoga sp.
1, 2 Unknown
101, 102 Unknown
103, 104 Unknown
105, 106 Unknown
107, 108 Unknown
11, 12 Unknown
111, 112 Unknown
113, 114 Unknown
115, 116 Unknown
117, 118 Unknown
119, 120 Unknown
121, 122 Unknown
123, 124 Unknown
125, 126 Unknown
127, 128 Unknown
129, 130 Unknown
13, 14 Unknown
131, 132 Unknown
133, 134 Unknown
135, 136 Unknown
137, 138 Unknown
139, 140 Unknown
141, 142 Unknown
143, 144 Unknown
145, 146 Unknown
147, 148 Unknown
149, 150 Unknown
15, 16 Unknown
151, 152 Unknown
153, 154 Unknown
155, 156 Unknown
157, 158 Unknown
159, 160 Unknown
163, 164 Unknown
165, 166 Unknown
167, 168 Unknown
169, 170 Unknown
17, 18 Unknown
171, 172 Unknown
173, 174 Unknown
177, 178 Unknown
179, 180 Unknown
181, 182 Unknown
183, 184 Unknown
185, 186 Unknown
187, 188 Unknown
189, 190 Unknown
19, 20 Unknown
191, 192 Unknown
193, 194 Unknown
195, 196 Unknown
197, 198 Unknown
199, 200 Unknown
201, 202 Unknown
203, 204 Unknown
205, 206 Unknown
207, 208 Unknown
209, 210 Unknown
21, 22 Unknown
211, 212 Unknown
213, 214 Unknown
215, 216 Unknown
217, 218 Unknown
219, 220 Unknown
221, 222 Unknown
223, 224 Unknown
225, 226 Unknown
227, 228 Unknown
23, 24 Unknown
233, 234 Unknown
235, 236 Unknown
237, 238 Unknown
239, 240 Unknown
241, 242 Unknown
243, 244 Unknown
245, 246 Unknown
247, 248 Unknown
249, 250 Unknown
25, 26 Unknown
251, 252 Unknown
253, 254 Unknown
255, 256 Unknown
257, 258 Unknown
259, 260 Unknown
265, 266 Unknown
267, 268 Unknown
269, 270 Unknown
27, 28 Unknown
271, 272 Unknown
275, 276 Unknown
279, 280 Unknown
281, 282 Unknown
283, 284 Unknown
285, 286 Unknown
289, 290 Unknown
29, 30 Unknown
297, 298 Unknown
299, 300 Unknown
3, 4 Unknown
301, 302 Unknown
303, 304 Unknown
305, 306 Unknown
307, 308 Unknown
309, 310 Unknown
31, 32 Unknown
311, 312 Unknown
313, 314 Unknown
315, 316 Unknown
317, 318 Unknown
319, 320 Unknown
321, 322 Unknown
323, 324 Unknown
325, 326 Unknown
327, 328 Unknown
329, 330 Unknown
33, 34 Unknown
335, 336 Unknown
337, 338 Unknown
339, 340 Unknown
341, 342 Unknown
343, 344 Unknown
345, 346 Unknown
347, 348 Unknown
349, 350 Unknown
35, 36 Unknown
351, 352 Unknown
353, 354 Unknown
355, 356 Unknown
357, 358 Unknown
359, 360 Unknown
361, 362 Unknown
37, 38 Unknown
371, 372 Unknown
373, 374 Unknown
375, 376 Unknown
377, 378 Unknown
379, 380 Unknown
381, 382 Unknown
383, 384 Unknown
385, 386 Unknown
387, 388 Unknown
389, 390 Unknown
39, 40 Unknown
391, 392 Unknown
393, 394 Unknown
403, 404 Unknown
405, 406 Unknown
407, 408 Unknown
409, 410 Unknown
41, 42 Unknown
411, 412 Unknown
413, 414 Unknown
415, 416 Unknown
417, 418 Unknown
419, 420 Unknown
421, 422 Unknown
423, 424 Unknown
425, 426 Unknown
429, 430 Unknown
43, 44 Unknown
431, 432 Unknown
437, 438 Unknown
441, 442 Unknown
443, 444 Unknown
445, 446 Unknown
45, 46 Unknown
451, 452 Unknown
453, 454 Unknown
457, 458 Unknown
459, 460 Unknown
461, 462 Unknown
463, 464 Artificial
465, 466 Unknown
467, 468 Unknown
469, 470 Unknown
47, 48 Unknown
471, 472 Unknown
473, 474 Unknown
475, 476 Unknown
477, 478 Unknown
481, 482 Unknown
487, 488 Unknown
489, 490 Unknown
49, 50 Unknown
491, 492 Unknown
493, 494 Unknown
497, 498 Unknown
501, 502 Unknown
503, 504 Unknown
505, 506 Unknown
507, 508 Unknown
509, 510 Unknown
51, 52 Unknown
511, 512 Unknown
513, 514 Unknown
515, 516 Unknown
53, 54 Unknown
55, 56 Unknown
57, 58 Unknown
61, 62 Unknown
63, 64 Unknown
65, 66 Unknown
69, 70 Unknown
7, 8 Unknown
71, 72 Unknown
73, 74 Unknown
77, 78 Unknown
79, 80 Unknown
81, 82 Unknown
83, 84 Unknown
85, 86 Unknown
87, 88 Unknown
89, 90 Unknown
9, 10 Unknown
91, 92 Unknown
93, 94 Unknown
95, 96 Unknown
97, 98 Unknown
99, 100 Unknown
[0161] In one aspect, the invention provides glucanase-encoding nucleic
acids, and the polypeptides encoded by them, with a common novelty in
that they are derived from a common source, e.g., an environmental or an
archaeal source.
[0162] In practicing the methods of the invention, homologous genes can be
modified by manipulating a template nucleic acid, as described herein.
The invention can be practiced in conjunction with any method or protocol
or device known in the art, which are well described in the scientific
and patent literature.
[0163] One aspect of the invention is an isolated nucleic acid comprising
one of the sequences of the invention, or a fragment comprising at least
10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500
consecutive bases of a nucleic acid of the invention. The isolated,
nucleic acids may comprise DNA, including cDNA, genomic DNA and synthetic
DNA. The DNA may be double-stranded or single-stranded and if single
stranded may be the coding strand or non-coding (anti-sense) strand.
Alternatively, the isolated nucleic acids may comprise RNA.
[0164] The isolated nucleic acids of the invention may be used to prepare
one of the polypeptides of the invention, or fragments comprising at
least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive
amino acids of one of the polypeptides of the invention.
[0165] Accordingly, another aspect of the invention is an isolated nucleic
acid which encodes one of the polypeptides of the invention, or fragments
comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids of one of the polypeptides of the invention. The
coding sequences of these nucleic acids may be identical to one of the
coding sequences of one of the nucleic acids of the invention or may be
different coding sequences which encode one of the of the invention
having at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids of one of the polypeptides of the invention, as a
result of the redundancy or degeneracy of the genetic code. The genetic
code is well known to those of skill in the art and can be obtained, for
example, on page 214 of B. Lewin, Genes VI, Oxford University Press,
1997.
[0166] The isolated nucleic acid which encodes one of the polypeptides of
the invention, but is not limited to: only the coding sequence of a
nucleic acid of the invention and additional coding sequences, such as
leader sequences or proprotein sequences and non-coding sequences, such
as introns or non-coding sequences 5' and/or 3' of the coding sequence.
Thus, as used herein, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only the coding sequence for
the polypeptide as well as a polynucleotide which includes additional
coding and/or non-coding sequence.
[0167] Alternatively, the nucleic acid sequences of the invention, may be
mutagenized using conventional techniques, such as site directed
mutagenesis, or other techniques familiar to those skilled in the art, to
introduce silent changes into the polynucleotides of the invention. As
used herein, "silent changes" include, for example, changes which do not
alter the amino acid sequence encoded by the polynucleotide. Such changes
may be desirable in order to increase the level of the polypeptide
produced by host cells containing a vector encoding the polypeptide by
introducing codons or codon pairs which occur frequently in the host
organism.
[0168] The invention also relates to polynucleotides which have nucleotide
changes which result in amino acid substitutions, additions, deletions,
fusions and truncations in the polypeptides of the invention. Such
nucleotide changes may be introduced using techniques such as site
directed mutagenesis, random chemical mutagenesis, exonuclease m deletion
and other recombinant DNA techniques. Alternatively, such nucleotide
changes may be naturally occurring allelic variants which are isolated by
identifying nucleic acids which specifically hybridize to probes
comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200,
300, 400, or 500 consecutive bases of one of the sequences of the
invention (or the sequences complementary thereto) under conditions of
high, moderate, or low stringency as provided herein.
[0169] General Techniques
[0170] The nucleic acids used to practice this invention, whether RNA,
iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or
hybrids thereof, may be isolated from a variety of sources, genetically
engineered, amplified, and/or expressed/generated recombinantly.
Recombinant polypeptides (e.g., glucanases, mannanases, or xylanases)
generated from these nucleic acids can be individually isolated or cloned
and tested for a desired activity. Any recombinant expression system can
be used, including bacterial, mammalian, yeast, insect or plant cell
expression systems.
[0171] Alternatively, these nucleic acids can be synthesized in vitro by
well-known chemical synthesis techniques, as described in, e.g., Adams
(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.
25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers
(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;
Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859;
U.S. Pat. No. 4,458,066.
[0172] Techniques for the manipulation of nucleic acids, such as, e.g.,
subcloning, labeling probes (e.g., random-primer labeling using Klenow
polymerase, nick translation, amplification), sequencing, hybridization
and the like are well described in the scientific and patent literature,
see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND
ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York
(1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY:
HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid
Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
[0173] Another useful means of obtaining and manipulating nucleic acids
used to practice the methods of the invention is to clone from genomic
samples, and, if desired, screen and re-clone inserts isolated or
amplified from, e.g., genomic clones or cDNA clones. Sources of nucleic
acid used in the methods of the invention include genomic or cDNA
libraries contained in, e.g., mammalian artificial chromosomes (MACs),
see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155; human artificial
chromosomes, see, e.g., Rosenfeld (1997) Nat. Genet. 15:333-335; yeast
artificial chromosomes (YAC); bacterial artificial chromosomes SAC); P1
artificial chromosomes, see, e.g., Woon (1998) Genomics 50:306-316;
P1-derived vectors (PACs), see, e.g., Kern (1997) Biotechniques
23:120-124; cosmids, recombinant viruses, phages or plasmids.
[0174] In one aspect, a nucleic acid encoding a polypeptide of the
invention is assembled in appropriate phase with a leader sequence
capable of directing secretion of the translated polypeptide or fragment
thereof.
[0175] The invention provides fusion proteins and nucleic acids encoding
them. A polypeptide of the invention can be fused to a heterologous
peptide or polypeptide, such as N-terminal identification peptides which
impart desired characteristics, such as increased stability or simplified
purification. Peptides and polypeptides of the invention can also be
synthesized and expressed as fusion proteins with one or more additional
domains linked thereto for, e.g., producing a more immunogenic peptide,
to more readily isolate a recombinantly synthesized peptide, to identify
and isolate antibodies and antibody-expressing B cells, and the like.
Detection and purification facilitating domains include, e.g., metal
chelating peptides such as polyhistidine tracts and histidine-tryptophan
modules that allow purification on immobilized metals, protein A domains
that allow purification on immobilized immunoglobulin, and the domain
utilized in the FLAGS extension/affinity purification system (Immunex
Corp, Seattle Wash.). The inclusion of a cleavable linker sequences such
as Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between a
purification domain and the motif-comprising peptide or polypeptide to
facilitate purification. For example, an expression vector can include an
epitope-encoding nucleic acid sequence linked to six histidine residues
followed by a thioredoxin and an enterokinase cleavage site (see e.g.,
Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr.
Purif. 12:404-414). The histidine residues facilitate detection and
purification while the enterokinase cleavage site provides a means for
purifying the epitope from the remainder of the fusion protein.
Technology pertaining to vectors encoding fusion proteins and application
of fusion proteins are well described in the scientific and patent
literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.
[0176] Transcriptional and Translational Control Sequences
[0177] The invention provides nucleic acid (e.g., DNA) sequences of the
invention operatively linked to expression (e.g., transcriptional or
translational) control sequence(s), e.g., promoters or enhancers, to
direct or modulate RNA synthesis/expression. The expression control
sequence can be in an expression vector. Exemplary bacterial promoters
include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplary
eukaryotic promoters include CMV immediate early, HSV thymidine kinase,
early and late SV40, LTRs from retrovirus, and mouse metallothionein I.
[0178] Promoters suitable for expressing a polypeptide in bacteria include
the E. coli lac or trp promoters, the lacI promoter, the lacZ promoter,
the T3 promoter, the T7 promoter, the gpt promoter, the lambda PR
promoter, the lambda PL promoter, promoters from operons encoding
glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), and the acid
phosphatase promoter. Eukaryotic promoters include the CMV immediate
early promoter, the HSV thymidine kinase promoter, heat shock promoters,
the early and late SV40 promoter, LTRs from retroviruses, and the mouse
metallothionein-I promoter. Other promoters known to control expression
of genes in prokaryotic or eukaryotic cells or their viruses may also be
used. Promoters suitable for expressing the polypeptide or fragment
thereof in bacteria include the E. coli lac or trp promoters, the lacI
promoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gpt
promoter, the lambda P.sub.R promoter, the lambda P.sub.L promoter,
promoters from operons encoding glycolytic enzymes such as
3-phosphoglycerate kinase (PGK) and the acid phosphatase promoter. Fungal
promoters include the .A-inverted. factor promoter. Eukaryotic promoters
include the CMV immediate early promoter, the HSV thymidine kinase
promoter, heat shock promoters, the early and late SV40 promoter, LTRs
from retroviruses and the mouse metallothionein-I promoter. Other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses may also be used.
[0179] Tissue-Specific Plant Promoters
[0180] The invention provides expression cassettes that can be expressed
in a tissue-specific manner, e.g., that can express a glucanase of the
invention in a tissue-specific manner. The invention also provides plants
or seeds that express a glucanase of the invention in a tissue-specific
manner. The tissue-specificity can be seed specific, stem specific, leaf
specific, root specific, fruit specific and the like.
[0181] In one aspect, a constitutive promoter such as the CaMV 35S
promoter can be used for expression in specific parts of the plant or
seed or throughout the plant. For example, for overexpression, a plant
promoter fragment can be employed which will direct expression of a
nucleic acid in some or all tissues of a plant, e.g., a regenerated
plant. Such promoters are referred to herein as "constitutive" promoters
and are active under most environmental conditions and states of
development or cell differentiation. Examples of constitutive promoters
include the cauliflower mosaic virus (CaMV) 35S transcription initiation
region, the 1'- or 2'-promoter derived from T-DNA of Agrobacterium
tumefaciens, and other transcription initiation regions from various
plant genes known to those of skill. Such genes include, e.g., ACT11 from
Arabidopsis (Huang (1996) Plant Mol. Biol. 33:125-139); Cat3 from
Arabidopsis (GenBank No. U43147, Zhong (1996) Mol. Gen. Genet.
251:196-203); the gene encoding stearoyl-acyl carrier protein desaturase
from Brassica napus (Genbank No. X74782, Solocombe (1994) Plant Physiol.
104:1167-1176); GPc1 from maize (GenBank No. X15596; Martinez (1989) J.
Mol. Biol. 208:551-565); the Gpc2 from maize (GenBank No. U45855,
Manjunath (1997) Plant Mol. Biol. 33:97-112); plant promoters described
in U.S. Pat. Nos. 4,962,028; 5,633,440.
[0182] The invention uses tissue-specific or constitutive promoters
derived from viruses which can include, e.g., the tobamovirus subgenomic
promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; the
rice tungro bacilliform virus (RTBV), which replicates only in phloem
cells in infected rice plants, with its promoter which drives strong
phloem-specific reporter gene expression; the cassaya vein mosaic virus
(CVMV) promoter, with highest activity in vascular elements, in leaf
mesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.
31:1129-1139).
[0183] Alternatively, the plant promoter may direct, expression of
glucanase-expressing nucleic acid in a specific tissue, organ or cell
type (i.e. tissue-specific promoters) or may be otherwise under more
precise environmental or developmental control or under the control of an
inducible promoter. Examples of environmental conditions that may affect
transcription include anaerobic conditions, elevated temperature, the
presence of light, or sprayed with chemicals/hormones. For example, the
invention incorporates the drought-inducible promoter of maize (Busk
(1997) supra); the cold, drought, and high salt inducible promoter from
potato (Kirch (1997) Plant Mol. Biol. 33:897 909).
[0184] Tissue-specific promoters can promote transcription only within a
certain time frame of developmental stage within that tissue. See, e.g.,
Blazquez (1998) Plant Cell 10:791-800, characterizing the Arabidopsis
LEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77, describing
the transcription factor SPL3, which recognizes a conserved sequence
motif in the promoter region of the A. thaliana floral meristem identity
gene AP1; and Mandel (1995) Plant Molecular Biology, Vol. 29, pp
995-1004, describing the meristem promoter eIF4. Tissue specific
promoters which are active throughout the life cycle of a particular
tissue can be used. In one aspect, the nucleic acids of the invention are
operably linked to a promoter active primarily only in cotton fiber
cells. In one aspect, the nucleic acids of the invention are operably
linked to a promoter active primarily during the stages of cotton fiber
cell elongation, e.g., as described by Rinehart (1996) supra. The nucleic
acids can be operably linked to the Fbl2A gene promoter to be
preferentially expressed in cotton fiber cells (Ibid). See also, John
(1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Pat.
Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promoters
and methods for the construction of transgenic cotton plants.
Root-specific promoters may also be used to express the nucleic acids of
the invention. Examples of root-specific promoters include the promoter
from the alcohol dehydrogenase gene (DeLisle (1990) Int Rev. Cytol.
123:39-60). Other promoters that can be used to express the nucleic acids
of the invention include, e.g., ovule-specific, embryo-specific,
endosperm-specific, integument-specific, seed coat-specific promoters, or
some combination thereof; a leaf-specific promoter (see, e.g., Busk
(1997) Plant J. 11:1285 1295, describing a leaf-specific promoter in
maize); the ORF13 promoter from Agrobacterium rhizogenes (which exhibits
high activity in roots, see, e.g., Hansen (1997) supra); a maize pollen
specific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224:161
168); a tomato promoter active during fruit ripening, senescence and
abscission of leaves and, to a lesser extent, of flowers can be used
(see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specific promoter
from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol. Biol.
35:425 431); the Blec4 gene from pea, which is active in epidermal tissue
of vegetative and floral shoot apices of transgenic alfalfa making it a
useful tool to target the expression of foreign genes to the epidermal
layer of actively growing shoots or fibers; the ovule-specific BEL1 gene
(see, e.g., Reiser (1995) Cell 83:735-742, GenBank No. U39944); and/or,
the promoter in Klee, U.S. Pat. No. 5,589,583, describing a plant
promoter region is capable of conferring high levels of transcription in
meristematic tissue and/or rapidly dividing cells.
[0185] Alternatively, plant promoters which are inducible upon exposure to
plant hormones, such as auxins, are used to express the nucleic acids of
the invention. For example, the invention can use the auxin-response
elements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.)
(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsive Arabidopsis
GST6 promoter (also responsive to salicylic acid and hydrogen peroxide)
(Chen (1996) Plant J. 10: 955-966); the auxin-inducible parC promoter
from tobacco (Sakai (1996) 37:906-913); a plant biotin response element
(Streit (1997) Mol. Plant. Microbe Interact. 10:933-937); and, the
promoter responsive to the stress hormone abscisic acid (Sheen (1996)
Science 274:1900-1902).
[0186] The nucleic acids of the invention can also be operably linked to
plant promoters which are inducible upon exposure to chemicals reagents
which can be applied to the plant, such as herbicides or antibiotics. For
example, the maize In2-2 promoter, activated by benzenesulfonamide
herbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol.
38:568-577); application of different herbicide safeners induces distinct
gene expression patterns, including expression in the root, hydathodes,
and the shoot apical meristem. Coding sequence can be under the control
of, e.g., a tetracycline-inducible promoter, e.g., as described with
transgenic tobacco plants containing the Aveiza sativa L. (oat) arginine
decarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylic
acid-responsive element ((1997) Plant J. 11:1315-1324). Using
chemically--(e.g., hormone- or pesticide-) induced promoters, i.e.,
promoter responsive to a chemical which can be applied to the transgenic
plant in the field, expression of a polypeptide of the invention can be
induced at a particular stage of development of the plant. Thus, the
invention also provides for transgenic plants containing an inducible
gene encoding for polypeptides of the invention whose host range is
limited to target plant species, such as corn, rice, barley, wheat,
potato or other crops, inducible at any stage of development of the crop.
[0187] One of skill will recognize that a tissue-specific plant promoter
may drive expression of operably linked sequences in tissues other than
the target tissue. Thus, a tissue-specific promoter is one that drives
expression preferentially in the target tissue or cell type, but may also
lead to some expression in other tissues as well.
[0188] The nucleic acids of the invention can also be operably linked to
plant promoters which are inducible upon exposure to chemicals reagents.
These reagents include, e.g., herbicides, synthetic auxins, or
antibiotics which can be applied, e.g., sprayed, onto transgenic plants.
Inducible expression of the glucanase-producing nucleic acids of the
invention will allow the grower to select plants with the optimal
glucanase expression and/or activity. The development of plant parts can
thus controlled. In this way the invention provides the means to
facilitate the harvesting of plants and plant parts. For example, in
various embodiments, the maize In2-2 promoter, activated by
benzenesulfonamide herbicide safeners, is used (De Veylder (1997) Plant
Cell Physiol. 38:568-577); application of different herbicide safeners
induces distinct gene expression patterns, including expression in the
root, hydathodes, and the shoot apical meristem. Coding sequences of the
invention are also under the control of a tetracycline-inducible
promoter, e.g., as described with transgenic tobacco plants containing
the Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997)
Plant J. 11:465-473); or, a salicylic acid-responsive element (Stange
(1997) Plant J. 11:1315-1324).
[0189] In some aspects, proper polypeptide expression may require
polyadenylation region at the 3'-end of the coding region. The
polyadenylation region can be derived from the natural gene, from a
variety of other plant (or animal or other) genes, or from genes in the
Agrobacterial T-DNA.
[0190] Expression Vectors and Cloning Vehicles
[0191] The invention provides expression vectors and cloning vehicles
comprising nucleic acids of the invention, e.g., sequences encoding the
glucanases, mannanases, or xylanases of the invention. Expression vectors
and cloning vehicles of the invention can comprise viral particles,
baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial
artificial chromosomes, viral DNA (e.g., vaccinia; adenovirus, foul pox
virus, pseudorabies and derivatives of SV40), P1-based artificial
chromosomes, yeast plasmids, yeast artificial chromosomes, and any other
vectors specific for specific hosts of interest (such as Bacillus,
Aspergillus and yeast). Vectors of the invention can include chromosomal,
non-chromosomal and synthetic DNA sequences. Large numbers of suitable
vectors are known to those of skill in the art, and are commercially
available. Exemplary vectors are include: bacterial: pQE vectors
(Qiagen), pBluescript plasmids, pNH vectors, (lambda-ZAP vectors
(Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic:
pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia).
However, any other plasmid or other vector may be used so long as they
are replicable and viable in the host. Low copy number or high copy
number vectors may be employed with the present invention.
[0192] The expression vector can comprise a promoter, a ribosome binding
site for translation initiation and a transcription terminator. The
vector may also include appropriate sequences for amplifying expression.
Mammalian expression vectors can comprise an origin of replication, any
necessary ribosome binding sites, a polyadenylation site, splice donor
and acceptor sites, transcriptional termination sequences, and 5'
flanking non-transcribed sequences. In some aspects, DNA sequences
derived from the SV40 splice and polyadenylation sites may be used to
provide the required non-transcribed genetic elements.
[0193] In one aspect, the expression vectors contain one or more
selectable marker genes to permit selection of host cells containing the
vector. Such selectable markers include genes encoding dihydrofolate
reductase or genes conferring neomycin resistance for eukaryotic cell
culture, genes conferring tetracycline or ampicillin resistance in E.
coli, and the S. cerevisiae TP1 gene. Promoter regions can be selected
from any desired gene using chloramphenicol transferase (CAT) vectors or
other vectors with selectable markers.
[0194] Vectors for expressing the polypeptide or fragment thereof in
eukaryotic cells can also contain enhancers to increase expression
levels. Enhancers are cis-acting elements of DNA, usually from about 10
to about 300 bp in length that act on a promoter to increase its
transcription. Examples include the SV40 enhancer on the late side of the
replication origin bp 100 to 270, the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and the adenovirus enhancers.
[0195] A nucleic acid sequence can be inserted into a vector by a variety
of procedures. In general, the sequence is ligated to the desired
position in the vector following digestion of the insert and the vector
with appropriate restriction endonucleases. Alternatively, blunt ends in
both the insert and the vector may be ligated. A variety of cloning
techniques are known in the art, e.g., as described in Ausubel and
Sambrook. Such procedures and others are deemed to be within the scope of
those skilled in the art.
[0196] The vector can be in the form of a plasmid, a viral particle, or a
phage. Other vectors include chromosomal, non-chromosomal and synthetic,
DNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,
baculovirus, yeast plasmids, vectors derived from combinations of
plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox
virus, and pseudorabies. A variety of cloning and expression vectors for
use with prokaryotic and eukaryotic hosts are described by, e.g.,
Sambrook.
[0197] Particular bacterial vectors which can be used include the
commercially available plasmids comprising genetic elements of the well
known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine
Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison, Wis., USA)
pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174 pBluescript II KS, pNH8A,
pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540,
pRIT5 (Pharmacia), pKK232-8 and pCM7. Particular eukaryotic vectors
include pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and
pSVL (Pharmacia). However, any other vector may be used as long as it is
replicable and viable in the host cell.
[0198] The nucleic acids of the invention can be expressed in expression
cassettes, vectors or viruses and transiently or stably expressed in
plant cells and seeds. One exemplary transient expression system uses
episomal expression systems, e.g., cauliflower mosaic virus (CaMV) viral
RNA generated in the nucleus by transcription of an episomal
mini-chromosome containing supercoiled DNA, see, e.g., Covey (1990) Proc.
Natl. Acad. Sci. USA 87:1633-1637. Alternatively, coding sequences, i.e.,
all or sub-fragments of sequences of the invention can be inserted into a
plant host cell genome becoming an integral part of the host chromosomal
DNA. Sense or antisense transcripts can be expressed in this manner. A
vector comprising the sequences (e.g., promoters or coding regions) from
nucleic acids of the invention can comprise a marker gene that confers a
selectable phenotype on a plant cell or a seed. For example, the marker
may encode biocide resistance, particularly antibiotic resistance, such
as resistance to kanamycin, G418, bleomycin, hygromycin, or herbicide
resistance, such as resistance to chlorosulfuron or Basta.
[0199] Expression vectors capable of expressing nucleic acids and proteins
in plants are well known in the art, and can include, e.g., vectors from
Agrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J.
16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene
173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology
169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology
234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993)
Microbiol Immunol. 37:471-476), cauliflower mosaic virus (see, e.g.,
Cecchini (1997) Mol. Plant. Microbe Interact. 10:1094-1101), maize Ac/Ds
transposable element (see, e.g., Rubin (1997) Mol. Cell. Biol.
17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204:161-194),
and the maize suppressor-mutator (Spm) transposable element (see, e.g.,
Schlappi (1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.
[0200] In one aspect, the expression vector can have two replication
systems to allow it to be maintained in two organisms, for example in
mammalian or insect cells for expression and in a prokaryotic host for
cloning and amplification. Furthermore, for integrating expression
vectors, the expression vector can contain at least one sequence
homologous to the host cell genome. It can contain two homologous
sequences which flank the expression construct. The integrating vector
can be directed to a specific locus in the host cell by selecting the
appropriate homologous sequence for inclusion in the vector. Constructs
for integrating vectors are well known in the art.
[0201] Expression vectors of the invention may also include a selectable
marker gene to allow for the selection of bacterial strains that have
been transformed, e.g., genes which render the bacteria resistant to
drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin,
neomycin and tetracycline. Selectable markers can also include
biosynthetic genes, such as those in the histidine, tryptophan and
leucine biosynthetic pathways.
[0202] The DNA sequence in the expression vector is operatively linked to
an appropriate expression control sequence(s) (promoter) to direct RNA
synthesis. Particular named bacterial promoters include lacI, lacZ, T3,
T7, gpt, lambda P.sub.R, P.sub.L and trp. Eukaryotic promoters include
CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from
retrovirus and mouse metallothionein-I. Selection of the appropriate
vector and promoter is well within the level of ordinary skill in the
art. The expression vector also contains a ribosome binding site for
translation initiation and a transcription terminator. The vector may
also include appropriate sequences for amplifying expression. Promoter
regions can be selected from any desired gene using chloramphenicol
transferase (CAT) vectors or other vectors with selectable markers. In
addition, the expression vectors in one aspect contain one or more
selectable marker genes to provide a phenotypic trait for selection of
transformed host cells such as dihydrofolate reductase or neomycin
resistance for eukaryotic cell culture, or such as tetracycline or
ampicillin resistance in E. coli.
[0203] Mammalian expression vectors may also comprise an origin of
replication, any necessary ribosome binding sites, a polyadenylation
site, splice donor and acceptor sites, transcriptional termination
sequences- and 5' flanking nontranscribed sequences. In some aspects, DNA
sequences derived from the SV40 splice and polyadenylation sites may be
used to provide the required nontranscribed genetic elements.
[0204] Vectors for expressing the polypeptide or fragment thereof in
eukaryotic cells may also contain enhancers to increase expression
levels. Enhancers are cis-acting elements of DNA, usually from about 10
to about 300 bp in length that act on a promoter to increase its
transcription. Examples include the SV40 enhancer on the late side of the
replication origin bp 100 to 270, the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication origin
and the adenovirus enhancers.
[0205] In addition, the expression vectors typically contain one or more
selectable marker genes to permit selection of host cells containing the
vector. Such selectable markers include genes encoding dihydrofolate
reductase or genes conferring neomycin resistance for eukaryotic cell
culture, genes conferring tetracycline or ampicillin resistance in E.
coli and the S. cerevisiae TRP1 gene.
[0206] In some aspects, the nucleic acid encoding one of the polypeptides
of the invention, or fragments comprising at least about 5, 10, 15, 20,
25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof is
assembled in appropriate phase with a leader sequence capable of
directing secretion of the translated polypeptide or fragment thereof.
Optionally, the nucleic acid can encode a fusion polypeptide in which one
of the polypeptides of the invention, or fragments comprising at least 5,
10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids
thereof is fused to heterologous peptides or polypeptides, such as
N-terminal identification peptides which impart desired characteristics,
such as increased stability or simplified purification.
[0207] The appropriate DNA sequence may be inserted into the vector by a
variety of procedures. In general, the DNA sequence is ligated to the
desired position in the vector following digestion of the insert and the
vector with appropriate restriction endonucleases. Alternatively, blunt
ends in both the insert and the vector may be ligated. A variety of
cloning techniques are disclosed in Ausubel et al. Current Protocols in
Molecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook et al,
Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor
Laboratory Press (1989. Such procedures and others are deemed to be
within the scope of those skilled in the art.
[0208] The vector may be, for example, in the form of a plasmid, a viral
particle, or a phage. Other vectors include chromosomal, nonchromosomal
and synthetic DNA sequences, derivatives of SV40; bacterial plasmids,
phage DNA, baculovirus, yeast plasmids, vectors derived from combinations
of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl
pox virus and pseudorabies. A variety of cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by Sambrook,
et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring
Harbor, N.Y., (1989).
[0209] Host Cells and Transformed Cells
[0210] The invention also provides a transformed cell comprising a nucleic
acid sequence of the invention, e.g., a sequence encoding a glucanase of
the invention, or a vector of the invention. The host cell may be any of
the host cells familiar to those skilled in the art, including
prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal
cells, yeast cells, mammalian cells, insect cells, or plant cells.
Exemplary bacterial cells include E. coli, Lactococcus lactis,
Streptomyces, Bacillus subtilis, Bacillus cereus, Salmonella typhimurium
or any species within the genera Bacillus, Streptomyces and
Staphylococcus. Exemplary insect cells include Drosophila S2 and
Spodoptera Sf9. Exemplary yeast cells include Pichia pastoris,
Saccharomyces cerevisiae or Schizosaccharomyces pombe. Exemplary animal
cells include CHO, COS or Bowes melanoma or any mouse or human cell line.
The selection of an appropriate host is within the abilities of those
skilled in the art. Techniques for transforming a wide variety of higher
plant species are well known and described in the technical and
scientific literature. See, e.g., Weising (1988) Ann. Rev. Genet.
22:421-477; U.S. Pat. No. 5,750,870.
[0211] The vector can be introduced into the host cells using any of a
variety of techniques, including transformation, transfection,
transduction, viral infection, gene guns, or Ti-mediated gene transfer.
Particular methods include calcium phosphate transfection, DEAE-Dextran
mediated transfection, lipofection, or electroporation (Davis, L.,
Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
[0212] In one aspect, the nucleic acids or vectors of the invention are
introduced into the cells for screening, thus, the nucleic acids enter
the cells in a manner suitable for subsequent expression of the nucleic
acid. The method of introduction is largely dictated by the targeted cell
type. Exemplary methods include CaPO.sub.4 precipitation, liposome
fusion, lipofection (e.g., LIPOFECTIN.TM.), electroporation, viral
infection, etc. The candidate nucleic acids may stably integrate into the
genome of the host cell (for example, with retroviral introduction) or
may exist either transiently or stably in the cytoplasm (i.e. through the
use of traditional plasmids, utilizing standard regulatory sequences,
selection markers, etc.). As many pharmaceutically important screens
require human or model mammalian cell targets, retroviral vectors capable
of transfecting such targets can be used.
[0213] Where appropriate, the engineered host cells can be cultured in
conventional nutrient media modified as appropriate for activating
promoters, selecting transformants or amplifying the genes of the
invention. Following transformation of a suitable host strain and growth
of the host strain to an appropriate cell density, the selected promoter
may be induced by appropriate means (e.g., temperature shift or chemical
induction) and the cells may be cultured for an additional period to
allow them to produce the desired polypeptide or fragment thereof.
[0214] Cells can be harvested by centrifugation, disrupted by physical or
chemical means, and the resulting crude extract is retained for further
purification. Microbial cells employed for expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents. Such
methods are well known to those skilled in the art. The expressed
polypeptide or fragment thereof can be recovered and purified from
recombinant cell cultures by methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite chromatography
and lectin chromatography. Protein refolding steps can be used, as
necessary, in completing configuration of the polypeptide. If desired,
high performance liquid chromatography (HPLC) can be employed for final
purification steps.
[0215] The constructs in host cells can be used in a conventional manner
to produce the gene product encoded by the recombinant sequence.
Depending upon the host employed in a recombinant production procedure,
the polypeptides produced by host cells containing the vector may be
glycosylated or may be non-glycosylated. Polypeptides of the invention
may or may not also include an initial methionine amino acid residue.
[0216] Cell-free translation systems can also be employed to produce a
polypeptide of the invention. Cell-free translation systems can use mRNAs
transcribed from a DNA construct comprising a promoter operably linked to
a nucleic acid encoding the polypeptide or fragment thereof. In some
aspects, the DNA construct may be linearized prior to conducting an in
vitro transcription reaction. The transcribed mRNA is then incubated with
an appropriate cell-free translation extract, such as a rabbit
reticulocyte extract, to produce the desired polypeptide or fragment
thereof.
[0217] The expression vectors can contain one or more selectable marker
genes to provide a phenotypic trait for selection of transformed host
cells such as dihydrofolate reductase or neomycin resistance for
eukaryotic cell culture, or such as tetracycline or ampicillin resistance
in E. coli.
[0218] Host cells containing the polynucleotides of interest, e.g.,
nucleic acids of the invention, can be cultured in conventional nutrient
media modified as appropriate for activating promoters, selecting
transformants or amplifying genes. The culture conditions, such as
temperature, pH and the like, are those previously used with the host
cell selected for expression and will be apparent to the ordinarily
skilled artisan. The clones which are identified as having the specified
enzyme activity may then be sequenced to identify the polynucleotide
sequence encoding an enzyme having the enhanced activity.
[0219] The invention provides a method for overexpressing a recombinant
glucanase in a cell comprising expressing a vector comprising a nucleic
acid of the invention, e.g., a nucleic acid comprising a nucleic acid
sequence with at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more
sequence identity to an exemplary sequence of the invention over a region
of at least about 100 residues, wherein the sequence identities are
determined by analysis with a sequence comparison algorithm or by visual
inspection, or, a nucleic acid that hybridizes under stringent conditions
to a nucleic acid sequence of the invention. The overexpression can be
effected by any means, e.g., use of a high activity promoter, a
dicistronic vector or by gene amplification of the vector.
[0220] The nucleic acids of the invention can be expressed, or
overexpressed, in any in vitro or in vivo expression system. Any cell
culture systems can be employed to express, or over-express, recombinant
protein, including bacterial, insect, yeast, fungal or mammalian
cultures. Over-expression can be effected by appropriate choice of
promoters, enhancers, vectors (e.g., use of replicon vectors, dicistronic
vectors (see, e.g., Gurtu (1996) Biochem. Biophys. Res. Commun.
229:295-8), media, culture systems and the like. In one aspect, gene
amplification using selection markers, e.g., glutamine synthetase (see,
e.g., Sanders (1987) Dev. Biol. Stand. 66:55-63), in cell systems are
used to overexpress the polypeptides of the invention.
[0221] Additional details regarding this approach are in the public
literature and/or are known to the skilled artisan. In a particular
non-limiting exemplification, such publicly available literature includes
EP 0659215 (WO 9403612 A1) (Nevalainen et al); Lapidot, A., Mechaly, A.,
Shoham, Y., "Overexpression and single-step purification of a
thermostable glucanase from Bacillus stearothermophilus T-6," J.
Biotechnol. November 51:259-64 (1996); Luthi, E., Jasmat, N. B.,
Bergquist, P. L., "Endoglucanase from the extremely thermophilic
bacterium Caldocellum saccharolyticum: overexpression of the gene in
Escherichia coli and characterization of the gene product," Appl.
Environ. Microbiol. September 56:2677-83 (1990); and Sung, W. L., Luk, C.
K., Zahab, D. M., Wakarchuk, W., "Overexpression of the Bacillus subtilis
and circulans endoglucanases in Escherichia coli," Protein Expr. Purif.
June 4:200-6 (1993), although these references do not teach the inventive
enzymes of the instant application.
[0222] The host cell may be any of the host cells familiar to those
skilled in the art, including prokaryotic cells, eukaryotic cells,
mammalian cells, insect cells, or plant cells. As representative examples
of appropriate hosts, there may be mentioned: bacterial cells, such as E.
coli, Streptomyces, Bacillus subtilis, Salmonella typhimurium and various
species within the genera Streptomyces and Staphylococcus, fungal cells,
such as yeast, insect cells such as Drosophila S2 and Spodoptera Sf9,
animal cells such as CHO, COS or Bowes melanoma and adenoviruses. The
selection of an appropriate host is within the abilities of those skilled
in the art.
[0223] The vector may be introduced into the host cells using any of a
variety of techniques, including transformation, transfection,
transduction, viral infection, gene guns, or Ti-mediated gene transfer.
Particular methods include calcium phosphate transfection, DEAE-Dextran
mediated transfection, lipofection, or electroporation (Davis, L.,
Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
[0224] Where appropriate, the engineered host cells can be cultured in
conventional nutrient media modified as appropriate for activating
promoters, selecting transformants or amplifying the genes of the
invention. Following transformation of a suitable host strain and growth
of the host strain to an appropriate cell density, the selected promoter
may be induced by appropriate means (e.g., temperature shift or chemical
induction) and the cells may be cultured for an additional period to
allow them to produce the desired polypeptide or fragment thereof.
[0225] Cells are typically harvested by centrifugation, disrupted by
physical or chemical means and the resulting crude extract is retained
for further purification. Microbial cells employed for expression of
proteins can be disrupted by any convenient method, including freeze-thaw
cycling, sonication, mechanical disruption, or use of cell lysing agents.
Such methods are well known to those skilled in the art. The expressed
polypeptide or fragment thereof can be recovered and purified from
recombinant cell cultures by methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite chromatography
and lectin chromatography. Protein refolding steps can be used, as
necessary, in completing configuration of the polypeptide. If desired,
high performance liquid chromatography (HPLC) can be employed for final
purification steps.
[0226] Various mammalian cell culture systems can also be employed to
express recombinant protein. Examples of mammalian expression systems
include the COS-7 lines of monkey kidney fibroblasts (described by
Gluzman, Cell, 23:175, 1981) and other cell lines capable of expressing
proteins from a compatible vector, such as the C127, 3T3, CHO, HeLa and
BHK cell lines.
[0227] The constructs in host cells can be used in a conventional manner
to produce the gene product encoded by the recombinant sequence.
Depending upon the host employed in a recombinant production procedure,
the polypeptides produced by host cells containing the vector may be
glycosylated or may be non-glycosylated. Polypeptides of the invention
may or may not also include an initial methionine amino acid residue.
[0228] Alternatively, the polypeptides of the invention, or fragments
comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids thereof can be synthetically produced by
conventional peptide synthesizers. In other aspects, fragments or
portions of the polypeptides may be employed for producing the
corresponding full-length polypeptide by peptide synthesis; therefore,
the fragments may be employed as intermediates for producing the
full-length polypeptides.
[0229] Cell-free translation systems can also be employed to produce one
of the polypeptides of the invention, or fragments comprising at least 5,
10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids
thereof using mRNAs transcribed from a DNA construct comprising a
promoter operably linked to a nucleic acid encoding the polypeptide or
fragment thereof. In some aspects, the DNA construct may be linearized
prior to conducting an in vitro transcription reaction. The transcribed
mRNA is then incubated with an appropriate cell-free translation extract,
such as a rabbit reticulocyte extract, to produce the desired polypeptide
or fragment thereof.
[0230] Amplification of Nucleic Acids
[0231] In practicing the invention, nucleic acids of the invention and
nucleic acids encoding the glucanases, mannanases, or xylanases of the
invention, or modified nucleic acids of the invention, can be reproduced
by amplification. Amplification can also be used to clone or modify the
nucleic acids of the invention. Thus, the invention provides
amplification primer sequence pairs for amplifying nucleic acids of the
invention. One of skill in the art can design amplification primer
sequence pairs for any part of or the full length of these sequences.
[0232] In one aspect, the invention provides a nucleic acid amplified by a
primer pair of the invention, e.g., a primer pair as set forth by about
the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or
25 residues of a nucleic acid of the invention, and about the first (the
5') 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of the
complementary strand.
[0233] The invention provides an amplification primer sequence pair for
amplifying a nucleic acid encoding a polypeptide having a glucanase
activity, wherein the primer pair is capable of amplifying a nucleic acid
comprising a sequence of the invention, or fragments or subsequences
thereof. One or each member of the amplification primer sequence pair can
comprise an oligonucleotide comprising at least about 10 to 50
consecutive bases of the sequence, or about 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, or 25 consecutive bases of the sequence. The
invention provides amplification primer pairs, wherein the primer pair
comprises a first member having a sequence as set forth by about the
first (the 5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25
residues of a nucleic acid of the invention, and a second member having a
sequence as set forth by about the first (the 5') 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, or 25 residues of the complementary strand of
the first member. The invention provides glucanases, mannanases, or
xylanases generated by amplification, e.g., polymerase chain reaction
(PCR), using an amplification primer pair of the invention. The invention
provides methods of making glucanases, mannanases, or xylanases by
amplification, e.g., polymerase chain reaction (PCR), using an
amplification primer pair of the invention. In one aspect, the
amplification primer pair amplifies a nucleic acid from a library, e.g.,
a gene library, such as an environmental library.
[0234] Amplification reactions can also be used to quantify the amount of
nucleic acid in a sample (such as the amount of message in a cell
sample), label the nucleic acid (e.g., to apply it to an array or a
blot), detect the nucleic acid, or quantify the amount of a specific
nucleic acid in a sample. In one aspect of the invention, message
isolated from a cell or a cDNA library are amplified.
[0235] The skilled artisan can select and design suitable oligonucleotide
amplification primers. Amplification methods are also well known in the
art, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCR
PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic
Press, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,
Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics
4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);
tanscription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad. Sci.
USA 86:1173); and, self-sustained sequence replication (see, e.g.,
Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicase
amplification (see, e.g., Smith (1997) J. Clin. Microbiol. 35:1477-1491),
automated Q-beta replicase amplification assay (see, e.g., Burg (1996)
Mol. Cell. Probes 10:257-271) and other RNA polymerase mediated
techniques (e.g., NASBA, Cangene, Mississauga, Ontario); see also Berger
(1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S. Pat. Nos.
4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology 13:563-564.
Determining the Degree of Sequence Identity
[0236] The invention provides nucleic acids comprising sequences having at
least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete
(100%) sequence identity to an exemplary nucleic acid of the invention
(e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,
SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID
NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID
NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID
NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID
NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID
NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID
NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID
NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID
NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ
ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119,
SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID
NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ
ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147,
SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID
NO:157, SEQ ID NO:199, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ
ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175,
SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID
NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ
ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203,
SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID
NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ
ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231,
SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID
NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ
ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259,
SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID
NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ
ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ ID NO:287,
SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID
NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ
ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315,
SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ ID
NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ
ID NO:335, SEQ ID NO:337, SEQ ID NO:339, SEQ ID NO:341, SEQ ID NO:343,
SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:349, SEQ ID NO:351, SEQ ID
NO:353, SEQ ID NO:355, SEQ ID NO:357, SEQ ID NO:359, SEQ ID NO:361, SEQ
ID NO:363, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:369, SEQ ID NO:371,
SEQ ID NO:373, SEQ ID NO:375, SEQ ID NO:377, SEQ ID NO:379, SEQ ID
NO:381, SEQ ID NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQ ID NO:389, SEQ
ID NO:391, SEQ ID NO:393, SEQ ID NO:395, SEQ ID NO:397, SEQ ID NO:399,
SEQ ID NO:401, SEQ ID NO:403, SEQ ID NO:405, SEQ ID NO:407, SEQ ID
NO:409, SEQ ID NO:411, SEQ ID NO:413, SEQ ID NO:415, SEQ ID NO:417, SEQ
ID NO:419, SEQ ID NO:421, SEQ ID NO:423, SEQ ID NO:425, SEQ ID NO:427,
SEQ ID NO:429, SEQ ID NO:431, SEQ ID NO:433, SEQ ID NO:435, SEQ ID
NO:437, SEQ ID NO:439, SEQ ID NO:441, SEQ ID NO:443, SEQ ID NO:445, SEQ
ID NO:447, SEQ ID NO:449, SEQ ID NO:451, SEQ ID NO:453, SEQ ID NO:455,
SEQ ID NO:457, SEQ ID NO:459, SEQ ID NO:461, SEQ ID NO:463, SEQ ID
NO:465, SEQ ID NO:467, SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ
ID NO:475, SEQ ID NO:477, SEQ ID NO:479, SEQ ID NO:481, SEQ ID NO:483,
SEQ ID NO:485, SEQ ID NO:487, SEQ ID NO:489, SEQ ID NO:491, SEQ ID
NO:493, SEQ ID NO:495, SEQ ID NO:497, SEQ ID NO:499, SEQ ID NO:501, SEQ
ID NO:503, SEQ ID NO:505, SEQ ID NO:507, SEQ ID NO:509, SEQ ID NO:511,
SEQ ID NO:513, SEQ ID NO:515, SEQ ID NO:517) over a region of at least
about 10, 20, 30, 40, 50, 60, 70, 75, 100, 150, 200, 250, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,
1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more, residues.
The invention provides polypeptides comprising sequences having at least
about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,
63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)
sequence identity to an exemplary polypeptide of the invention. The
extent of sequence identity (homology) may be determined using any
computer program and associated parameters, including those described
herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with the default
parameters.
[0237] Nucleic acid sequences of the invention can comprise at least 10,
15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500
consecutive nucleotides of an exemplary sequence of the invention and
sequences substantially identical thereto. Homologous sequences and
fragments of nucleic acid sequences of the invention can refer to a
sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%,
70%, 65%, 60%, 55%, or 50% homology to these sequences. Homology may be
determined using any of the computer programs and parameters described
herein, including FASTA version 3.0t78 with the default parameters.
Homologous sequences also include RNA sequences in which uridines replace
the thymines in the nucleic acid sequences of the invention. The
homologous sequences may be obtained using any of the procedures
described herein or may result from the correction of a sequencing error.
It will be appreciated that the nucleic acid sequences of the invention
can be represented in the traditional single character format (See the
inside back cover of Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman
& Co., New York) or in any other format which records the identity of the
nucleotides in a sequence.
[0238] Various sequence comparison programs identified elsewhere in this
patent specification are particularly contemplated for use in this aspect
of the invention. Protein and/or nucleic acid sequence homologies may be
evaluated using any of the variety of sequence comparison algorithms and
programs known in the art. Such algorithms and programs include, but are
by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW
(Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988;
Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Thompson et al.,
Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., Methods
Enzymol. 266:383-402, 1996; Altschul et al., J. Mol. Biol.
215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993).
[0239] Homology or identity is often measured using sequence analysis
software (e.g., Sequence Analysis Software Package of the Genetics
Computer Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705). Such software matches similar
sequences by assigning degrees of homology to various deletions,
substitutions and other modifications. The terms "homology" and
"identity" in the context of two or more nucleic acids or polypeptide
sequences, refer to two or more sequences or subsequences that are the
same or have a specified percentage of amino acid residues or nucleotides
that are the same when compared and aligned for maximum correspondence
over a comparison window or designated region as measured using any
number of sequence comparison algorithms or by manual alignment and
visual inspection.
[0240] For sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a sequence
comparison algorithm, test and reference sequences are entered into a
computer, subsequence coordinates are designated, if necessary and
sequence algorithm program parameters are designated. Default program
parameters can be used, or alternative parameters can be designated. The
sequence comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference sequence,
based on the program parameters.
[0241] A "comparison window", as used herein, includes reference to a
segment of any one of the number of contiguous positions selected from
the group consisting of from 20 to 600, usually about 50 to about 200,
more usually about 100 to about 150 in which a sequence may be compared
to a reference sequence of the same number of contiguous positions after
the two sequences are optimally aligned. Methods of alignment of sequence
for comparison are well-known in the art. Optimal alignment of sequences
for comparison can be conducted, e.g. by the local homology algorithm of
Smith & Waterman, Adv. Appl. Math. 2:48.sup.2, 1981, by the homology
alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by
the search for similarity method of person & Lipman, Proc. Nat'l. Acad.
Sci. USA 85:2444, 1988, by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group, 575 Science Dr., Madison,
Wis.), or by manual alignment and visual inspection. Other algorithms for
determining homology or identity include, for example, in addition to a
BLAST program (Basic Local Alignment Search Tool at the National Center
for Biological Information), ALIGN, AMAS (Analysis of Multiply Aligned
Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET (Aligned
Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN
(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProved
Searcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,
CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, Las
Vegas algorithm, FNAT (forced Nucleotide Alignment Tool), Framealign,
Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence Analysis
Package), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC
(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP
(Local Content Program), MACAW (Multiple Alignment Construction &
Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN, PIMA
(Pattern-Induced Multi-sequence Alignment), SAGA (Sequence Alignment by
Genetic Algorithm) and WHAT-IF. Such alignment programs can also be used
to screen genome databases to identify polynucleotide sequences having
substantially identical sequences. A number of genome databases are
available, for example, a substantial portion of the human genome is
available as part of the Human Genome Sequencing Project (J. Roach,
http://weber.u.Washington.edu/.about.roach/human_genome_progress 2.html)
(Gibbs, 1995). At least twenty-one other genomes have already been
sequenced, including, for example, M. genitalium (Fraser et al., 1995),
M. jannaschii (Bult et al., 1996), H. influenzae (Fleiscmann et al.,
1995), E. coli (Blattner et al., 1997) and yeast (S. cerevisiae) (Mewes
et al., 1997) and D. melanogaster (Adams et al., 2000). Significant
progress has also been made in sequencing the genomes of model organism,
such as mouse, C. elegans and Arabadopsis sp. Several databases
containing genomic information annotated with some functional information
are maintained by different organization and are accessible via the
internet One example of a useful algorithm is BLAST and BLAST 2.0
algorithms, which are described in Altschul et al., Nuc. Acids Res.
25:3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215:403-410, 1990,
respectively. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information. This
algorithm involves first identifying high scoring sequence pairs (HSPs)
by identifying short words of length W in the query sequence, which
either match or satisfy some positive-valued threshold score T when
aligned with a word of the same length in a database sequence. T is
referred to as the neighborhood word score threshold (Altschul et al.,
supra). These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are extended
in both directions along each sequence for as far as the cumulative
alignment score can be increased. Cumulative scores are calculated using,
for nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0). For amino acid sequences, a scoring
matrix is used to calculate the cumulative score. Extension of the word
hits in each direction are halted when: the cumulative alignment score
falls off by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the accumulation of one or
more negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T and X determine the
sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W of 11, an
expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For
amino acid sequences, the BLASTP program uses as defaults a wordlength of
3 and expectations (E) of 10 and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)
alignments (B) of 50, expectation (E) of 10, M=5, N=-4 and a comparison
of both strands.
[0242] The BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul, Proc.
Natl. Acad. Sci. USA 90:5873, 1993). One measure of similarity provided
by BLAST algorithm is the smallest sum probability (P(N)), which provides
an indication of the probability by which a match between two nucleotide
or amino acid sequences would occur by chance. For example, a nucleic
acid is considered similar to a references sequence if the smallest sum
probability in a comparison of the test nucleic acid to the reference
nucleic acid is less than about 0.2, more in one aspect less than about
0.01 and most in one aspect less than about 0.001.
[0243] In one aspect, protein and nucleic acid sequence homologies are
evaluated using the Basic Local Alignment Search Tool ("BLAST") In
particular, five specific BLAST programs are used to perform the
following task: [0244] (1) BLASTP and BLAST3 compare an amino acid
query sequence against a protein sequence database; [0245] (2) BLASTN
compares a nucleotide query sequence against a nucleotide sequence
database; t [0246] (3) BLASTX compares the six-frame conceptual
translation products of a query nucleotide sequence (both strands)
against a protein sequence database; [0247] (4) TBLASTN compares a query
protein sequence against a nucleotide sequence database translated in all
six reading frames (both strands); and [0248] (5) TBLASTX compares the
six-frame translations of a nucleotide query sequence against the
six-frame translations of a nucleotide sequence database.
[0249] The BLAST programs identify homologous sequences by identifying
similar segments, which are referred to herein as "high-scoring segment
pairs," between a query amino or nucleic acid sequence and a test
sequence which is in one aspect obtained from a protein or nucleic acid
sequence database. High-scoring segment pairs are in one aspect
identified (i.e., aligned) by means of a scoring matrix, many of which
are known in the art. In one aspect, the scoring matrix used is the
BLOSLUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992; Henikoff
and Henikoff, Proteins 17:49-61, 1993). Less in one aspect, the PAM or
PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds.,
1978, Matrices for Detecting Distance Relationships: Atlas of Protein
Sequence and Structure, Washington: National Biomedical Research
Foundation). BLAST programs are accessible through the U.S. National
Library of Medicine.
[0250] The parameters used with the above algorithms may be adapted
depending on the sequence length and degree of homology studied. In some
aspects, the parameters may be the default parameters used by the
algorithms in the absence of instructions from the user.
Computer Systems and Computer Program Products
[0251] To determine and identify sequence identities, structural
homologies, motifs and the like in silico, a nucleic acid or polypeptide
sequence of the invention can be stored, recorded, and manipulated on any
medium which can be read and accessed by a computer.
[0252] Accordingly, the invention provides computers, computer systems,
computer readable mediums, computer programs products and the like
recorded or stored thereon the nucleic acid and polypeptide sequences of
the invention. As used herein, the words "recorded" and "stored" refer to
a process for storing information on a computer medium. A skilled artisan
can readily adopt any known methods for recording information on a
computer readable medium to generate manufactures comprising one or more
of the nucleic acid and/or polypeptide sequences of the invention.
[0253] The polypeptides of the invention include the polypeptide sequences
of the invention, e.g., the exemplary sequences of the invention, and
sequences substantially identical thereto, and fragments of any of the
preceding sequences. Substantially identical, or homologous, polypeptide
sequences refer to a polypeptide sequence having at least 50%, 51%, 52%,
53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,
67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to
an exemplary sequence of the invention.
[0254] Homology may be determined using any of the computer programs and
parameters described herein, including FASTA version 3.0t78 with the
default parameters or with any modified parameters. The homologous
sequences may be obtained using any of the procedures described herein or
may result from the correction of a sequencing error. The polypeptide
fragments comprise at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75,
100, 150, 200, 250, 300, 350, 400, 450, 500 or more consecutive amino
acids of the polypeptides of the invention. It will be appreciated that
the polypeptide codes as set forth in amino acid sequences of the
invention, can be represented in the traditional single character format
or three letter format (See the inside back cover of Stryer, Lubert.
Biochemistry. 3rd Ed., W. H Freeman & Co., New York) or in any other
format which relates the identity of the polypeptides in a sequence.
[0255] A nucleic acid or polypeptide sequence of the invention can be
stored, recorded and manipulated on any medium which can be read and
accessed by a computer. As used herein, the words "recorded" and "stored"
refer to a process for storing information on a computer medium. A
skilled artisan can readily adopt any of the presently known methods for
recording information on a computer readable medium to generate
manufactures comprising one or more of the nucleic acid sequences of the
invention, one or more of the polypeptide sequences of the invention.
Another aspect of the invention is a computer readable medium having
recorded thereon at least 2, 5, 10, 15, or 20 or more nucleic acid
sequences of the invention.
[0256] Another aspect of the invention is a computer readable medium
having recorded thereon one or more of the nucleic acid sequences of the
invention. Another aspect of the invention is a computer readable medium
having recorded thereon one or more of the polypeptide sequences of the
invention. Another aspect of the invention is a computer readable medium
having recorded thereon at least 2, 5, 10, 15, or 20 or more of the
sequences as set forth above.
[0257] Computer readable media include magnetically readable media,
optically readable media, electronically readable media and
magnetic/optical media. For example, the computer readable media may be a
hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk
(DVD), Random Access Memory (RAM), or Read Only Memory (ROM as well as
other types of other media known to those skilled in the art.
[0258] Aspects of the invention include systems (e.g. internet based
systems), particularly computer systems which store and manipulate the
sequence information described herein. One example of a computer system
100 is illustrated in block diagram form in FIG. 1. As used herein, "a
computer system" refers to the hardware components, software components
and data storage components used to analyze a nucleotide sequence of a
nucleic acid sequence of the invention, or a polypeptide sequence of the
invention. The computer system 100 typically includes a processor for
processing, accessing and manipulating the sequence data. The processor
105 can be any well-known type of central processing unit, such as, for
example, the Pentium III from Intel Corporation, or similar processor
from Sun, Motorola, Compaq, AMD or International Business Machines.
[0259] Typically the computer system 100 is a general purpose system that
comprises the processor 105 and one or more internal data storage
components 110 for storing data and one or more data retrieving devices
for retrieving the data stored on the data storage components. A skilled
artisan can readily appreciate that any one of the currently available
computer systems are suitable.
[0260] In one particular aspect, the computer system 100 includes a
processor 105 connected to a bus which is connected to a main memory 115
(in one aspect implemented as RAM) and one or more internal data storage
devices 110, such as a hard drive and/or other computer readable media
having data recorded thereon. In some aspects, the computer system 100
further includes one or more data retrieving device 118 for reading the
data stored on the internal data storage devices 110.
[0261] The data retrieving device 118 may represent, for example, a floppy
disk drive, a compact disk drive, a magnetic tape drive, or a modem
capable of connection to a remote data storage system (e.g., via the
internet) etc. In some aspects, the internal data storage device 110 is a
removable computer readable medium such as a floppy disk, a compact disk,
a magnetic tape, etc. containing control logic and/or data recorded
thereon. The computer system 100 may advantageously include or be
programmed by appropriate software for reading the control logic and/or
the data from the data storage component once inserted in the data
retrieving device.
[0262] The computer system 100 includes a display 120 which is used to
display output to a computer user. It should also be noted that the
computer system 100 can be linked to other computer systems 125a-c in a
network or wide area network to provide centralized access to the
computer system 100.
[0263] Software for accessing and processing the nucleotide sequences of a
nucleic acid sequence of the invention, or a polypeptide sequence of the
invention, (such as search tools, compare tools and modeling tools etc.)
may reside in main memory 115 during execution.
[0264] In some aspects, the computer system 100 may further comprise a
sequence comparison algorithm for comparing a nucleic acid sequence of
the invention, or a polypeptide sequence of the invention, stored on a
computer readable medium to a reference nucleotide or polypeptide
sequence(s) stored on a computer readable medium. A "sequence comparison
algorithm" refers to one or more programs which are implemented (locally
or remotely) on the computer system 100 to compare a nucleotide sequence
with other nucleotide sequences and/or compounds stored within a data
storage means. For example, the sequence comparison algorithm may compare
the nucleotide sequences of a nucleic acid sequence of the invention, or
a polypeptide sequence of the invention, stored on a computer readable
medium to reference sequences stored on a computer readable medium to
identify homologies or structural motifs.
[0265] FIG. 2 is a flow diagram illustrating one aspect of a process 200
for comparing a new nucleotide or protein sequence with a database of
sequences in order to determine the homology levels between the new
sequence and the sequences in the database. The database of sequences can
be a private database stored within the computer system 100, or a public
database such as GENBANK that is available through the Internet.
[0266] The process 200 begins at a start state 201 and then moves to a
state 202 wherein the new sequence to be compared is stored to a memory
in a computer system 100. As discussed above, the memory could be any
type of memory, including RAM or an internal storage device.
[0267] The process 200 then moves to a state 204 wherein a database of
sequences is opened for analysis and comparison. The process 200 then
moves to a state 206 wherein the first sequence stored in the database is
read into a memory on the computer. A comparison is then performed at a
state 210 to determine if the first sequence is the same as the second
sequence. It is important to note that this step is not limited to
performing an exact comparison between the new sequence and the first
sequence in the database. Well-known methods are known to those of skill
in the art for comparing two nucleotide or protein sequences, even if
they are not identical. For example, gaps can be introduced into one
sequence in order to raise the homology level between the two tested
sequences. The parameters that control whether gaps or other features are
introduced into a sequence during comparison are normally entered by the
user of the computer system.
[0268] Once a comparison of the two sequences has been performed at the
state 210, a determination is made at a decision state 210 whether the
two sequences are the same. Of course, the term "same" is not limited to
sequences that are absolutely identical. Sequences that are within the
homology parameters entered by the user will be marked as "same" in the
process 200.
[0269] If a determination is made that the two sequences are the same, the
process 200 moves to a state 214 wherein the name of the sequence from
the database is displayed to the user. This state notifies the user that
the sequence with the displayed name fulfills the homology constraints
that were entered. Once the name of the stored sequence is displayed to
the user, the process 200 moves to a decision state 218 wherein a
determination is made whether more sequences exist in the database. If no
more sequences exist in the database, then the process 200 terminates at
an end state 220. However, if more sequences do exist in the database,
then the process 200 moves to a state 224 wherein a pointer is moved to
the next sequence in the database so that it can be compared to the new
sequence. In this manner, the new sequence is aligned and compared with
every sequence in the database.
[0270] It should be noted that if a determination had been made at the
decision state 212 that the sequences were not homologous, then the
process 200 would move immediately to the decision state 218 in order to
determine if any other sequences were available in the database for
comparison.
[0271] Accordingly, one aspect of the invention is a computer system
comprising a processor, a data storage device having stored thereon a
nucleic acid sequence of the invention, or a polypeptide sequence of the
invention, a data storage device having retrievably stored thereon
reference nucleotide sequences or polypeptide sequences to be compared to
a nucleic acid sequence of the invention, or a polypeptide sequence of
the invention and a sequence comparer for conducting the comparison. The
sequence comparer may indicate a homology level between the sequences
compared or identify structural motifs in the above described nucleic
acid code a nucleic acid sequence of the invention, or a polypeptide
sequence of the invention, or it may identify structural motifs in
sequences which are compared to these nucleic acid codes and polypeptide
codes. In some aspects, the data storage device may have stored thereon
the sequences of at least 2, 5, 10, 15, 20, 25, 30 or 40 or more of the
nucleic acid sequences of the invention, or the polypeptide sequences of
the invention.
[0272] Another aspect of the invention is a method for determining the
level of homology between a nucleic acid sequence of the invention, or a
polypeptide sequence of the invention and a reference nucleotide
sequence. The method including reading the nucleic acid, code or the
polypeptide code and the reference nucleotide or polypeptide sequence
through the use of a computer program which determines homology levels
and determining homology between the nucleic acid code or polypeptide
code and the reference nucleotide or polypeptide sequence with the
computer program. The computer program may be any of a number of computer
programs for determining homology levels, including those specifically
enumerated herein, (e.g., BLAST2N with the default parameters or with any
modified parameters). The method may be implemented using the computer
systems described above. The method may also be performed by reading at
least 2, 5, 10, 15, 20, 25, 30 or 40 or more of the above described
nucleic acid sequences of the invention, or the polypeptide sequences of
the invention through use of the computer program and determining
homology between the nucleic acid codes or polypeptide codes and
reference nucleotide sequences or polypeptide sequences.
[0273] FIG. 3 is a flow diagram illustrating one aspect of a process 250
in a computer for determining whether two sequences are homologous. The
process 250 begins at a start state 252 and then moves to a state 254
wherein a first sequence to be compared is stored to a memory. The second
sequence to be compared is then stored to a memory at a state 256. The
process 250 then moves to a state 260 wherein the first character in the
first sequence is read and then to a state 262 wherein the first
character of the second sequence is read. It should be understood that if
the sequence is a nucleotide sequence, then the character would normally
be either A, T, C, G or U. If the sequence is a protein sequence, then it
is in one aspect in the single letter amino acid code so that the first
and sequence sequences can be easily compared.
[0274] A determination is then made at a decision state 264 whether the
two characters are the same. If they are the same, then the process 250
moves to a state 268 wherein the next characters in the first and second
sequences are read. A determination is then made whether the next
characters are the same. If they are, then the process 250 continues this
loop until two characters are not the same. If a determination is made
that the next two characters are not the same, the process 250 moves to a
decision state 274 to determine whether there are any more characters
either sequence to read.
[0275] If there are not any more characters to read, then the process 250
moves to a state 276 wherein the level of homology between the first and
second sequences is displayed to the user. The level of homology is
determined by calculating the proportion of characters between the
sequences that were the same out of the total number of sequences in the
first sequence. Thus, if every character in a first 100 nucleotide
sequence aligned with a every character in a second sequence, the
homology level would be 100%.
[0276] Alternatively, the computer program may be a computer program which
compares the nucleotide sequences of a nucleic acid sequence as set forth
in the invention, to one or more reference nucleotide sequences in order
to determine whether the nucleic acid code of the invention, differs from
a reference nucleic acid sequence at one or more positions. Optionally
such a program records the length and identity of inserted, deleted or
substituted nucleotides with respect to the sequence of either the
reference polynucleotide or a nucleic acid sequence of the invention. In
one aspect, the computer program may be a program which determines
whether a nucleic acid sequence of the invention, contains a single
nucleotide polymorphism (SNP) with respect to a reference nucleotide
sequence.
[0277] Accordingly, another aspect of the invention is a method for
determining whether a nucleic acid sequence of the invention, differs at
one or more nucleotides from a reference nucleotide sequence comprising
the steps of reading the nucleic acid code and the reference nucleotide
sequence through use of a computer program which identifies differences
between nucleic acid sequences and identifying differences between the
nucleic acid code and the reference nucleotide sequence with the computer
program. In some aspects, the computer program is a program which
identifies single nucleotide polymorphisms. The method may be implemented
by the computer systems described above and the method illustrated in
FIG. 3. The method may also be performed by reading at least 2, 5, 10,
15, 20, 25, 30, or 40 or more of the nucleic acid sequences of the
invention and the reference nucleotide sequences through the use of the
computer program and identifying differences between the nucleic acid
codes and the reference nucleotide sequences with the computer program.
[0278] In other aspects the computer based system may further comprise an
identifier for identifying features within a nucleic acid sequence of the
invention or a polypeptide sequence of the invention.
[0279] An "identifier" refers to one or more programs which identifies
certain features within a nucleic acid sequence of the invention, or a
polypeptide sequence of the invention. In one aspect, the identifier may
comprise a program which identifies an open reading frame in a nucleic
acid sequence of the invention.
[0280] FIG. 4 is a flow diagram illustrating one aspect of an identifier
process 300 for detecting the presence of a feature in a sequence. The
process 300 begins at a start state 302 and then moves to a state 304
wherein a first sequence that is to be checked for features is stored to
a memory 115 in the computer system 100. The process 300 then moves to a
state 306 wherein a database of sequence features is opened. Such a
database would include a list of each feature's attributes along with the
name of the feature. For example, a feature name could be "Initiation
Codon" and the attribute would be "ATG". Another example would be the
feature name "TAATAA Box" and the feature attribute would be "TAATAA". An
example of such a database is produced by the University of Wisconsin
Genetics Computer Group. Alternatively, the features may be structural
polypeptide motifs such as alpha helices, beta sheets, or functional
polypeptide motifs such as enzymatic catalytic domains (CDs), or, active
sites, helix-turn-helix motifs or other motifs known to those skilled in
the art.
[0281] Once the database of features is opened at the state 306, the
process 300 moves to a state 308 wherein the first feature is read from
the database. A comparison of the attribute of the first feature with the
first sequence is then made at a state 310. A determination is then made
at a decision state 316 whether the attribute of the feature was found in
the first sequence. If the attribute was found, then the process 300
moves to a state 318 wherein the name of the found feature is displayed
to the user.
[0282] The process 300 then moves to a decision state 320 wherein a
determination is made whether move features exist in the database. If no
more features do exist, then the process 300 terminates at an end state
324. However, if more features do exist in the database, then the process
300 reads the next sequence feature at a state 326 and loops back to the
state 310 wherein the attribute of the next feature is compared against
the first sequence. It should be noted, that if the feature attribute is
not found in the first sequence at the decision state 316, the process
300 moves directly to the decision state 320 in order to determine if any
more features exist in the database.
[0283] Accordingly, another aspect of the invention is a method of
identifying a feature within a nucleic acid sequence of the invention, or
a polypeptide sequence of the invention, comprising reading the nucleic
acid code(s) or polypeptide code(s) through the use of a computer program
which identifies features therein and identifying features within the
nucleic acid code(s) with the computer program. In one aspect, computer
program comprises a computer program which identifies open reading
frames. The method may be performed by reading a single sequence or at
least 2, 5, 10, 15, 20, 25, 30, or 40 of the nucleic acid sequences of
the invention, or the polypeptide sequences of the invention, through the
use of the computer program and identifying features within the nucleic
acid codes or polypeptide codes with the computer program.
[0284] A nucleic acid sequence of the invention, or a polypeptide sequence
of the invention, may be stored and manipulated in a variety of data
processor programs in a variety of formats. For example, a nucleic acid
sequence of the invention, or a polypeptide sequence of the invention,
may be stored as text in a word processing file, such as Microsoft
WORD.TM. or WORDPERFECT.TM. or as an ASCII file in a variety of database
programs familiar to those of skill in the art, such as DB2.TM.,
SYBASE.TM., or ORACLE.TM.. In addition, many computer programs and
databases may be used as sequence comparison algorithms, identifiers, or
sources of reference nucleotide sequences or polypeptide sequences to be
compared to a nucleic acid sequence of the invention, or a polypeptide
sequence of the invention. The following list is intended not to limit
the invention but to provide guidance to programs and databases which are
useful with the nucleic acid sequences of the invention, or the
polypeptide sequences of the invention.
[0285] The programs and databases which may be used include, but are not
limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications
Group), GeneMine (Molecular Applications Group), Look (Molecular
Applications Group), MacLook (Molecular Applications Group), BLAST and
BLAST-2 (NCBI), BLASIN and BLASIX (Altschul et al, J. Mol. Biol. 215:403,
1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444,
1988), FASTDB (Brudag et al. Comp. App. Biosci. 6:237-245, 1990),
Catalyst Molecular Simulations Inc.), Catalyst/SHAPE (Molecular
Simulations Inc.), Cerius.sup.2.DBAccess (Molecular Simulations Inc.),
HypoGen (Molecular Simulations Inc.), Insight II, (Molecular Simulations
Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular
Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi, (Molecular
Simulations Inc.), QuanteMM, (Molecular Simulations Inc.), Homology
(Molecular Simulations Inc.), Modeler (Molecular Simulations Inc.), ISIS
(Molecular Simulations Inc.), Quanta/Protein Design (Molecular
Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity
Explorer (Molecular Simulations Inc.), Gene Explorer Molecular
Simulations Inc.), SeqFold (Molecular Simulations Inc.), the MDL
Available Chemicals Directory database, the MDL Drug Data Report data
base, the Comprehensive Medicinal Chemistry database, Derwents's World
Drug Index database, the BioByteMasterFile database, the Genbank database
and the Genseqn database. Many other programs and data bases would be
apparent to one of skill in the art given the present disclosure.
[0286] Motifs which may be detected using the above programs include
sequences encoding leucine zippers, helix-turn-helix motifs,
glycosylation sites, ubiquitination sites, alpha helices and beta sheets,
signal sequences encoding signal peptides which direct the secretion of
the encoded proteins, sequences implicated in transcription regulation
such as homeoboxes, acidic stretches, enzymatic active sites (catalytic
domains (CDs)), substrate binding sites and enzymatic cleavage sites.
Hybridization of Nucleic Acids
[0287] The invention provides isolated, synthetic or recombinant nucleic
acids that hybridize under stringent conditions to an exemplary sequence
of the invention (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID
NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID
NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID
NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID
NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID
NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID
NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID
NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID
NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID
NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ
ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125,
SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID
NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ
ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153,
SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:199, SEQ ID NO:161, SEQ ID
NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ
ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181,
SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID
NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ
ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209,
SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID
NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ
ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237,
SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID
NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ
ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265,
SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID
NO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ
ID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293,
SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID
NO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ
ID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321,
SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID
NO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:337, SEQ ID NO:339, SEQ
ID NO:341, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:349,
SEQ ID NO:351, SEQ ID NO:353, SEQ ID NO:355, SEQ ID NO:357, SEQ ID
NO:359, SEQ ID NO:361, SEQ ID NO:363, SEQ ID NO:365, SEQ ID NO:367, SEQ
ID NO:369, SEQ ID NO:371, SEQ ID NO:373, SEQ ID NO:375, SEQ ID NO:377,
SEQ ID NO:379, SEQ ID NO:381, SEQ ID NO:383, SEQ ID NO:385, SEQ ID
NO:387, SEQ ID NO:389, SEQ ID NO:391, SEQ ID NO:393, SEQ ID NO:395, SEQ
ID NO:397, SEQ ID NO:399, SEQ ID NO:401, SEQ ID NO:403, SEQ ID NO:405,
SEQ ID NO:407, SEQ ID NO:409, SEQ ID NO:411, SEQ ID NO:413, SEQ ID
NO:415, SEQ ID NO:417, SEQ ID NO:419, SEQ ID NO:421, SEQ ID NO:423, SEQ
ID NO:425, SEQ ID NO:427, SEQ ID NO:429, SEQ ID NO:431, SEQ ID NO:433,
SEQ ID NO:435, SEQ ID NO:437, SEQ ID NO:439, SEQ ID NO:441, SEQ ID
NO:443, SEQ ID NO:445, SEQ ID NO:447, SEQ ID NO:449, SEQ ID NO:451, SEQ
ID NO:453, SEQ ID NO:455, SEQ ID NO:457, SEQ ID NO:459, SEQ ID NO:461,
SEQ ID NO:463, SEQ ID NO:465, SEQ ID NO:467, SEQ ID NO:469, SEQ ID
NO:471, SEQ ID NO:473, SEQ ID NO:475, SEQ ID NO:477, SEQ ID NO:479, SEQ
ID NO:481, SEQ ID NO:483, SEQ ID NO:485, SEQ ID NO:487, SEQ ID NO:489,
SEQ ID NO:491, SEQ ID NO:493, SEQ ID NO:495, SEQ ID NO:497, SEQ ID
NO:499, SEQ ID NO:501, SEQ ID NO:503, SEQ ID NO:505, SEQ ID NO:507, SEQ
ID NO:509, SEQ ID NO:511, SEQ ID NO:513, SEQ ID NO:515, SEQ ID NO:517).
The stringent conditions can e highly stringent conditions, medium
stringent conditions and/or low stringent conditions, including the high
and reduced stringency conditions described herein. In one aspect, it is
the stringency of the wash conditions that set forth the conditions which
determine whether a nucleic acid is within the scope of the invention, as
discussed below.
[0288] In alternative aspects, nucleic acids of the invention as defined
by their ability to hybridize under stringent conditions can be between
about five residues and the full length of nucleic acid of the invention;
e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65,
70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,
650, 700, 750, 800, 850, 900, 950, 1000, or more, residues in length.
Nucleic acids shorter than full length are also included. These nucleic
acids can be useful as, e.g., hybridization probes, labeling probes, PCR
oligonucleotide probes, iRNA (single or double stranded), antisense or
sequences encoding antibody binding peptides (epitopes), motifs, active
sites (catalytic domains (CDs)) and the like.
[0289] In one aspect, nucleic acids of the invention are defined by their
ability to hybridize under high stringency comprises conditions of about
50% formamide at about 37.degree. C. to 42.degree. C. In one aspect,
nucleic acids of the invention are defined by their ability to hybridize
under reduced stringency comprising conditions in about 35% to 25%
formamide at about 30.degree. C. to 35.degree. C.
[0290] Alternatively, nucleic acids of the invention are defined by their
ability to hybridize under high stringency comprising conditions at
42.degree. C. in 50% formamide, 5.times.SSPE, 0.3% SDS, and a repetitive
sequence blocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g.,
200 n/ml sheared and denatured salmon sperm DNA). In one aspect, nucleic
acids of the invention are defined by their ability to hybridize under
reduced stringency conditions comprising 35% formamide at a reduced
temperature of 35.degree. C.
[0291] In nucleic acid hybridization reactions, the conditions used to
achieve a particular level of stringency will vary, depending on the
nature of the nucleic acids being hybridized. For example, the length,
degree of complementarity, nucleotide sequence composition (e.g., GC v.
AT content) and nucleic acid type (e.g., RNA v. DNA) of the hybridizing
regions of the nucleic acids can be considered in selecting hybridization
conditions. An additional consideration is whether one of the nucleic
acids is immobilized, for example, on a filter.
[0292] Hybridization may be carried out under conditions of low
stringency, moderate stringency or high stringency. As an example of
nucleic acid hybridization, a polymer membrane containing immobilized
denatured nucleic acids is first prehybridized for 30 minutes at
45.degree. C. in a solution consisting of 0.9 M NaCl, 50 mM
NaH.sub.2PO.sub.4, pH 7.0, 5.0 mM Na.sub.2EDTA, 0.5% SDS,
10.times.Denhardt's and 0.5 mg/ml polyriboadenylic acid. Approximately
2.times.10.sup.7 cpm (specific activity 4-9.times.10.sup.8 cpm/ug) of
.sup.32P end-labeled oligonucleotide probe are then added to the
solution. After 12-16 hours of incubation, the membrane is washed for 30
minutes at room temperature in 1.times.SET (150 mM NaCl, 20 mM Tris
hydrochloride, pH 7.8, 1 mM Na.sub.2EDTA) containing 0.5% SDS, followed
by a 30 minute wash in fresh 1.times.SET at T.sub.m-10.degree. C. for the
oligonucleotide probe. The membrane is then exposed to auto-radiographic
film for detection of hybridization signals.
[0293] All of the foregoing hybridizations would be considered to be under
conditions of high stringency.
[0294] Following hybridization, a filter can be washed to remove any
non-specifically bound detectable probe. The stringency used to wash the
filters can also be varied depending on the nature of the nucleic acids
being hybridized, the length of the nucleic acids being hybridized, the
degree of complementarity, the nucleotide sequence composition (e.g., GC
v. AT content) and the nucleic acid type (e.g., RNA v. DNA). Examples of
progressively higher stringency condition washes are as follows:
2.times.SSC, 0.1% SDS at room temperature for 15 minutes (low
stringency); 0.1.times.SSC, 0.5% SDS at room temperature for 30 minutes
to 1 hour (moderate stringency); 0.1.times.SSC, 0.5% SDS for 15 to 30
minutes at between the hybridization temperature and 68.degree. C. (high
stringency); and 0.15M NaCl for 15 minutes at 72.degree. C. (very high
stringency). A final low stringency wash can be conducted in
0.1.times.SSC at room temperature. The examples above are merely
illustrative of one set of conditions that can be used to wash filters.
One of skill in the art would know that there are numerous recipes for
different stringency washes. Some other examples are given below.
[0295] Nucleic acids which have hybridized to the probe are identified by
autoradiography or other conventional techniques.
[0296] The above procedure may be modified to identify nucleic acids
having decreasing levels of homology to the probe sequence. For example,
to obtain nucleic acids of decreasing homology to the detectable probe,
less stringent conditions may be used. For example, the hybridization
temperature may be decreased in increments of 5.degree. C. from
68.degree. C. to 42.degree. C. in a hybridization buffer having a Na+
concentration of approximately 1M. Following hybridization, the filter
may be washed with 2.times.SSC, 0.5% SDS at the temperature of
hybridization. These conditions are considered to be "moderate"
conditions above 50.degree. C. and "low" conditions below 50.degree. C. A
specific example of "moderate" hybridization conditions is when the above
hybridization is conducted at 55.degree. C. A specific example of "low
stringency" hybridization conditions is when the above hybridization is
conducted at 45.degree. C.
[0297] Alternatively, the hybridization may be carried out in buffers,
such as 6.times.SSC, containing formamide at a temperature of 42.degree.
C. In this case, the concentration of formamide in the hybridization
buffer may be reduced in 5% increments from 50% to 0% to identify clones
having decreasing levels of homology to the probe. Following
hybridization, the filter may be washed with 6.times.SSC, 0.5% SDS at
50.degree. C. These conditions are considered to be "moderate" conditions
above 25% formamide and "low" conditions below 25% formamide. A specific
example of "moderate" hybridization conditions is when the above
hybridization is conducted at 30% formamide. A specific example of "low
stringency" hybridization conditions is when the above hybridization is
conducted at 10% formamide.
[0298] However, the selection of a hybridization format is not
critical--it is the stringency of the wash conditions that set forth the
conditions which determine whether a nucleic acid is within the scope of
the invention. Wash conditions used to identify nucleic acids within the
scope of the invention include, e.g.: a salt concentration of about 0.02
molar at pH 7 and a temperature of at least about 50.degree. C. or about
55.degree. C. to about 60.degree. C.; or, a salt concentration of about
0.15 M NaCl at 72.degree. C. for about 15 minutes; or, a salt
concentration of about 0.2.times.SSC at a temperature of at least about
50.degree. C. or about 55.degree. C. to about 60.degree. C. for about 15
to about 20 minutes; or, the hybridization complex is washed twice with a
solution with a salt concentration of about 2.times.SSC containing 0.1%
SDS at room temperature for 15 minutes and then washed twice by
0.1.times.SSC containing 0.1% SDS at 68.degree. C. for 15 minutes; or,
equivalent conditions. See Sambrook, Tijssen and Ausubel for a
description of SSC buffer and equivalent conditions.
[0299] These methods may be used to isolate nucleic acids of the
invention. For example, the preceding methods may be used to isolate
nucleic acids having a sequence with at least about 97%, at least 95%, at
least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at
least 65%, at least 60%, at least 55%, or at least 50% homology to a
nucleic acid sequence selected from the group consisting of one of the
sequences of the invention, or fragments comprising at least about 10,
15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500
consecutive bases thereof and the sequences complementary thereto.
Homology may be measured using the alignment algorithm. For example, the
homologous polynucleotides may have a coding sequence which is a
naturally occurring allelic variant of one of the coding sequences
described herein. Such allelic variants may have a substitution, deletion
or addition of one or more nucleotides when compared to the nucleic acids
of the invention.
[0300] Additionally, the above procedures may be used to isolate nucleic
acids which encode polypeptides having at least about 99%, 95%, at least
90%, at least 85%, at least 80%, at least 75%, at least 70%, at least
65%, at least 60%, at least 55%, or at least 50% homology to a
polypeptide of the invention, or fragments comprising at least 5, 10, 15,
20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof
as determined using a sequence alignment algorithm (e.g., such as the
FASTA version 3.0t78 algorithm with the default parameters).
Oligonucleotides Probes and Methods for Using them
[0301] The invention also provides nucleic acid probes that can be used,
e.g., for identifying nucleic acids encoding a polypeptide with a
glucanase activity or fragments thereof or for identifying glucanase
genes. In one aspect, the probe comprises at least 10 consecutive bases
of a nucleic acid of the invention. Alternatively, a probe of the
invention can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80,
90, 100, 110, 120, 130, 150 or about 10 to 50, about 20 to 60 about 30 to
70, consecutive bases of a sequence as set forth in a nucleic acid of the
invention. The probes identify a nucleic acid by binding and/or
hybridization. The probes can be used in arrays of the invention, see
discussion below, including, e.g., capillary arrays. The probes of the
invention can also be used to isolate other nucleic acids or
polypeptides.
[0302] The isolated nucleic acids of the invention, the sequences
complementary thereto, or a fragment comprising at least 10, 15, 20, 25,
30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases of
one of the sequences of the invention, or the sequences complementary
thereto may also be used as probes to determine whether a biological
sample, such as a soil sample, contains an organism having a nucleic acid
sequence of the invention or an organism from which the nucleic acid was
obtained. In such procedures, a biological sample potentially harboring
the organism from which the nucleic acid was isolated is obtained and
nucleic acids are obtained from the sample. The nucleic acids are
contacted with the probe under conditions which permit the probe to
specifically hybridize to any complementary sequences from which are
present therein.
[0303] Where necessary, conditions which permit the probe to specifically
hybridize to complementary sequences may be determined by placing the
probe in contact with complementary sequences from samples known to
contain the complementary sequence as well as control sequences which do
not contain the complementary sequence. Hybridization conditions, such as
the salt concentration of the hybridization buffer, the formamide
concentration of the hybridization buffer, or the hybridization
temperature, may be varied to identify conditions which allow the probe
to hybridize specifically to complementary nucleic acids.
[0304] If the sample contains the organism from which the nucleic acid was
isolated, specific hybridization of the probe is then detected.
Hybridization may be detected by labeling the probe with a detectable
agent such as a radioactive isotope, a fluorescent dye or an enzyme
capable of catalyzing the formation of a detectable product.
[0305] Many methods for using the labeled probes to detect the presence of
complementary nucleic acids in a sample are familiar to those skilled in
the art. These include Southern Blots, Northern Blots, colony
hybridization procedures and dot blots. Protocols for each of these
procedures are provided in Ausubel et al. Current Protocols in Molecular
Biology, John Wiley 503 Sons, Inc. (1997) and Sambrook et al., Molecular
Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press
(1989.
[0306] Alternatively, more than one probe (at least one of which is
capable of specifically hybridizing to any complementary sequences which
are present in the nucleic acid sample), may be used in an amplification
reaction to determine whether the sample contains an organism containing
a nucleic acid sequence of the invention (e.g., an organism from which
the nucleic acid was isolated). Typically, the probes comprise
oligonucleotides. In one aspect, the amplification reaction may comprise
a PCR reaction. PCR protocols are described in Ausubel and Sambrook,
supra. Alternatively, the amplification may comprise a ligase chain
reaction, 3SR, or strand displacement reaction. (See Barany, F., "The
Ligase Chain Reaction in a PCR World", PCR Methods and Applications
1:5-16, 1991; E. Fahy et al., "Self-sustained Sequence Replication (3SR):
An Isothermal Transcription-based Amplification System Alternative to
PCR", PCR Methods and Applications 1:25-33, 1991; and Walker G. T. et
al., "Strand Displacement Amplification--an Isothermal in vitro DNA
Amplification Technique", Nucleic Acid Research 20:1691-1696, 1992). In
such procedures, the nucleic acids in the ample are contacted with the
probes, the amplification reaction is performed and any resulting
amplification product is detected. The amplification product may be
detected by performing gel electrophoresis on the reaction products and
staining the gel with an intercalator such as ethidium bromide.
Alternatively, one or more of the probes may be labeled with a
radioactive isotope and the presence of a radioactive amplification
product may be detected by autoradiography after gel electrophoresis.
[0307] Probes derived from sequences near the ends of the sequences of the
invention, may also be used in chromosome walking procedures to identify
clones containing genomic sequences located adjacent to the sequences of
the invention. Such methods allow the isolation of genes which encode
additional proteins from the host organism.
[0308] The isolated nucleic acids of the invention, the sequences
complementary thereto, or a fragment comprising at least 10, 15, 20, 25,
30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive bases of
one of the sequences of the invention, or the sequences complementary
thereto may be used as probes to identify and isolate related nucleic
acids. In some aspects, the related nucleic acids may be cDNAs or genomic
DNAs from organisms other than the one from which the nucleic acid was
isolated. For example, the other organisms may be related organisms. In
such procedures, a nucleic acid sample is contacted with the probe under
conditions which permit the probe to specifically hybridize to related
sequences. Hybridization of the probe to nucleic acids from the related
organism is then detected using any of the methods described above.
[0309] By varying the stringency of the hybridization conditions used to
identify nucleic acids, such as cDNAs or genomic DNAs, which hybridize to
the detectable probe, nucleic acids having different levels of homology
to the probe can be identified and isolated. Stringency may be varied by
conducting the hybridization at varying temperatures below the melting
temperatures of the probes. The melting temperature, T.sub.m, is the
temperature (under defined ionic strength and pH) at which 50% of the
target sequence hybridizes to a perfectly complementary probe. Very
stringent conditions are selected to be equal to or about 5.degree. C.
lower than the T.sub.m for a particular probe. The melting temperature of
the probe may be calculated using the following formulas:
[0310] For probes between 14 and 70 nucleotides in length the melting
temperature (T.sub.m) is calculated using the formula:
T.sub.m=81.5+16.6(log [Na+])+0.41(fraction G+C)-(600/N) where N is the
length of the probe.
[0311] If the hybridization is carried out in a solution containing
formamide, the melting temperature may be calculated using the equation:
T.sub.m-81.5+16.6(log [Na+])+0.41(fraction G+C)-(0.63% formamide)-(600/N)
where N is the length of the probe.
[0312] Prehybridization may be carried out in 6.times.SSC,
5.times.Denhardts reagent, 0.5% SDS, 100 .mu.g denatured fragmented
salmon sperm DNA or 6.times.SSC, 5.times.Denhardt's reagent, 0.5% SDS,
100 .mu.g denatured fragmented salmon sperm DNA, 50% formamide. The
formulas for SSC and Denhardt's solutions are listed in Sambrook et al.,
supra.
[0313] Hybridization is conducted by adding the detectable probe to the
prehybridization solutions listed above. Where the probe comprises double
stranded DNA, it is denatured before addition to the hybridization
solution. The filter is contacted with the hybridization solution for a
sufficient period of time to allow the probe to hybridize to cDNAs or
genomic DNAs containing sequences complementary thereto or homologous
thereto. For probes over 200 nucleotides in length, the hybridization may
be carried out at 15-25.degree. C. below the T.sub.m. For shorter probes,
such as oligonucleotide probes, the hybridization may be conducted at
5-10.degree. C. below the T.sub.m. Typically, for hybridizations in
6.times.SSC, the hybridization is conducted at approximately 68.degree.
C. Usually, for hybridizations in 50% formamide containing solutions, the
hybridization is conducted at approximately 42.degree. C.
Inhibiting Expression of Enzymes (Glucanases)
[0314] The invention provides nucleic acids complementary to (e.g.,
antisense sequences to) the nucleic acids of the invention, e.g.,
endoglucanase-, mannanase-, or xylanase-encoding nucleic acids. Antisense
sequences are capable of inhibiting the transport, splicing or
transcription of glucanase-encoding, endoglucanase-, mannanase-, or
xylanase-encoding genes. The inhibition can be effected through the
targeting of genomic DNA or messenger RNA. The transcription or function
of targeted nucleic acid can be inhibited, for example, by hybridization
and/or cleavage. One particularly useful set of inhibitors provided by
the present invention includes oligonucleotides which are able to either
bind glucanase, mannanase, or xylanase gene or message, in either case
preventing or inhibiting the production or function of glucanase,
mannanase, or xylanase. The association can be through sequence specific
hybridization. Another useful class of inhibitors includes
oligonucleotides which cause inactivation or cleavage of glucanase,
mannanase, or xylanase message. The oligonucleotide can have enzyme
activity which causes such cleavage, such as ribozymes. The
oligonucleotide can be chemically modified or conjugated to an enzyme or
composition capable of cleaving the complementary nucleic acid. A pool of
many different such oligonucleotides can be screened for those with the
desired activity. Thus, the invention provides various compositions for
the inhibition of glucanase, mannanase, or xylanase expression on a
nucleic acid and/or protein level, e.g., antisense, iRNA and ribozymes
comprising glucanase, mannanase, or xylanase sequences of the invention
and the anti-glucanase, mannanase, or xylanase antibodies of the
invention.
[0315] Inhibition of glucanase, mannanase, or xylanase expression can have
a variety of industrial applications. For example, inhibition of
glucanase, mannanase, or xylanase expression can slow or prevent
spoilage. Spoilage can occur when polysaccharides, e.g., structural
polysaccharides, are enzymatically degraded. This can lead to the
deterioration, or rot, of fruits and vegetables. In one aspect, use of
compositions of the invention that inhibit the expression and/or activity
of glucanases, mannanase, or xylanase, e.g., antibodies, antisense
oligonucleotides, ribozymes and RNAi, are used to slow or prevent
spoilage. Thus, in one aspect, the invention provides methods and
compositions comprising application onto a plant or plant product (e.g.,
a cereal, a grain, a fruit, seed, root, leaf, etc.) antibodies, antisense
oligonucleotides, ribozymes and RNAi of the invention to slow or prevent
spoilage. These compositions also can be expressed by the plant (e.g., a
transgenic plant) or another organism (e.g., a bacterium or other
microorganism transformed with a glucanase, mannanase, or xylanase gene
of the invention).
[0316] The compositions of the invention for the inhibition of glucanase,
mannanase, or xylanase expression (e.g., antisense, iRNA, ribozymes,
antibodies) can be used as pharmaceutical compositions, e.g., as
anti-pathogen agents or in other therapies, e.g., as anti-microbials for,
e.g., Salmonella.
[0317] Antisense Oligonucleotides
[0318] The invention provides antisense oligonucleotides capable of
binding glucanase, mannanase, or xylanase message or gene which can
inhibit a target gene or message to, e.g., inhibit a glucan hydrolase
activity (e.g., catalyzing hydrolysis of internal .beta.-1,4-xylosidic
linkages) by targeting mRNA. Strategies for designing antisense
oligonucleotides are well described in the scientific and patent
literature, and the skilled artisan can design such glucanase, mannanase,
or xylanase oligonucleotides using the novel reagents of the invention.
For example, gene walking/RNA mapping protocols to screen for effective
antisense oligonucleotides are well known in the art, see, e.g., Ho
(2000) Methods Enzymol. 314:168-183, describing an RNA mapping assay,
which is based on standard molecular techniques to provide an easy and
reliable method for potent antisense sequence selection. See also Smith
(2000) Eur. S. Pharm. Sci. 11:191-198.
[0319] Naturally occurring nucleic acids are used as antisense
oligonucleotides. The antisense oligonucleotides can be of any length;
for example, in alternative aspects, the antisense oligonucleotides are
between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.
The optimal length can be determined by routine screening. The antisense
oligonucleotides can be present at any concentration. The optimal
concentration can be determined by routine screening. A wide variety of
synthetic, non-naturally occurring nucleotide and nucleic acid analogues
are known which can address this potential problem. For example, peptide
nucleic acids (PNAs) containing non-ionic backbones, such as
N-(2-aminoethyl)glycine units can be used. Antisense oligonucleotides
having phosphorothioate linkages can also be used, as described in WO
97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;
Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).
Antisense oligonucleotides having synthetic DNA backbone analogues
provided by the invention can also include phosphoro-dithioate,
methylphosphonate, phosphoranidate, alkyl phosphotriester, sulfamate,
3'-thioacetal, methylene(methylimino), 3'-N-carbamate, and morpholino
carbamate nucleic acids, as described above.
[0320] Combinatorial chemistry methodology can be used to create vast
numbers of oligonucleotides that can be rapidly screened for specific
oligonucleotides that have appropriate binding affinities and
specificities toward any target, such as the sense and antisense
glucanase, mannanase, or xylanase sequences of the invention (see, e.g.,
Gold (1995) J. of Biol. Chem. 270:13581-13584).
[0321] Inhibitory Ribozymes
[0322] The invention provides ribozymes capable of binding glucanase,
mannanase, or xylanase message or genes. These ribozymes can inhibit
glucanase, mannanase, or xylanase activity by, e.g., targeting mRNA.
Strategies for designing ribozymes and selecting the glucanase-,
mannanase-, or xylanase-specific antisense sequence for targeting are
well described in the scientific and patent literature, and the skilled
artisan can design such ribozymes using the novel reagents of the
invention. Ribozymes act by binding to a target RNA through the target
RNA binding portion of a ribozyme which is held in close proximity to an
enzymatic portion of the RNA that cleaves the target RNA. Thus, the
ribozyme recognizes and binds a target RNA through complementary
base-pairing, and once bound to the correct site, acts enzymatically to
cleave and inactivate the target RNA. Cleavage of a target RNA in such a
manner will destroy its ability to direct synthesis of an encoded protein
if the cleavage occurs in the coding sequence. After a ribozyme has bound
and cleaved its RNA target, it can be released from that RNA to bind and
cleave new targets repeatedly.
[0323] In some circumstances, the enzymatic nature of a ribozyme can be
advantageous over other technologies, such as antisense technology (where
a nucleic acid molecule simply binds to a nucleic acid target to block
its transcription, translation or association with another molecule) as
the effective concentration of ribozyme necessary to effect a therapeutic
treatment can be lower than that of an antisense oligonucleotide. This
potential advantage reflects the ability of the ribozyme to act
enzymatically. Thus, a single ribozyme molecule is able to cleave many
molecules of target RNA. In addition, a ribozyme is typically a highly
specific inhibitor, with the specificity of inhibition depending not only
on the base pairing mechanism of binding, but also on the mechanism by
which the molecule inhibits the expression of the RNA to which it binds.
That is, the inhibition is caused by cleavage of the RNA target and so
specificity is defined as the ratio of the rate of cleavage of the
targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage
mechanism is dependent upon factors additional to those involved in base
pairing. Thus, the specificity of action of a ribozyme can be greater
than that of antisense oligonucleotide binding the same RNA site.
[0324] The ribozyme of the invention, e.g., an enzymatic ribozyme RNA
molecule, can be formed in a hammerhead motif, a hairpin motif, as a
hepatitis delta virus motif, a group I intron motif and/or an RNaseP-like
RNA in association with an RNA guide sequence. Examples of hammerhead
motifs are described by, e.g., Rossi (1992) Aids Research and Human
Retroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry 28:4929,
and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis delta virus motif
by Perrotta (1992) Biochemistry 31:16; the RNaseP motif by
Guerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S.
Pat. No. 4,987,071. The recitation of these specific motifs is not
intended to be limiting. Those skilled in the art will recognize that a
ribozyme of the invention, e.g., an enzymatic RNA molecule of this
invention, can have a specific substrate binding site complementary to
one or more of the target gene RNA regions. A ribozyme of the invention
can have a nucleotide sequence within or surrounding that substrate
binding site which imparts an RNA cleaving activity to the molecule.
[0325] RNA Interference (RNAi)
[0326] In one aspect, the invention provides an RNA inhibitory molecule, a
so-called "RNAi" molecule, comprising a glucanase, mannanase, or xylanase
sequence of the invention. The RNAi molecule comprises a double-stranded
RNA (dsRNA) molecule. The RNAi can inhibit expression of a glucanase,
mannanase, or xylanase gene. In one aspect, the RNAi is about 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.
While the invention is not limited by any particular mechanism of action,
the RNAi can enter a cell and cause the degradation of a single-stranded
RNA (ssRNA) of similar or identical sequences, including endogenous
mRNAs. When a cell is exposed to double-stranded RNA (dsRNA), mRNA from
the homologous gene is selectively degraded by a process called RNA
interference (RNAi). A possible basic mechanism behind RNAi is the
breaking of a double-stranded RNA (dsRNA) matching a specific gene
sequence into short pieces called short interfering RNA, which trigger
the degradation of mRNA that matches its sequence. In one aspect, the
RNAi's of the invention are used in gene-silencing therapeutics, see,
e.g., Shuey (2002) Drug Discov. Today 7:1040-1046. In one aspect, the
invention provides methods to selectively degrade RNA using the RNAi's of
the invention. The process may be practiced in vitro, ex vivo or in vivo.
In one aspect, the RNAi molecules of the invention can be used to
generate a loss-of-function mutation in a cell, an organ or an animal.
Methods for making and using RNAi molecules for selectively degrade RNA
are well known in the art, see, e.g., U.S. Pat. Nos. 6,506,559;
6,511,824; 6,515,109; 6,489,127.
Modification of Nucleic Acids
[0327] The invention provides methods of generating variants of the
nucleic acids of the invention, e.g., those encoding a glucanase,
mannanase, or xylanase. These methods can be repeated or used in various
combinations to generate glucanases, mannanases, or xylanases having an
altered or different activity or an altered or different stability from
that of a glucanase, mannanase, or xylanase encoded by the template
nucleic acid. These methods also can be repeated or used in various
combinations, e.g., to generate variations in gene/message expression,
message translation or message stability. In another aspect, the genetic
composition of a cell is altered by, e.g., modification of a homologous
gene ex vivo, followed by its reinsertion into the cell.
[0328] A nucleic acid of the invention can be altered by any means. For
example, random or stochastic methods, or, non-stochastic, or "directed
evolution," methods, see, e.g., U.S. Pat. No. 6,361,974. Methods for
random mutation of genes are well known in the art, see, e.g., U.S. Pat.
No. 5,830,696. For example, mutagens can be used to randomly mutate a
gene. Mutagens include, e.g., ultraviolet light or gamma irradiation, or
a chemical mutagen, e.g., mitomycin, nitrous acid, photoactivated
psoralens, alone or in combination, to induce DNA breaks amenable to
repair by recombination. Other chemical mutagens include, for example,
sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.
Other mutagens are analogues of nucleotide precursors, e.g.,
nitrosoguanidime, 5-bromouracil, 2-aminopurine, or acridine. These agents
can be added to a PCR reaction in place of the nucleotide precursor
thereby mutating the sequence. Intercalating agents such as proflavine,
acriflavine, quinacrine and the like can also be used.
[0329] Any technique in molecular biology can be used, e.g., random PCR
mutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA
89:5467-5471; or, combinatorial multiple cassette mutagenesis, see, e.g.,
Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleic acids,
e.g., genes, can be reassembled after random, or "stochastic,"
fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862; 6,287,861;
5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. In alternative
aspects, modifications, additions or deletions are introduced by
error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,
assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette
mutagenesis, recursive ensemble mutagenesis, exponential ensemble
mutagenesis, site-specific mutagenesis, gene reassembly, Gene Site
Saturation Mutagenesis.TM. (GSSM.TM., synthetic ligation reassembly
(SLR), recombination, recursive sequence recombination,
phosphothioate-modified DNA mutagenesis, uracil-containing template
mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification mutagenesis,
artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid
multimer creation, and/or a combination of these and other methods.
[0330] The following publications describe a variety of recursive
recombination procedures and/or methods which can be incorporated into
the methods of the invention: Stemmer (1999) "Molecular breeding of
viruses for targeting and other clinical properties" Tumor Targeting
4:1-4; Ness (1999) Nature Biotechnology 17:893-896; Chang (1999)
"Evolution of a cytokine using DNA family shuffling" Nature Biotechnology
17:793-797; Minshull (1999) "Protein evolution by molecular breeding"
Current Opinion in Chemical Biology 3:284-290; Christians (1999)
"Directed evolution of thymidine kinase for AZT phosphorylation using DNA
family shuffling" Nature Biotechnology 17:259-264; Crameri (1998) "DNA
shuffling of a family of genes from diverse species accelerates directed
evolution" Nature 391:288-291; Crameri (1997) "Molecular evolution of an
arsenate detoxification pathway by DNA shuffling," Nature Biotechnology
15:436-438; Zhang (1997) "Directed evolution of an effective fucosidase
from a galactosidase by DNA shuffling and screening" Proc. Natl. Acad.
Sci. USA 94:4504-4509; Patten et al. (1997) "Applications of DNA
Shuffling to Pharmaceuticals and Vaccines" Current Opinion in
Biotechnology 8:724-733; Crameri et al. (1996) "Construction and
evolution of antibody-phage libraries by DNA shuffling" Nature Medicine
2:100-103; Gates et al. (1996) "Affinity selective isolation of ligands
from peptide libraries through display on a lac repressor `headpiece
dimer`" Journal of Molecular Biology 255:373-386; Stemmer (1996) "Sexual
PCR and Assembly PCR" In: The Encyclopedia of Molecular Biology. VCH
Publishers, New York. pp. 447-457; Crameri and Stemmer (1995)
"Combinatorial multiple cassette mutagenesis creates all the permutations
of mutant and wildtype cassettes" BioTechniques 18:194-195; Stemmer et
al. (1995) "Single-step assembly of a gene and entire plasmid form large
numbers of oligodeoxyribonucleotides" Gene, 164:49-53; Stemmer (1995)
"The Evolution of Molecular Computation" Science 270: 1510; Stemmer
(1995) "Searching Sequence Space" Bio/Technology 13:549-553; Stemmer
(1994) "Rapid evolution of a protein in vitro by DNA shuffling" Nature
370:389-391; and Stemmer (1994) "DNA shuffling by random fragmentation
and reassembly: In vitro recombination for molecular evolution." Proc,
Natl. Acad. Sci. USA 91:10747-10751.
[0331] Mutational methods of generating diversity include, for example,
site-directed mutagenesis (Ling et al. (1997) "Approaches to DNA
mutagenesis: an overview" Anal Biochem. 254(2): 157-178; Dale et al.
(1996) "Oligonucleotide-directed random mutagenesis using the
phosphorothioate method" Methods Mol. Biol. 57:369-374; Smith (1985) "In
vitro mutagenesis" Ann. Rev. Genet. 19:423-462; Botstein & Shortle (1985)
"Strategies and applications of in vitro mutagenesis" Science
229:1193-1201; Carter (1986) "Site-directed mutagenesis" Biochem. J.
237:1-7; and Kunkel (1987) "The efficiency of oligonucleotide directed
mutagenesis" in Nucleic Acids & Molecular Biology (Eckstein, F. and
Lilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis using
uracil containing templates (Kunkel (1985) "Rapid and efficient
site-specific mutagenesis without phenotypic selection" Proc. Natl. Acad.
Sci. USA 82:488-492; Kunkel et al. (1987) "Rapid and efficient
site-specific mutagenesis without phenotypic selection" Methods in
Enzymol. 154, 367-382; and Bass et al. (1988) "Mutant Trp repressors with
new DNA-binding specificities" Science 242:240-245);
oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500
(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller (1982)
"Oligonucleotide-directed mutagenesis using M13-derived vectors: an
efficient and general procedure for the production of point mutations in
any DNA fragment" Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)
"Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13
vectors" Methods in Enzymol. 100:468-500; and Zoller (1987)
Oligonucleotide-directed mutagenesis: a simple method using two
oligonucleotide primers and a single-stranded DNA template" Methods in
Enzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor
(1985) "The use of phosphorothioate-modified DNA in restriction enzyme
reactions to prepare nicked DNA" Nucl. Acids Res. 13: 8749-8764; Taylor
(1985) "The rapid generation of oligonucleotide-directed mutations at
high frequency using phosphorothioate-modified DNA" Nucl. Acids Res. 13:
8765-8787 (1985); Nakamaye (1986) "Inhibition of restriction endonuclease
Nci I cleavage by phosphorothioate groups and its application to
oligonucleotide-directed mutagenesis" Nucl. Acids Res. 14: 9679-9698;
Sayers (1988) "Y-T Exonucleases in phosphorothioate-based
oligonucleotide-directed mutagenesis" Nucl. Acids Res. 16:791-802; and
Sayers et al. (1988) "Strand specific cleavage of
phosphorothioate-containing DNA by reaction with restriction
endonucleases in the presence of ethidium bromide" Nucl. Acids Res. 16:
803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) "The
gapped duplex DNA approach to oligonucleotide-directed mutation
construction" Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)
Methods in Enzymol. "Oligonucleotide-directed construction of mutations
via gapped duplex DNA" 154:350-367; Kramer (1988) "Improved enzymatic in
vitro reactions in the gapped duplex DNA approach to
oligonucleotide-directed construction of mutations" Nucl. Acids Res. 16:
7207; and Fritz (1988) "Oligonucleotide-directed construction of
mutations: a gapped duplex DNA procedure without enzymatic reactions in
vitro" Nucl. Acids Res. 16: 6987-6999).
[0332] Additional protocols that can be used to practice the invention
include point mismatch repair (Kramer (1984) "Point Mismatch Repair" Cell
38:879-887), mutagenesis using repair-deficient host strains (Carter et
al. (1985) "Improved oligonucleotide site-directed mutagenesis using M13
vectors" Nucl. Acids Res. 13: 4431-4443; and Carter (1987) "Improved
oligonucleotide-directed mutagenesis using M13 vectors" Methods in
Enzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) "Use
of oligonucleotides to generate large deletions" Nucl. Acids Res. 14:
5115), restriction-selection and restriction-selection and
restriction-purification (Wells et al. (1986) "Importance of
hydrogen-bond formation in stabilizing the transition state of
subtilisin" Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis by
total gene synthesis (Nambiar et al. (1984) "Total synthesis and cloning
of a gene coding for the ribonuclease S protein" Science 223: 1299-1301;
Sakamar and Khorana (1988) "Total synthesis and expression of a gene for
the a-subunit of bovine rod outer segment guanine nucleotide-binding
protein (transducin)" Nucl. Acids Res. 14: 6361-6372; Wells et al. (1985)
"Cassette mutagenesis: an efficient method for generation of multiple
mutations at defined sites" Gene 34:315-323; and Grundstrom et al. (1985)
"Oligonucleotide-directed mutagenesis by microscale `shot-gun` gene
synthesis" Nucl. Acids Res. 13: 3305-3316), double-strand break repair
(Mandecki (1986); Arnold (1993) "Protein engineering for unusual
environments" Current Opinion in Biotechnology 4:450-455.
"Oligonucleotide-directed double-strand break repair in plasmids of
Escherichia coli: a method for site-specific mutagenesis" Proc. Natl.
Acad. Sci. USA, 83:7177-7181). Additional details on many of the above
methods can be found in Methods in Enzymology Volume 154, which also
describes useful controls for trouble-shooting problems with various
mutagenesis methods.
[0333] Protocols that can be used to practice the invention are described,
e.g., in U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), "Methods for
In Vitro Recombination;" U.S. Pat. No. 5,811,238 to Stemmer et al. (Sep.
22, 1998) "Methods for Generating Polynucleotides having Desired
Characteristics by Iterative Selection and Recombination;" U.S. Pat. No.
5,830,721 to Stemmer et al. (Nov. 3, 1998), "DNA Mutagenesis by Random
Fragmentation and Reassembly;" U.S. Pat. No. 5,834,252 to Stemmer, et al.
(Nov. 10, 1998) "End-Complementary Polymerase Reaction;" U.S. Pat. No.
5,837,458 to Minshull, et al. (Nov. 17, 1998), "Methods and Compositions
for Cellular and Metabolic Engineering;" WO 95/22625, Stemmer and
Crameri, "Mutagenesis by Random Fragmentation and Reassembly;" WO
96/33207 by Stemmer and Lipschutz "End Complementary Polymerase Chain
Reaction;" WO 97/20078 by Stemmer and Crameri "Methods for Generating
Polynucleotides having Desired Characteristics by Iterative Selection and
Recombination;" WO 97/35966 by Minshull and Stemmer, "Methods and
Compositions for Cellular and Metabolic Engineering;" WO 99/41402 by
Punnonen et al. "Targeting of Genetic Vaccine Vectors;" WO 99/41383 by
Punnonen et al. "Antigen Library Immunization;" WO 99/41369 by Punnonen
et al. "Genetic Vaccine Vector Engineering;" WO 99/41368 by Punnonen et
al. "Optimization of Immunomodulatory Properties of Genetic Vaccines;" EP
752008 by Stemmer and Crameri, "DNA Mutagenesis by Random Fragmentation
and Reassembly;" EP 0932670 by Stemmer "Evolving Cellular DNA Uptake by
Recursive Sequence Recombination;" WO 99/23107 by Stemmer et al.,
"Modification of Virus Tropism and Host Range by Viral Genome Shuffling;"
WO 99/21979 by Apt et al., "Human Papillomavirus Vectors;" WO 98/31837 by
del Cardayre et al. "Evolution of Whole Cells and Organisms by Recursive
Sequence Recombination;" WO 98/27230 by Patten and Stemmer, "Methods and
Compositions for Polypeptide Engineering;" WO 98/27230 by Stemmer et al.,
"Methods for Optimization of Gene Therapy by Recursive Sequence Shuffling
and Selection," WO 00/00632, "Methods for Generating Highly Diverse
Libraries," WO 00/09679, "Methods for Obtaining in Vitro Recombined
Polynucleotide Sequence Banks and Resulting Sequences," WO 98/42832 by
Arnold et al., "Recombination of Polynucleotide Sequences Using Random or
Defined Primers," WO 99/29902 by Arnold et al., "Method for Creating
Polynucleotide and Polypeptide Sequences," WO 98/41653 by Vind, "An in
Vitro Method for Construction of a DNA Library," WO 98/41622 by Borchert
et al., "Method for Constructing a Library Using DNA Shuffling," and WO
98/42727 by Pati and Zarling, "Sequence Alterations using Homologous
Recombination."
[0334] Protocols that can be used to practice the invention (providing
details regarding various diversity generating methods) are described,
e.g., in U.S. patent application Ser. No. 09/407,800, "SHUFFLING OF CODON
ALTERED GENES" by Patten et al. filed Sep. 28, 1999; "EVOLUTION OF WHOLE
CELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION" by del Cardayre
et al., U.S. Pat. No. 6,379,964; "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID
RECOMBINATION" by Crameri et al., U.S. Pat. Nos. 6,319,714; 6,368,861;
6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; "USE OF CODON-VARIED
OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING" by Welch et al., U.S.
Pat. No. 6,436,675; "METHODS FOR MAKING CHARACTER STRINGS,
POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTCS" by
Selifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and, e.g.
"METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES
HAVING DESIRED CHARACTERISTICS" by Selifonov et al., filed Jul. 18, 2000
(U.S. Ser. No. 09/618,579); "METHODS OF POPULATING DATA STRUCTURES FOR
USE IN EVOLUTIONARY SIMULATIONS" by Selifonov and Stemmer, filed Jan. 18,
2000 (PCT/US00/01138); and "SINGLE-STRANDED NUCLEIC ACID
TEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION" by
Affholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and U.S. Pat.
Nos. 6,177,263; 6,153,410.
[0335] Non-stochastic, or "directed evolution," methods include, e.g.,
Gene Site Saturation Mutagenesis.TM. (GSSM.TM.), synthetic ligation
reassembly (SLR), or a combination thereof are used to modify the nucleic
acids of the invention to generate glucanases, mannanases, or xylanases
with new or altered properties (e.g., activity under highly acidic or
alkaline conditions, high or low temperatures, and the like).
Polypeptides encoded by the modified nucleic acids can be screened for an
activity before testing for glucan or other polysaccharide hydrolysis or
other activity. Any testing modality or protocol can be used, e.g., using
a capillary array platform. See, e.g., U.S. Pat. Nos. 6,361,974;
6,280,926; 5,939,250.
[0336] Saturation Mutagenesis, or, GSSM.TM.
[0337] In one aspect, codon primers containing a degenerate N,N,G/T
sequence are used to introduce point mutations into a polynucleotide,
e.g., a glucanase, mannanase, or xylanase or an antibody of the
invention, so as to generate a set of progeny polypeptides in which a
full range of single amino acid substitutions is represented at each
amino acid position, e.g., an amino acid residue in an enzyme active site
(catalytic domains (CDs)) or ligand binding site targeted to be modified.
These oligonucleotides can comprise a contiguous first homologous
sequence, a degenerate N,N,G/T sequence, and, optionally, a second
homologous sequence. The downstream progeny translational products from
the use of such oligonucleotides include all possible amino acid changes
at each amino acid site along the polypeptide, because the degeneracy of
the N,N,G/T sequence includes codons for all 20 amino acids. In one
aspect, one such degenerate oligonucleotide (comprised of, e.g., one
degenerate N,N,G/T cassette) is used for subjecting each original codon
in a parental polynucleotide template to a full range of codon
substitutions. In another aspect, at least two degenerate cassettes are
used--either in the same oligonucleotide or not, for subjecting at least
two original codons in a parental polynucleotide template to a full range
of codon substitutions. For example, more than one N,N,G/T sequence can
be contained in one oligonucleotide to introduce amino acid mutations at
more than one site. This plurality of N,N,G/T sequences can be directly
contiguous, or separated by one or more additional nucleotide
sequence(s). In another aspect, oligonucleotides serviceable for
introducing additions and deletions can be used either alone or in
combination with the codons containing an N,N,G/T sequence, to introduce
any combination or permutation of amino acid additions, deletions, and/or
substitutions.
[0338] In one aspect, simultaneous mutagenesis of two or more contiguous
amino acid positions is done using an oligonucleotide that contains
contiguous N,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In
another aspect, degenerate cassettes having less degeneracy than the
N,N,G/T sequence are used. For example, it may be desirable in some
instances to use (e.g. in an oligonucleotide) a degenerate triplet
sequence comprised of only one N, where said N can be in the first second
or third position of the triplet. Any other bases including any
combinations and permutations thereof can be used in the remaining two
positions of the triplet. Alternatively, it may be desirable in some
instances to use (e.g. in an oligo) a degenerate N,N,N triplet sequence.
[0339] In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)
allows for systematic and easy generation of a full range of possible
natural amino acids (for a total of 20 amino acids) into each and every
amino acid position in a polypeptide (in alternative aspects, the methods
also include generation of less than all possible substitutions per amino
acid residue, or codon, position). For example, for a 100 amino acid
polypeptide, 2000 distinct species (i.e. 20 possible amino acids per
position.times.100 amino acid positions) can be generated. Through the
use of an oligonucleotide or set of oligonucleotides containing a
degenerate N,N,G/T triplet, 32 individual sequences can code for all 20
possible natural amino acids. Thus, in a reaction vessel in which a
parental polynucleotide sequence is subjected to saturation mutagenesis
using at least one such oligonucleotide, there are generated 32 distinct
progeny polynucleotides encoding 20 distinct polypeptides. In contrast,
the use of a non-degenerate oligonucleotide in site-directed mutagenesis
leads to only one progeny polypeptide product per reaction vessel.
Nondegenerate oligonucleotides can optionally be used in combination with
degenerate primers disclosed; for example, nondegenerate oligonucleotides
can be used to generate specific point mutations in a working
polynucleotide. This provides one means to generate specific silent point
mutations, point mutations leading to corresponding amino acid changes,
and point mutations that cause the generation of stop codons and the
corresponding expression of polypeptide fragments.
[0340] In one aspect, each saturation mutagenesis reaction vessel contains
polynucleotides encoding at least 20 progeny polypeptide (e.g.,
glucanases, mannanases, or xylanases) molecules such that all 20 natural
amino acids are represented at the one specific amino acid position
corresponding to the codon position mutagenized in the parental
polynucleotide (other aspects use less than all 20 natural combinations).
The 32-fold degenerate progeny polypeptides generated from each
saturation mutagenesis reaction vessel can be subjected to clonal
amplification (e.g. cloned into a suitable host, e.g., E. coli host,
using, e.g., an expression vector) and subjected to expression screening.
When an individual progeny polypeptide is identified by screening to
display a favorable change in property (when compared to the parental
polypeptide, such as increased glucan hydrolysis activity under alkaline
or acidic conditions), it can be sequenced to identify the
correspondingly favorable amino acid substitution contained therein.
[0341] In one aspect, upon mutagenizing each and every amino acid position
in a parental polypeptide using saturation mutagenesis as disclosed
herein, favorable amino acid changes may be identified at more than one
amino acid position. One or more new progeny molecules can be generated
that contain a combination of all or part of these favorable amino acid
substitutions. For example, if 2 specific favorable amino acid changes
are identified in each of 3 amino acid positions in a polypeptide, the
permutations include 3 possibilities at each position (no change from the
original amino acid, and each of two favorable changes) and 3 positions.
Thus, there are 3.times.3.times.3 or 27 total possibilities, including 7
that were previously examined--6 single point mutations (i.e. 2 at each
of three positions) and no change at any position.
[0342] In yet another aspect, site-saturation mutagenesis can be used
together with shuffling, chimerization, recombination and other
mutagenizing processes, along with screening. This invention provides for
the use of any mutagenizing process(es), including saturation
mutagenesis, in an iterative manner. In one exemplification, the
iterative use of any mutagenizing process(es) is used in combination with
screening.
[0343] The invention also provides for the use of proprietary codon
primers (containing a degenerate N,N,N sequence) to introduce point
mutations into a polynucleotide, so as to generate a set of progeny
polypeptides in which a full range of single amino acid substitutions is
represented at each amino acid position (Gene Site Saturation
Mutagenesis.TM. (GSSM.TM.). The oligos used are comprised contiguously of
a first homologous sequence, a degenerate N,N,N sequence and in one
aspect but not necessarily a second homologous sequence. The downstream
progeny translational products from the use of such oligos include all
possible amino acid changes at each amino acid site along the
polypeptide, because the degeneracy of the N,N,N sequence includes codons
for all 20 amino acids.
[0344] In one aspect, one such degenerate oligo (comprised of one
degenerate N,N,N cassette) is used for subjecting each original codon in
a parental polynucleotide template to a full range of codon
substitutions. In another aspect, at least two degenerate N,N,N cassettes
are used--either in the same oligo or not, for subjecting at least two
original codons in a parental polynucleotide template to a full range of
codon substitutions. Thus, more than one N,N,N sequence can be contained
in one oligo to introduce amino acid mutations at more than one site.
This plurality of N,N,N sequences can be directly contiguous, or
separated by one or more additional nucleotide sequence(s). In another
aspect, oligos serviceable for introducing additions and deletions can be
used either alone or in combination with the codons containing an N,N,N
sequence, to introduce any combination or permutation of amino acid
additions, deletions and/or substitutions.
[0345] In a particular exemplification, it is possible to simultaneously
mutagenize two or more contiguous amino acid positions using an oligo
that contains contiguous N,N,N triplets, i.e. a degenerate (N,N,N).sub.n
sequence.
[0346] In another aspect, the present invention provides for the use of
degenerate cassettes having less degeneracy than the N,N,N sequence. For
example, it may be desirable in some instances to use (e.g. in an oligo)
a degenerate triplet sequence comprised of only one N, where the N can be
in the first second or third position of the triplet. Any other bases
including any combinations and permutations thereof can be used in the
remaining two positions of the triplet. Alternatively, it may be
desirable in some instances to use (e.g., in an oligo) a degenerate N,N,N
triplet sequence, N,N,G/T, or an N,N, G/C triplet sequence.
[0347] It is appreciated, however, that the use of a degenerate triplet
(such as N,N,G/T or an N,N, G/C triplet sequence) as disclosed in the
instant invention is advantageous for several reasons. In one aspect,
this invention provides a means to systematically and fairly easily
generate the substitution of the full range of possible amino acids (for
a total of 20 amino acids) into each and every amino acid position in a
polypeptide. Thus, for a 100 amino acid polypeptide, the invention
provides a way to systematically and fairly easily generate 2000 distinct
species (i.e., 20 possible amino acids per position times 100 amino acid
positions). It is appreciated that there is provided, through the use of
an oligo containing a degenerate N,N,G/T or an N,N,G/C triplet sequence,
32 individual sequences that code for 20 possible amino acids. Thus, in a
reaction vessel in which a parental polynucleotide sequence is subjected
to saturation mutagenesis using one such oligo, there are generated 32
distinct progeny polynucleotides encoding 20 distinct polypeptides. In
contrast, the use of a non-degenerate oligo in site-directed mutagenesis
leads to only one progeny polypeptide product per reaction vessel.
[0348] This invention also provides for the use of nondegenerate oligos,
which can optionally be used in combination with degenerate primers
disclosed. It is appreciated that in some situations, it is advantageous
to use nondegenerate oligos to generate specific point mutations in a
working polynucleotide. This provides a means to generate specific silent
point mutations, point mutations leading to corresponding amino acid
changes and point mutations that cause the generation of stop codons and
the corresponding expression of polypeptide fragments.
[0349] Thus, in one aspect of this invention, each saturation mutagenesis
reaction vessel contains polynucleotides encoding at least 20 progeny
polypeptide molecules such that all 20 amino acids are represented at the
one specific amino acid position corresponding to the codon position
mutagenized in the parental polynucleotide. The 32-fold degenerate
progeny polypeptides generated from each saturation mutagenesis reaction
vessel can be subjected to clonal amplification (e.g., cloned into a
suitable E. coli host using an expression vector) and subjected to
expression screening. When an individual progeny polypeptide is
identified by screening to display a favorable change in property (when
compared to the parental polypeptide), it can be sequenced to identify
the correspondingly favorable amino acid substitution contained therein.
[0350] It is appreciated that upon mutagenizing each and every amino acid
position in a parental polypeptide using saturation mutagenesis as
disclosed herein, favorable amino acid changes may be identified at more
than one amino acid position. One or more new progeny molecules can be
generated that contain a combination of all or part of these favorable
amino acid substitutions. For example, if 2 specific favorable amino acid
changes are identified in each of 3 amino acid positions in a
polypeptide, the permutations include 3 possibilities at each position
(no change from the original amino acid and each of two favorable
changes) and 3 positions. Thus, there are 3.times.3.times.3 or 27 total
possibilities, including 7 that were previously examined--6 single point
mutations (i.e., 2 at each of three positions) and no change at any
position.
[0351] Thus, in a non-limiting exemplification, this invention provides
for the use of saturation mutagenesis in combination with additional
mutagenization processes, such as process where two or more related
polynucleotides are introduced into a suitable host cell such that a
hybrid polynucleotide is generated by recombination and reductive
reassortment.
[0352] In addition to performing mutagenesis along the entire sequence of
a gene, the instant invention provides that mutagenesis can be use to
replace each of any number of bases in a polynucleotide sequence, wherein
the number of bases to be mutagenized is in one aspect every integer from
15 to 100,000. Thus, instead of mutagenizing every position along a
molecule, one can subject every or a discrete number of bases (in one
aspect a subset totaling from 15 to 100,000) to mutagenesis. In one
aspect, a separate nucleotide is used for mutagenizing each position or
group of positions along a polynucleotide sequence. A group of 3
positions to be mutagenized may be a codon. The mutations can be
introduced using a mutagenic primer, containing a heterologous cassette,
also referred to as a mutagenic cassette. Exemplary cassettes can have
from 1 to 500 bases. Each nucleotide position in such heterologous
cassettes be N, A, C, G, T, A/C, A/G, A/T, C/G, C/T, G/T, C/G/T, A/G/T,
A/C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E can
be referred to as a designer oligo).
[0353] In a general sense, saturation mutagenesis is comprised of
mutagenizing a complete set of mutagenic cassettes (wherein each cassette
is in one aspect about 1-500 bases in length) in defined polynucleotide
sequence to be mutagenized (wherein the sequence to be mutagenized is in
one aspect from about 15 to 100,000 bases in length). Thus, a group of
mutations (ranging from 1 to 100 mutations) is introduced into each
cassette to be mutagenized. A grouping of mutations to be introduced into
one cassette can be different or the same from a second grouping of
mutations to be introduced into a second cassette during the application
of one round of saturation mutagenesis. Such groupings are exemplified by
deletions, additions, groupings of particular codons and groupings of
particular nucleotide cassettes.
[0354] Defined sequences to be mutagenized include a whole gene, pathway,
cDNA, an entire open reading frame (ORE) and entire promoter, enhancer,
repressor/transactivator, origin of replication, intron, operator, or any
polynucleotide functional group. Generally, a "defined sequences" for
this purpose may be any polynucleotide that a 15 base-polynucleotide
sequence and polynucleotide sequences of lengths between 15 bases and
15,000 bases (this invention specifically names every integer in
between). Considerations in choosing groupings of codons include types of
amino acids encoded by a degenerate mutagenic cassette.
[0355] In one exemplification a grouping of mutations that can be
introduced into a mutagenic cassette, this invention specifically
provides for degenerate codon substitutions (using degenerate oligos)
that code for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19 and 20 amino acids at each position and a library of polypeptides
encoded thereby.
[0356] Synthetic Ligation Reassembly (SLR)
[0357] The invention provides a non-stochastic gene modification system
termed "synthetic ligation reassembly," or simply "SLR," a "directed
evolution process," to generate polypeptides, e.g., glucanases,
mannanases, or xylanases or antibodies of the invention, with new or
altered properties.
[0358] SLR is a method of ligating oligonucleotide fragments together
non-stochastically. This method differs from stochastic oligonucleotide
shuffling in that the nucleic acid building blocks are not shuffled,
concatenated or chimerized randomly, but rather are assembled
non-stochastically. See, e.g., U.S. patent application Ser. No.
09/332,835 entitled "Synthetic Ligation Reassembly in Directed Evolution"
and filed on Jun. 14, 1999 ("U.S. Ser. No. 09/332,835"). In one aspect,
SLR comprises the following steps: (a) providing a template
polynucleotide, wherein the template polynucleotide comprises sequence
encoding a homologous gene; (b) providing a plurality of building block
polynucleotides, wherein the building block polynucleotides are designed
to cross-over reassemble with the template polynucleotide at a
predetermined sequence, and a building block polynucleotide comprises a
sequence that is a variant of the homologous gene and a sequence
homologous to the template polynucleotide flanking the variant sequence;
(c) combining a building block polynucleotide with a template
polynucleotide such that the building block polynucleotide cross-over
reassembles with the template polynucleotide to generate polynucleotides
comprising homologous gene sequence variations.
[0359] SLR does not depend on the presence of high levels of homology
between polynucleotides to be rearranged. Thus, this method can be used
to non-stochastically generate libraries (or sets) of progeny molecules
comprised of over 10.sup.100 different chimeras. SLR can be used to
generate libraries comprised of over 10.sup.1000 different progeny
chimeras. Thus, aspects of the present invention include non-stochastic
methods of producing a set of finalized chimeric nucleic acid molecule
shaving an overall assembly order that is chosen by design. This method
includes the steps of generating by design a plurality of specific
nucleic acid building blocks having serviceable mutually compatible
ligatable ends, and assembling these nucleic acid building blocks, such
that a designed overall assembly order is achieved.
[0360] The mutually compatible ligatable ends of the nucleic acid building
blocks to be assembled are considered to be "serviceable" for this type
of ordered assembly if they enable the building blocks to be coupled in
predetermined orders. Thus, the overall assembly order in which the
nucleic acid building blocks can be coupled is specified by the design of
the ligatable ends. If more than one assembly step is to be used, then
the overall assembly order in which the nucleic acid building blocks can
be coupled is also specified by the sequential order of the assembly
step(s). In one aspect, the annealed building pieces are treated with an
enzyme, such as a ligase (e.g. T4 DNA ligase), to achieve covalent
bonding of the building pieces.
[0361] In one aspect, the design of the oligonucleotide building blocks is
obtained by analyzing a set of progenitor nucleic acid sequence templates
that serve as a basis for producing a progeny set of finalized chimeric
polynucleotides. These parental oligonucleotide templates thus serve as a
source of sequence information that aids in the design of the nucleic
acid building blocks that are to be mutagenized, e.g., chimerized or
shuffled. In one aspect of this method, the sequences of a plurality of
parental nucleic acid templates are aligned in order to select one or
more demarcation points. The demarcation points can be located at an area
of homology, and are comprised of one or more nucleotides. These
demarcation points are in one aspect shared by at least two of the
progenitor templates. The demarcation points can thereby be used to
delineate the boundaries of oligonucleotide building blocks to be
generated in order to rearrange the parental polynucleotides. The
demarcation points identified and selected in the progenitor molecules
serve as potential chimerization points in the assembly of the final
chimeric progeny molecules. A demarcation point can be an area of
homology (comprised of at least one homologous nucleotide base) shared by
at least two parental polynucleotide sequences. Alternatively, a
demarcation point can be an area of homology that is shared by at least
half of the parental polynucleotide sequences, or, it can be an area of
homology that is shared by at least two thirds of the parental
polynucleotide sequences. Even more in one aspect a serviceable
demarcation points is an area of homology that is shared by at least
three fourths of the parental polynucleotide sequences, or, it can be
shared by at almost all of the parental polynucleotide sequences. In one
aspect, a demarcation point is an area of homology that is shared by all
of the parental polynucleotide sequences.
[0362] In one aspect, a ligation reassembly process is performed
exhaustively in order to generate an exhaustive library of progeny
chimeric polynucleotides. In other words, all possible ordered
combinations of the nucleic acid building blocks are represented in the
set of finalized chimeric nucleic acid molecules. At the same time, in
another aspect, the assembly order (i.e. the order of assembly of each
building block in the 5' to 3 sequence of each finalized chimeric nucleic
acid) in each combination is by design (or non-stochastic) as described
above. Because of the non-stochastic nature of this invention, the
possibility of unwanted side products is greatly reduced.
[0363] In another aspect, the ligation reassembly method is performed
systematically. For example, the method is performed in order to generate
a systematically compartmentalized library of progeny molecules, with
compartments that can be screened systematically, e.g. one by one. In
other words this invention provides that, through the selective and
judicious use of specific nucleic acid building blocks, coupled with the
selective and judicious use of sequentially stepped assembly reactions, a
design can be achieved where specific sets of progeny products are made
in each of several reaction vessels. This allows a systematic examination
and screening procedure to be performed. Thus, these methods allow a
potentially very large number of progeny molecules to be examined
systematically in smaller groups. Because of its ability to perform
chimerizations in a manner that is highly flexible yet exhaustive and
systematic as well, particularly when there is a low level of homology
among the progenitor molecules, these methods provide for the generation
of a library (or set) comprised of a large number of progeny molecules.
Because of the non-stochastic nature of the instant ligation reassembly
invention, the progeny molecules generated in one aspect comprise a
library of finalized chimeric nucleic acid molecules having an overall
assembly order that is chosen by design. The saturation mutagenesis and
optimized directed evolution methods also can be used to generate
different progeny molecular species. It is appreciated that the invention
provides freedom of choice and control regarding the selection of
demarcation points, the size and number of the nucleic acid building
blocks, and the size and design of the couplings. It is appreciated,
furthermore, that the requirement for intermolecular homology is highly
relaxed for the operability of this invention. In fact, demarcation
points can even be chosen in areas of little or no intermolecular
homology. For example, because of codon wobble, i.e. the degeneracy of
codons, nucleotide substitutions can be introduced into nucleic acid
building blocks without altering the amino acid originally encoded in the
corresponding progenitor template. Alternatively, a codon can be altered
such that the coding for an originally amino acid is altered. This
invention provides that such substitutions can be introduced into the
nucleic acid building block in order to increase the incidence of
intermolecular homologous demarcation points and thus to allow an
increased number of couplings to be achieved among the building blocks,
which in turn allows a greater number of progeny chimeric molecules to be
generated.
[0364] In one aspect, the present invention provides a non-stochastic
method termed synthetic gene reassembly, that is somewhat related to
stochastic shuffling, save that the nucleic acid building blocks are not
shuffled or concatenated or chimerized randomly, but rather are assembled
non-stochastically.
[0365] The synthetic gene reassembly method does not depend on the
presence of a high level of homology between polynucleotides to be
shuffled. The invention can be used to non-stochastically generate
libraries (or sets) of progeny molecules comprised of over 10100
different chimeras. Conceivably, synthetic gene reassembly can even be
used to generate libraries comprised of over 10.sup.1000 different
progeny chimeras.
[0366] Thus, in one aspect, the invention provides a non-stochastic method
of producing a set of finalized chimeric nucleic acid molecules having an
overall assembly order that is chosen by design, which method is
comprised of the steps of generating by design a plurality of specific
nucleic acid building blocks having serviceable mutually compatible
ligatable ends and assembling these nucleic acid building blocks, such
that a designed overall assembly order is achieved.
[0367] The mutually compatible ligatable ends of the nucleic acid building
blocks to be assembled are considered to be "serviceable" for this type
of ordered assembly if they enable the building blocks to be coupled in
predetermined orders. Thus, in one aspect, the overall assembly order in
which the nucleic acid building blocks can be coupled is specified by the
design of the ligatable ends and, if more than one assembly step is to be
used, then the overall assembly order in which the nucleic acid building
blocks can be coupled is also specified by the sequential order of the
assembly step(s). In a one aspect of the invention, the annealed building
pieces are treated with an enzyme, such as a ligase (e.g., T4 DNA ligase)
to achieve covalent bonding of the building pieces.
[0368] In a another aspect, the design of nucleic acid building blocks is
obtained upon analysis of the sequences of a set of progenitor nucleic
acid templates that serve as a basis for producing a progeny set of
finalized chimeric nucleic acid molecules. These progenitor nucleic acid
templates thus serve as a source of sequence information that aids in the
design of the nucleic acid building blocks that are to be mutagenized,
i.e. chimerized or shuffled.
[0369] In one exemplification, the invention provides for the
chimerization of a family of related genes and their encoded family of
related products. In a particular exemplification, the encoded products
are enzymes. The glucanases, mannanases, or xylanases of the present
invention can be mutagenized in accordance with the methods described
herein.
[0370] Thus according to one aspect of the invention, the sequences of a
plurality of progenitor nucleic acid templates (e.g., polynucleotides of
the invention) are aligned in order to select one or more demarcation
points, which demarcation points can be located at an area of homology.
The demarcation points can be used to delineate the boundaries of nucleic
acid building blocks to be generated. Thus, the demarcation points
identified and selected in the progenitor molecules serve as potential
chimerization points in the assembly of the progeny molecules.
[0371] Typically a serviceable demarcation point is an area of homology
(comprised of at least one homologous nucleotide base) shared by at least
two progenitor templates, but the demarcation point can be an area of
homology that is shared by at least half of the progenitor templates, at
least two thirds of the progenitor templates, at least three fourths of
the progenitor templates and in one aspect at almost all of the
progenitor templates. Even more in one aspect still a serviceable
demarcation point is an area of homology that is shared by all of the
progenitor templates.
[0372] In a one aspect, the gene reassembly process is performed
exhaustively in order to generate an exhaustive library. In other words,
all possible ordered combinations of the nucleic acid building blocks are
represented in the set of finalized chimeric nucleic acid molecules. At
the same time, the assembly order (i.e. the order of assembly of each
building block in the 5' to 3 sequence of each finalized chimeric nucleic
acid) in each combination is by design (or non-stochastic). Because of
the non-stochastic nature of the method, the possibility of unwanted side
products is greatly reduced.
[0373] In another aspect, the method provides that the gene reassembly
process is performed systematically, for example to generate a
systematically compartmentalized library, with compartments that can be
screened systematically, e.g., one by one. In other words the invention
provides that, through the selective and judicious use of specific
nucleic acid building blocks, coupled with the selective and judicious
use of sequentially stepped assembly reactions, an experimental design
can be achieved where specific sets of progeny products are made in each
of several reaction vessels. This allows a systematic examination and
screening procedure to be performed. Thus, it allows a potentially very
large number of progeny molecules to be examined systematically in
smaller groups.
[0374] Because of its ability to perform chimerizations in a manner that
is highly flexible yet exhaustive and systematic as well, particularly
when there is a low level of homology among the progenitor molecules, the
instant invention provides for the generation of a library (or set)
comprised of a large number of progeny molecules. Because of the
non-stochastic nature of the instant gene reassembly invention, the
progeny molecules generated in one aspect comprise a library of finalized
chimeric nucleic acid molecules having an overall assembly order that is
chosen by design. In a particularly aspect, such a generated library is
comprised of greater than 10.sup.3 to greater than 10.sup.1000 different
progeny molecular species.
[0375] In one aspect, a set of finalized chimeric nucleic acid molecules,
produced as described is comprised of a polynucleotide encoding a
polypeptide. According to one aspect, this polynucleotide is a gene,
which may be a man-made gene. According to another aspect, this
polynucleotide is a gene pathway, which may be a man-made gene pathway.
The invention provides that one or more man-made genes generated by the
invention may be incorporated into a man-made gene pathway, such as
pathway operable in a eukaryotic organism (including a plant).
[0376] In another exemplification, the synthetic nature of the step in
which the building blocks are generated allows the design and
introduction of nucleotides (e.g., one or more nucleotides, which may be,
for example, codons or introns or regulatory sequences) that can later be
optionally removed in an in vitro process (e.g., by mutagenesis) or in an
in vivo process (e.g., by utilizing the gene splicing ability of a host
organism). It is appreciated that in many instances the introduction of
these nucleotides may also be desirable for many other reasons in
addition to the potential benefit of creating a serviceable demarcation
point.
[0377] Thus, according to another aspect, the invention provides that a
nucleic acid building block can be used to introduce an intron. Thus, the
invention provides that functional introns may be introduced into a
man-made gene of the invention. The invention also provides that
functional introns may be introduced into a man-made gene pathway of the
invention. Accordingly, the invention provides for the generation of a
chimeric polynucleotide that is a man-made gene containing one (or more)
artificially introduced intron(s).
[0378] Accordingly, the invention also provides for the generation of a
chimeric polynucleotide that is a man-made gene pathway containing one
(or more) artificially introduced intron(s). In one aspect, the
artificially introduced intron(s) are functional in one or more host
cells for gene splicing much in the way that naturally-occurring introns
serve functionally in gene splicing. The invention provides a process of
producing man-made intron-containing polynucleotides to be introduced
into host organisms for recombination and/or splicing.
[0379] A man-made gene produced using the invention can also serve as a
substrate for recombination with another nucleic acid. Likewise, a
man-made gene pathway produced using the invention can also serve as a
substrate for recombination with another nucleic acid. In one aspect, the
recombination is facilitated by, or occurs at, areas of homology between
the man-made, intron-containing gene and a nucleic acid, which serves as
a recombination partner. In one aspect, the recombination partner may
also be a nucleic acid generated by the invention, including a man-made
gene or a man-made gene pathway. Recombination may be facilitated by or
may occur at areas of homology that exist at the one (or more)
artificially introduced intron(s) in the man-made gene.
[0380] The synthetic gene reassembly method of the invention utilizes a
plurality of nucleic acid building blocks, each of which in one aspect
has two ligatable ends. The two ligatable ends on each nucleic acid
building block may be two blunt ends (i.e. each having an overhang of
zero nucleotides), or in one aspect one blunt end and one overhang, or
more in one aspect still two overhangs.
[0381] A useful overhang for this purpose may be a 3' overhang or a 5'
overhang. Thus, a nucleic acid building block may have a 3' overhang or
alternatively a 5' overhang or alternatively two 3' overhangs or
alternatively two 5' overhangs. The overall order in which the nucleic
acid building blocks are assembled to form a finalized chimeric nucleic
acid molecule is determined by purposeful experimental design and is not
random.
[0382] In one aspect, a nucleic acid building block is generated by
chemical synthesis of two single-stranded nucleic acids (also referred to
as single-stranded oligos) and contacting them so as to allow them to
anneal to form a double-stranded nucleic acid building block.
[0383] A double-stranded nucleic acid building block can be of variable
size. The sizes of these building blocks can be small or large. Exemplary
sizes for building block range from 1 base pair (not including any
overhangs) to 100,000 base pairs (not including any overhangs). Other
exemplary size ranges are also provided, which have lower limits of from
1 bp to 10,000 bp (including every integer value in between) and upper
limits of from 2 bp to 100,000 bp (including every integer value in
between).
[0384] Many methods exist by which a double-stranded nucleic acid building
block can be generated that is serviceable for the invention; and these
are known in the art and can be readily performed by the skilled artisan.
[0385] According to one aspect, a double-stranded nucleic acid building
block is generated by first generating two single stranded nucleic acids
and allowing them to anneal to form a double-stranded nucleic acid
building block. The two strands of a double-stranded nucleic acid
building block may be complementary at every nucleotide apart from any
that form an overhang; thus containing no mismatches, apart from any
overhang(s). According to another aspect, the two strands of a
double-stranded nucleic acid building block are complementary at fewer
than every nucleotide apart from any that form an overhang. Thus,
according to this aspect, a double-stranded nucleic acid building block
can be used to introduce codon degeneracy. In one aspect the codon
degeneracy is introduced using the site-saturation mutagenesis described
herein, using one or more N,N,G/T cassettes or alternatively using one or
more N,N,N cassettes.
[0386] The in vivo recombination method of the invention can be performed
blindly on a pool of unknown hybrids or alleles of a specific
polynucleotide or sequence. However, it is not necessary to know the
actual DNA or RNA sequence of the specific polynucleotide.
[0387] The approach of using recombination within a mixed population of
genes can be useful for the generation of any useful proteins, for
example, interleukin I, antibodies, tPA and growth hormone. This approach
may be used to generate proteins having altered specificity or activity.
The approach may also be useful for the generation of hybrid nucleic acid
sequences, for example, promoter regions, introns, exons, enhancer
sequences, 31 untranslated regions or 51 untranslated regions of genes.
Thus this approach may be used to generate genes having increased rates
of expression. This approach may also be useful in the study of
repetitive DNA sequences. Finally, this approach may be useful to mutate
ribozymes or aptamers.
[0388] In one aspect the invention described herein is directed to the use
of repeated cycles of reductive reassortment, recombination and selection
which allow for the directed molecular evolution of highly complex linear
sequences, such as DNA, RNA or proteins thorough recombination.
[0389] Optimized Directed Evolution System
[0390] The invention provides a non-stochastic gene modification system
termed "optimized directed evolution system" to generate polypeptides,
e.g., glucanases, mannanases, or xylanases or antibodies of the
invention, with new or altered properties. Optimized directed evolution
is directed to the use of repeated cycles of reductive reassortment,
recombination and selection that allow for the directed molecular
evolution of nucleic acids through recombination. Optimized directed
evolution allows generation of a large population of evolved chimeric
sequences, wherein the generated population is significantly enriched for
sequences that have a predetermined number of crossover events.
[0391] A crossover event is a point in a chimeric sequence where a shift
in sequence occurs from one parental variant to another parental variant.
Such a point is normally at the juncture of where oligonucleotides from
two parents are ligated together to form a single sequence. This method
allows calculation of the correct concentrations of oligonucleotide
sequences so that the final chimeric population of sequences is enriched
for the chosen number of crossover events. This provides more control
over choosing chimeric variants having a predetermined number of
crossover events.
[0392] In addition, this method provides a convenient means for exploring
a tremendous amount of the possible protein variant space in comparison
to other systems. Previously, if one generated, for example, 10.sup.13
chimeric molecules during a reaction, it would be extremely difficult to
test such a high number of chimeric variants for a particular activity.
Moreover, a significant portion of the progeny population would have a
very high number of crossover events which resulted in proteins that were
less likely to have increased levels of a particular activity. By using
these methods, the population of chimerics molecules can be enriched for
those variants that have a particular number of crossover events. Thus,
although one can still generate 10.sup.13 chimeric molecules during a
reaction, each of the molecules chosen for further analysis most likely
has, for example, only three crossover events. Because the resulting
progeny population can be skewed to have a predetermined number of
crossover events, the boundaries on the functional variety between the
chimeric molecules is reduced. This provides a more manageable number of
variables when calculating which oligonucleotide from the original
parental polynucleotides might be responsible for affecting a particular
trait.
[0393] One method for creating a chimeric progeny polynucleotide sequence
is to create oligonucleotides corresponding to fragments or portions of
each parental sequence. Each oligonucleotide in one aspect includes a
unique region of overlap so that mixing the oligonucleotides together
results in a new variant that has each oligonucleotide fragment assembled
in the correct order. Additional information can also be found, e.g., in
U.S. Ser. No. 09/332,835; U.S. Pat. No. 6,361,974.
[0394] The number of oligonucleotides generated for each parental variant
bears a relationship to the total number of resulting crossovers in the
chimeric molecule that is ultimately created. For example, three parental
nucleotide sequence variants might be provided to undergo a ligation
reaction in order to find a chimeric variant having, for example, greater
activity at high temperature. As one example, a set of 50 oligonucleotide
sequences can be generated corresponding to each portions of each
parental variant. Accordingly, during the ligation reassembly process
there could be up to 50 crossover events within each of the chimeric
sequences. The probability that each of the generated chimeric
polynucleotides will contain oligonucleotides from each parental variant
in alternating order is very low. If each oligonucleotide fragment is
present in the ligation reaction in the same molar quantity it is likely
that in some positions oligonucleotides from the same parental
polynucleotide will ligate next to one another and thus not result in a
crossover event. If the concentration of each oligonucleotide from each
parent is kept constant during any ligation step in this example, there
is a 1/3 chance (assuming 3 parents) that an oligonucleotide from the
same parental variant will ligate within the chimeric sequence and
produce no crossover.
[0395] Accordingly, a probability density function (PDF) can be determined
to predict the population of crossover events that are likely to occur
during each step in a ligation reaction given a set number of parental
variants, a number of oligonucleotides corresponding to each variant, and
the concentrations of each variant during each step in the ligation
reaction. The statistics and mathematics behind determining the PDF is
described below. By utilizing these methods, one can calculate such a
probability density function, and thus enrich the chimeric progeny
population for a predetermined number of crossover events resulting from
a particular ligation reaction. Moreover, a target number of crossover
events can be predetermined, and the system then programmed to calculate
the starting quantities of each parental oligonucleotide during each step
in the ligation reaction to result in a probability density function that
centers on the predetermined number of crossover events. These methods
are directed to the use of repeated cycles of reductive reassortment,
recombination and selection that allow for the directed molecular
evolution of a nucleic acid encoding a polypeptide through recombination.
This system allows generation of a large population of evolved chimeric
sequences, wherein the generated population is significantly enriched for
sequences that have a predetermined number of crossover events. A
crossover event is a point in a chimeric sequence where a shift in
sequence occurs from one parental variant to another parental variant.
Such a point is normally at the juncture of where oligonucleotides from
two parents are ligated together to form a single sequence. The method
allows calculation of the correct concentrations of oligonucleotide
sequences so that the final chimeric population of sequences is enriched
for the chosen number of crossover events. This provides more control
over choosing chimeric variants having a predetermined number of
crossover events.
[0396] In addition, these methods provide a convenient means for exploring
a tremendous amount of the possible protein variant space in comparison
to other systems. By using the methods described herein, the population
of chimerics molecules can be enriched for those variants that have a
particular number of crossover events. Thus, although one can still
generate 10.sup.13 chimeric molecules during a reaction, each of the
molecules chosen for further analysis most likely has, for example, only
three crossover events. Because the resulting progeny population can be
skewed to have a predetermined number of crossover events, the boundaries
on the functional variety between the chimeric molecules is reduced. This
provides a more manageable number of variables when calculating which
oligonucleotide from the original parental polynucleotides might be
responsible for affecting a particular trait.
[0397] In one aspect, the method creates a chimeric progeny polynucleotide
sequence by creating oligonucleotides corresponding to fragments or
portions of each parental sequence. Each oligonucleotide in one aspect
includes a unique region of overlap so that mixing the oligonucleotides
together results in a new variant that has each oligonucleotide fragment
assembled in the correct order. See also U.S. Ser. No. 09/332,835.
[0398] Determining Crossover Events
[0399] Aspects of the invention include a system and software that receive
a desired crossover probability density function (PDF), the number of
parent genes to be reassembled, and the number of fragments in the
reassembly as inputs. The output of this program is a "fragment PDF" that
can be used to determine a recipe for producing reassembled genes, and
the estimated crossover PDF of those genes. The processing described
herein is in one aspect performed in MATLAB.TM. (The Mathworks, Natick,
Mass.) a programming language and development environment for technical
computing.
[0400] Iterative Processes
[0401] In practicing the invention, these processes can be iteratively
repeated. For example, a nucleic acid (or, the nucleic acid) responsible
for an altered or new glucanase, mannanase, or xylanase phenotype is
identified, re-isolated, again modified, re-tested for activity. This
process can be iteratively repeated until a desired phenotype is
engineered. For example, an entire biochemical anabolic or catabolic
pathway can be engineered into a cell, including, e.g., glucanase,
mannanase, or xylanase activity.
[0402] Similarly, if it is determined that a particular oligonucleotide
has no affect at all on the desired trait (e.g., a new glucanase,
mannanase, or xylanase phenotype), it can be removed as a variable by
synthesizing larger parental oligonucleotides that include the sequence
to be removed. Since incorporating the sequence within a larger sequence
prevents any crossover events, there will no longer be any variation of
this sequence in the progeny polynucleotides. This iterative practice of
determining which oligonucleotides are most related to the desired trait,
and which are unrelated, allows more efficient exploration all of the
possible protein variants that might be provide a particular trait or
activity.
[0403] In Vivo Shuffling
[0404] In vivo shuffling of molecules is use in methods of the invention
that provide variants of polypeptides of the invention, e.g., antibodies,
glucanases, mannanases, or xylanases and the like. In vivo shuffling can
be performed utilizing the natural property of cells to recombine
multimers. While recombination in vivo has provided the major natural
route to molecular diversity, genetic recombination remains a relatively
complex process that involves 1) the recognition of homologies; 2) strand
cleavage, strand invasion, and metabolic steps leading to the production
of recombinant chiasma; and finally 3) the resolution of chiasma into
discrete recombined molecules. The formation of the chiasma requires the
recognition of homologous sequences.
[0405] In another aspect, the invention includes a method for producing a
hybrid polynucleotide from at least a first polynucleotide and a second
polynucleotide. The invention can be used to produce a hybrid
polynucleotide by introducing at least a first polynucleotide and a
second polynucleotide which share at least one region of partial sequence
homology (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,
61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,
97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,
127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,
155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,
183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,
211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,
239, 241, 243, 245, 247, 249, 251, 253, 255, 257 and combinations
thereof) into a suitable host cell. The regions of partial sequence
homology promote processes which result in sequence reorganization
producing a hybrid polynucleotide. The term "hybrid polynucleotide", as
used herein, is any nucleotide sequence which results from the method of
the present invention and contains sequence from at least two original
polynucleotide sequences. Such hybrid polynucleotides can result from
intermolecular recombination events which promote sequence integration
between DNA molecules. In addition, such hybrid polynucleotides can
result from intramolecular reductive reassortment processes which utilize
repeated sequences to alter a nucleotide sequence within a DNA molecule.
[0406] In vivo reassortment is focused on "inter-molecular" processes
collectively referred to as "recombination" which in bacteria, is
generally viewed as a "RecA-dependent" phenomenon. The invention can rely
on recombination processes of a host cell to recombine and re-assort
sequences, or the cells' ability to mediate reductive processes to
decrease the complexity of quasi-repeated sequences in the cell by
deletion. This process of "reductive reassortment" occurs by an
"intra-molecular", RecA-independent process.
[0407] Therefore, in another aspect of the invention, novel
polynucleotides can be generated by the process of reductive
reassortment. The method involves the generation of constructs containing
consecutive sequences (original encoding sequences), their insertion into
an appropriate vector and their subsequent introduction into an
appropriate host cell. The reassortment of the individual molecular
identities occurs by combinatorial processes between the consecutive
sequences in the construct possessing regions of homology, or between
quasi-repeated units. The reassortment process recombines and/or reduces
the complexity and extent of the repeated sequences and results in the
production of novel molecular species. Various treatments may be applied
to enhance the rate of reassortment. These could include treatment with
ultra-violet light, or DNA damaging chemicals and/or the use of host cell
lines displaying enhanced levels of "genetic instability". Thus the
reassortment process may involve homologous recombination or the natural
property of quasi-repeated sequences to direct their own evolution.
[0408] Repeated or "quasi-repeated" sequences play a role in genetic
instability. In the present invention, "quasi-repeats" are repeats that
are not restricted to their original unit structure. Quasi-repeated units
can be presented as an array of sequences in a construct; consecutive
units of similar sequences. Once ligated, the junctions between the
consecutive sequences become essentially invisible and the
quasi-repetitive nature of the resulting construct is now continuous at
the molecular level. The deletion process the cell performs to reduce the
complexity of the resulting construct operates between the quasi-repeated
sequences. The quasi-repeated units provide a practically limitless
repertoire of templates upon which slippage events can occur. The
constructs containing the quasi-repeats thus effectively provide
sufficient molecular elasticity that deletion (and potentially insertion)
events can occur virtually anywhere within the quasi-repetitive units.
[0409] When the quasi-repeated sequences are all ligated in the same
orientation, for instance head to tail or vice versa, the cell cannot
distinguish individual units. Consequently, the reductive process can
occur throughout the sequences. In contrast, when for example, the units
are presented head to head, rather than head to tail, the inversion
delineates the endpoints of the adjacent unit so that deletion formation
will favor the loss of discrete units. Thus, it is preferable with the
present method that the sequences are in the same orientation. Random
orientation of quasi-repeated sequences will result in the loss of
reassortment efficiency, while consistent orientation of the sequences
will offer the highest efficiency. However, while having fewer of the
contiguous sequences in the same orientation decreases the efficiency, it
may still provide sufficient elasticity for the effective recovery of
novel molecules. Constructs can be made with the quasi-repeated sequences
in the same orientation to allow higher efficiency.
[0410] Sequences can be assembled in a head to tail orientation using any
of a variety of methods, including the following: [0411] a) Primers
that include a poly-A head and poly-T tail which when made
single-stranded would provide orientation can be utilized. This is
accomplished by having the first few bases of the primers made from RNA
and hence easily removed RNaseH. [0412] b) Primers that include unique
restriction cleavage sites can be utilized. Multiple sites, a battery of
unique sequences and repeated synthesis and ligation steps would be
required. [0413] c) The inner few bases of the primer could be thiolated
and an exonuclease used to produce properly tailed molecules.
[0414] The recovery of the re-assorted sequences relies on the
identification of cloning vectors with a reduced repetitive index (RI).
The re-assorted encoding sequences can then be recovered by
amplification. The products are re-cloned and expressed. The recovery of
cloning vectors with reduced RI can be affected by: [0415] 1) The use of
vectors only stably maintained when the construct is reduced in
complexity. [0416] 2) The physical recovery of shortened vectors by
physical procedures. In this case, the cloning vector would be recovered
using standard plasmid isolation procedures and size fractionated on
either an agarose gel, or column with a low molecular weight cut off
utilizing standard procedures. [0417] 3) The recovery of vectors
containing interrupted genes which can be selected when insert size
decreases. [0418] 4) The use of direct selection techniques with an
expression vector and the appropriate selection.
[0419] Encoding sequences (for example, genes) from related organisms may
demonstrate a high degree of homology and encode quite diverse protein
products. These types of sequences are particularly useful in the present
invention as quasi-repeats. However, while the examples illustrated below
demonstrate the reassortment of nearly identical original encoding
sequences (quasi-repeats), this process is not limited to such nearly
identical repeats.
[0420] The following example demonstrates a method of the invention.
Encoding nucleic acid sequences (quasi-repeats) derived from three (3)
unique species are described. Each sequence encodes a protein with a
distinct set of properties. Each of the sequences differs by a single or
a few base pairs at a unique position in the sequence. The quasi-repeated
sequences are separately or collectively amplified and ligated into
random assemblies such that all possible permutations and combinations
are available in the population of ligated molecules. The number of
quasi-repeat units can be controlled by the assembly conditions. The
average number of quasi-repeated units in a construct is defined as the
repetitive index (RI).
[0421] Once formed, the constructs may, or may not be size fractionated on
an agarose gel according to published protocols, inserted into a cloning
vector and transfected into an appropriate host cell. The cells are then
propagated and "reductive reassortment" is effected. The rate of the
reductive reassortment process may be stimulated by the introduction of
DNA damage if desired. Whether the reduction in RI is mediated by
deletion formation between repeated sequences by an "intra-molecular"
mechanism, or mediated by recombination-like events through
"inter-molecular" mechanisms is immaterial. The end result is a
reassortment of the molecules into all possible combinations.
[0422] Optionally, the method comprises the additional step of screening
the library members of the shuffled pool to identify individual shuffled
library members having the ability to bind or otherwise interact, or
catalyze a particular reaction (e.g., such as catalytic domain of an
enzyme) with a predetermined macromolecule, such as for example a
proteinaceous receptor, an oligosaccharide, virion, or other
predetermined compound or structure.
[0423] The polypeptides that are identified from such libraries can be
used for therapeutic, diagnostic, research and related purposes (e.g.,
catalysts, solutes for increasing osmolarity of an aqueous solution and
the like) and/or can be subjected to one or more additional cycles of
shuffling and/or selection.
[0424] In another aspect, it is envisioned that prior to or during
recombination or reassortment, polynucleotides generated by the method of
the invention can be subjected to agents or processes which promote the
introduction of mutations into the original polynucleotides. The
introduction of such mutations would increase the diversity of resulting
hybrid polynucleotides and polypeptides encoded therefrom. The agents or
processes which promote mutagenesis can include, but are not limited to:
(+)-CC-1065, or a synthetic analog such as (+)-CC-1065-(N-3-Adenine (See
Sun and Hurley, (1992); an N-acetylated or deacetylated
4'-fluoro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis
(See, for example, van de Poll et al. (1992)); or a N-acetylated or
deacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis
(See also, van de Poll et al. (1992), pp. 751-758); trivalent chromium, a
trivalent chromium salt, a polycyclic aromatic hydrocarbon (PAH) DNA
adduct capable of inhibiting DNA replication, such as
7-bromomethyl-benz[a]anthracene ("BMA"), tris(2,3-dibromopropyl)phosphate
("Tris-BP"), 1,2-dibromo-3-chloropropane ("DBCP"), 2-bromoacrolein (2BA),
benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide ("BPDE"), a platinum(II)
halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline
("N-hydroxy-IQ") and
N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine
("N-hydroxy-PhIP"). Exemplary means for slowing or halting PCR
amplification consist of UV light (+)-CC-1065 and
(+)-CC-1065-(N-3-Adenine). Particularly encompassed means are DNA adducts
or polynucleotides comprising the DNA adducts from the polynucleotides or
polynucleotides pool, which can be released or removed by a process
including heating the solution comprising the polynucleotides prior to
further processing.
[0425] In another aspect the invention is directed to a method of
producing recombinant proteins having biological activity by treating a
sample comprising double-stranded template polynucleotides encoding a
wild-type protein under conditions according to the invention which
provide for the production of hybrid or re-assorted polynucleotides.
[0426] Producing Sequence Variants
[0427] The invention also provides additional methods for making sequence
variants of the nucleic acid (e.g., glucanase, mannanase, or xylanase)
sequences of the invention. The invention also provides additional
methods for isolating glucanases, mannanases, or xylanases using the
nucleic acids and polypeptides of the invention. In one aspect, the
invention provides for variants of a glucanase, mannanase, or xylanase
coding sequence (e.g., a gene, cDNA or message) of the invention, which
can be altered by any means, including, e.g., random or stochastic
methods, or, non-stochastic, or "directed evolution," methods, as
described above.
[0428] The isolated variants may be naturally occurring. Variant can also
be created in vitro. Variants may be created using genetic engineering
techniques such as site directed mutagenesis, random chemical
mutagenesis, Exonuclease III deletion procedures, and standard cloning
techniques. Alternatively, such variants, fragments, analogs, or
derivatives may be created using chemical synthesis or modification
procedures. Other methods of making variants are also familiar to those
skilled in the art. These include procedures in which nucleic acid
sequences obtained from natural isolates are modified to generate nucleic
acids which encode polypeptides having characteristics which enhance
their value in industrial or laboratory applications. In such procedures,
a large number of variant sequences having one or more nucleotide
differences with respect to the sequence obtained from the natural
isolate are generated and characterized. These nucleotide differences can
result in amino acid changes with respect to the polypeptides encoded by
the nucleic acids from the natural isolates.
[0429] For example, variants may be created using error prone PCR. In
error prone PCR, PCR is performed under conditions where the copying
fidelity of the DNA polymerase is low, such that a high rate of point
mutations is obtained along the entire length of the PCR product Error
prone PCR is described, e.g., in Leung, D. W., et al., Technique,
1:11-15, 1989) and Caldwell, R. C. & Joyce G. F., PCR Methods Applic.,
2:28-33, 1992. Briefly, in such procedures, nucleic acids to be
mutagenized are mixed with PCR primers, reaction buffer, MgCl.sub.2,
MnCl.sub.2, Taq polymerase and an appropriate concentration of dNTPs for
achieving a high rate of point mutation along the entire length of the
PCR product. For example, the reaction may be performed using 20 fmoles
of nucleic acid to be mutagenized, 30 pmole of each PCR primer, a
reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01%
gelatin, 7 mM MgCl.sub.2, 0.5 mM MnCl.sub.2, 5 units of Taq polymerase,
0.2 mM dGTP, 0.2 mM dTP, 1 mM dCTP, and 1 mM dTTP. PCR may be performed
for 30 cycles of 94.degree. C. for 1 min, 45.degree. C. for 1 min, and
72.degree. C. for 1 min. However, it will be appreciated that these
parameters may be varied as appropriate. The mutagenized nucleic acids
are cloned into an appropriate vector and the activities of the
polypeptides encoded by the mutagenized nucleic acids are evaluated.
[0430] Variants may also be created using oligonucleotide directed
mutagenesis to generate site-specific mutations in any cloned DNA of
interest. Oligonucleotide mutagenesis is described, e.g., in
Reidhaar-Olson (1988) Science 241:53-57. Briefly, in such procedures a
plurality of double stranded oligonucleotides bearing one or more
mutations to be introduced into the cloned DNA are synthesized and
inserted into the cloned DNA to be mutagenized. Clones containing the
mutagenized DNA are recovered and the activities of the polypeptides they
encode are assessed.
[0431] Another method for generating variants is assembly PCR. Assembly
PCR involves the assembly of a PCR product from a mixture of small DNA
fragments. A large number of different PCR reactions occur in parallel in
the same vial, with the products of one reaction priming the products of
another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.
5,965,408.
[0432] Still another method of generating variants is sexual PCR
mutagenesis. In sexual PCR mutagenesis, forced homologous recombination
occurs between DNA molecules of different but highly related DNA sequence
in vitro, as a result of random fragmentation of the DNA molecule based
on sequence homology, followed by fixation of the crossover by primer
extension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,
in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, in
such procedures a plurality of nucleic acids to be recombined are
digested with DNase to generate fragments having an average size of
50-200 nucleotides. Fragments of the desired average size are purified
and resuspended in a PCR mixture. PCR is conducted under conditions which
facilitate recombination between the nucleic acid fragments. For example,
PCR may be performed by resuspending the purified fragments at a
concentration of 10-30 ng/l in a solution of 0.2 mM of each dNTP, 2.2 mM
MgCl.sub.2, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5
units of Taq polymerase per 100:1 of reaction mixture is added and PCR is
performed using the following regime: 94.degree. C. for 60 seconds,
94.degree. C. for 30 seconds, 50-55.degree. C. for 30 seconds, 72.degree.
C. for 30 seconds (3045 times) and 72.degree. C. for 5 minutes. However,
it will be appreciated that these parameters may be varied as
appropriate. In some aspects, oligonucleotides may be included in the PCR
reactions. In other aspects, the Klenow fragment of DNA polymerase I may
be used in a first set of PCR reactions and Taq polymerase may be used in
a subsequent set of PCR reactions. Recombinant sequences are isolated and
the activities of the polypeptides they encode are assessed.
[0433] Variants may also be created by in vivo mutagenesis. In some
aspects, random mutations in a sequence of interest are generated by
propagating the sequence of interest in a bacterial strain, such as an E.
coli strain, which carries mutations in one or more of the DNA repair
pathways. Such "mutator" strains have a higher random mutation rate than
that of a wild-type parent. Propagating the DNA in one of these strains
will eventually generate random mutations within the DNA. Mutator strains
suitable for use for in vivo mutagenesis are described in PCT Publication
No. WO 91/16427, published Oct. 31, 1991, entitled "Methods for Phenotype
Creation from Multiple Gene Populations".
[0434] Variants may also be generated using cassette mutagenesis. In
cassette mutagenesis a small region of a double stranded DNA molecule is
replaced with a synthetic oligonucleotide "cassette" that differs from
the native sequence. The oligonucleotide often contains completely and/or
partially randomized native sequence.
[0435] Recursive ensemble mutagenesis may also be used to generate
variants. Recursive ensemble mutagenesis is an algorithm for protein
engineering (protein mutagenesis) developed to produce diverse
populations of phenotypically related mutants whose members differ in
amino acid sequence. This method uses a feedback mechanism to control
successive rounds of combinatorial cassette mutagenesis. Recursive
ensemble mutagenesis is described in Arlin, A. P. and Youvan, D. C.,
PNAS, USA, 89:7811-7815, 1992.
[0436] In some aspects, variants are created using exponential ensemble
mutagenesis. Exponential ensemble mutagenesis is a process for generating
combinatorial libraries with a high percentage of unique and functional
mutants, wherein small groups of residues are randomized in parallel to
identify, at each altered position, amino acids which lead to functional
proteins. Exponential ensemble mutagenesis is described in Delegrave, S.
and Youvan, D. C., Biotechnology Research, 11:1548-1552, 1993. Random and
site-directed mutagenesis are described in Arnold, F. H., Current Opinion
in Biotechnology, 4:450-455, 1993.
[0437] In some aspects, the variants are created using shuffling
procedures wherein portions of a plurality of nucleic acids which encode
distinct polypeptides are fused together to create chimeric nucleic acid
sequences which encode chimeric polypeptides as described in U.S. Pat.
No. 5,965,408, filed Jul. 9, 1996, entitled, "Method of DNA Reassembly by
Interrupting Synthesis" and U.S. Pat. No. 5,939,250, filed May 22, 1996,
entitled, "Production of Enzymes Having Desired Activities by
Mutagenesis.
[0438] The variants of the polypeptides of the invention may be variants
in which one or more of the amino acid residues of the polypeptides of
the sequences of the invention are substituted with a conserved or
non-conserved amino acid residue (in one aspect a conserved amino acid
residue) and such substituted amino acid residue may or may not be one
encoded by the genetic code.
[0439] The invention provides alternative embodiments of the polypeptides
of the invention (and the nucleic acids that encode them) comprising at
least one conservative amino acid substitution, as discussed herein
(e.g., conservative amino acid substitutions are those that substitute a
given amino acid in a polypeptide by another amino acid of like
characteristics). The invention provides polypeptides (and the nucleic
acids that encode them) wherein any, some or all amino acids residues are
substituted by another amino acid of like characteristics, e.g., a
conservative amino acid substitution.
[0440] Conservative substitutions are those that substitute a given amino
acid in a polypeptide by another amino acid of like characteristics.
Typically seen as conservative substitutions are the following
replacements: replacements of an aliphatic amino acid such as Alanine,
Valine, Leucine and Isoleucine with another aliphatic amino acid;
replacement of a Serine with a Threonine or vice versa; replacement of an
acidic residue such as Aspartic acid and Glutamic acid with another
acidic residue; replacement of a residue beating an amide group, such as
Asparagine and Glutamine, with another residue bearing an amide group;
exchange of a basic residue such as Lysine and Arginine with another
basic residue; and replacement of an aromatic residue such as
Phenylalanine, Tyrosine with another aromatic residue. In alternative
aspects, these conservative substitutions can also be synthetic
equivalents of these amino acids.
[0441] Other variants are those in which one or more of the amino acid
residues of a polypeptide of the invention includes a substituent group.
[0442] Still other variants are those in which the polypeptide is
associated with another compound, such as a compound to increase the
half-life of the polypeptide (for example, polyethylene glycol).
[0443] Additional variants are those in which additional amino acids are
fused to the polypeptide, such as a leader sequence, a secretory
sequence, a proprotein sequence or a sequence which facilitates
purification, enrichment, or stabilization of the polypeptide.
[0444] In some aspects, the fragments, derivatives and analogs retain the
same biological function or activity as the polypeptides of the
invention. In other aspects, the fragment, derivative, or analog includes
a proprotein, such that the fragment, derivative, or analog can be
activated by cleavage of the proprotein portion to produce an active
polypeptide.
[0445] Optimizing Codons to Achieve High Levels of Protein Expression in
Host Cells
[0446] The invention provides methods for modifying glucanase-,
mannanase-, or xylanase-encoding nucleic acids to modify codon usage. In
one aspect, the invention provides methods for modifying codons in a
nucleic acid encoding a glucanase to increase or decrease its expression
in a host cell. The invention also provides nucleic acids encoding a
glucanase, mannanase, or xylanase modified to increase its expression in
a host cell, glucanase, mannanase, or xylanase so modified, and methods
of making the modified glucanase, mannanase, or xylanase. The method
comprises identifying a "non-preferred" or a "less preferred" codon in
glucanase-, mannanase, or xylanase encoding nucleic acid and replacing
one or more of these non-preferred or less preferred codons with a
"preferred codon" encoding the same amino acid as the replaced codon and
at least one non-preferred or less preferred codon in the nucleic acid
has been replaced by a preferred codon encoding the same amino acid. A
preferred codon is a codon over-represented in coding sequences in genes
in the host cell and a non-preferred or less preferred codon is a codon
under-represented in coding sequences in genes in the host cell.
[0447] Host cells for expressing the nucleic acids, expression cassettes
and vectors of the invention include bacteria, yeast, fungi, plant cells,
insect cells and mammalian cells. Thus, the invention provides methods
for optimizing codon usage in all of these cells, codon-altered nucleic
acids and polypeptides made by the codon-altered nucleic acids. Exemplary
host cells include gram negative bacteria, such as Escherichia coli; gram
positive bacteria, such as Streptomyces, Lactobacillus gasseri,
Lactococcus lactis, Lactococcus cremoris, Bacillus sp., Bacillus
subtilis, Bacillus cereus. Exemplary host cells also include eukaryotic
organisms, e.g., various yeast, such as Saccharomyces sp., including
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and
Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, and
mammalian cells and cell lines and insect cells and cell lines. Thus, the
invention also includes nucleic acids and polypeptides optimized for
expression in these organisms and species, e.g., the nucleic acids of the
invention are codon-optimized for expression in a host cell, e.g., a
Pichia sp., e.g., P. pastoris, a Saccharomyces sp., or a Bacillus sp., a
Streptomyces sp., and the like.
[0448] For example, the codons of a nucleic acid encoding a polypeptide of
the invention, e.g., a glucanase, mannanase, or xylanase, or a similar
enzyme isolated from a bacterial cell, are modified such that the nucleic
acid (encoding the enzyme) is optimally expressed in a bacterial cell
different from the bacteria from which the enzyme (e.g., glucanase,
mannanase, or xylanase) was derived, a yeast, a fungi, a plant cell, an
insect cell or a mammalian cell. Methods for optimizing codons are well
known in the art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int. J.
Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188; Narum
(2001) Infect. Immun. 69:7250-7253. See also Narum (2001) Infect. Immun.
69:7250-7253, describing optimizing codons in mouse systems; Outchkourov
(2002) Protein Expr. Purif. 24:18-24, describing optimizing codons in
yeast; Feng (2000) Biochemistry 39:15399-15409, describing optimizing
codons in E. coli; Humphreys (2000) Protein Expr. Purif. 20:252-264,
describing optimizing codon usage that affects secretion in E. coli; Gao
(2004) Biotechnol Prog. 20:443-448, describing "UpGene", an application
of a web-based DNA codon optimization algorithm.
[0449] For example, as discussed in Example 4, below, the nucleic acid
encoding the polypeptide having a sequence as set forth in SEQ ID NO:6
(e.g., SEQ ID NO:5) was subjected to codon optimization for optimal
expression in Pichia pastoris; the Pichia pastoris codon-optimized
enzyme-encoding nucleic acid is SEQ ID NO:463. The exemplary polypeptide
having a sequence as set forth as SEQ ID NO:464 at position 91 is alanine
(SEQ ID NO:464), and in an alternative aspect, valine (as in SEQ ID
NO:6). Similarly, the exemplary nucleic acid encoding SEQ ID NO:464
(i.e., SEQ ID NO:463) can, in alternative embodiments, encode either
alanine or valine (or another conservative substitution) at position 91.
Similarly, the exemplary nucleic acid encoding SEQ ID NO:6 (i.e., SEQ ID
NO:5) can, in alternative embodiments, encode either alanine or valine
(or another conservative substitution) at position 91. In fact, the
invention provides alternative embodiments of the polypeptides of the
invention (and the nucleic acids that encode them) comprising at least
one conservative amino acid substitution, as discussed herein (e.g.,
conservative amino acid substitutions are those that substitute a given
amino acid in a polypeptide by another amino acid of like
characteristics), as discussed herein.
Transgenic Non-Human Animals
[0450] The invention provides transgenic non-human animals comprising a
nucleic acid, a polypeptide (e.g., a glucanase, mannanase, or xylanase),
an expression cassette or vector or a transfected or transformed cell of
the invention. The invention also provides methods of making and using
these transgenic non-human animals.
[0451] The transgenic non-human animals can be, e.g., goats, rabbits,
sheep, pigs, cows, rats and mice, comprising the nucleic acids of the
invention. These animals can be used, e.g., as in vivo models to study
glucanase, mannanase, or xylanase activity, or, as models to screen for
agents that change the glucanase, mannanase, or xylanase activity in
vivo. The coding sequences for the polypeptides to be expressed in the
transgenic non-human animals can be designed to be constitutive, or,
under the control of tissue-specific, developmental-specific or inducible
transcriptional regulatory factors. Transgenic non-human animals can be
designed and generated using any method known in the art; see, e.g., U.S.
Pat. Nos. 6,211,428; 6,187,992; 6,156,952; 6,118,044; 6,111,166;
6,107,541; 5,959,171; 5,922,854; 5,892,070; 5,880,327; 5,891,698;
5,639,940; 5,573,933; 5,387,742; 5,087,571, describing making and using
transformed cells and eggs and transgenic mice, rats, rabbits, sheep,
pigs and cows. See also, e.g., Pollock (1999) J. Immunol. Methods
231:147-157, describing the production of recombinant proteins in the
milk of transgenic dairy animals; Baguisi (1999) Nat. Biotechnol.
17:456-461, demonstrating the production of transgenic goats. U.S. Pat.
No. 6,211,428, describes making and using transgenic non-human mammals
which express in their brains a nucleic acid construct comprising a DNA
sequence. U.S. Pat. No. 5,387,742, describes injecting cloned recombinant
or synthetic DNA sequences into fertilized mouse eggs, implanting the
injected eggs in pseudo-pregnant females, and growing to term transgenic
mice whose cells express proteins related to the pathology of Alzheimer's
disease. U.S. Pat. No. 6,187,992, describes making and using a transgenic
mouse whose genome comprises a disruption of the gene encoding amyloid
precursor protein (APP).
[0452] "Knockout animals" can also be used to practice the methods of the
invention. For example, in one aspect, the transgenic or modified animals
of the invention comprise a "knockout animal," e.g., a "knockout mouse,"
engineered not to express an endogenous gene, which is replaced with a
gene expressing a glucanase, mannanase, or xylanase of the invention, or,
a fusion protein comprising a glucanase, mannanase, or xylanase of the
invention.
Transgenic Plants and Seeds
[0453] The invention provides transgenic plants and seeds comprising a
nucleic acid, a polypeptide (e.g., a glucanase, mannanase, or xylanase),
an expression cassette or vector or a transfected or transformed cell of
the invention. The invention also provides plant products, e.g., oils,
seeds, leaves, extracts and the like, comprising a nucleic acid and/or a
polypeptide (e.g., a glucanase, mannanase, or xylanase) of the invention.
The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous
(a monocot). The invention also provides methods of making and using
these transgenic plants and seeds. The transgenic plant or plant cell
expressing a polypeptide of the present invention may be constructed in
accordance with any method known in the art. See, for example, U.S. Pat.
No. 6,309,872.
[0454] Nucleic acids and expression constructs of the invention can be
introduced into a plant cell by any means. For example, nucleic acids or
expression constructs can be introduced into the genome of a desired
plant host, or, the nucleic acids or expression constructs can be
episomes. Introduction into the genome of a desired plant can be such
that the host's glucanase, mannanase, or xylanase production is regulated
by endogenous transcriptional or translational control elements. The
invention also provides "knockout plants" where insertion of gene
sequence by, e.g., homologous recombination, has disrupted the expression
of the endogenous gene. Means to generate "knockout" plants are
well-known in the art, see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA
95:4368-4373; Miao (1995) Plant J 7:359-365. See discussion on transgenic
plants, below.
[0455] The nucleic acids of the invention can be used to confer desired
traits on essentially any plant, e.g., on starch-producing plants, such
as potato, wheat, rice, barley, and the like. Nucleic acids of the
invention can be used to manipulate metabolic pathways of a plant in
order to optimize or alter host's expression of glucanase, mannanase, or
xylanase. The can change glucanase, mannanase, or xylanase activity in a
plant. Alternatively, a glucanase, mannanase, or xylanase of the
invention can be used in production of a transgenic plant to produce a
compound not naturally produced by that plant. This can lower production
costs or create a novel product.
[0456] In one aspect, the first step in production of a transgenic plant
involves making an expression construct for expression in a plant cell.
These techniques are well known in the art. They can include selecting
and cloning a promoter, a coding sequence for facilitating efficient
binding of ribosomes to mRNA and selecting the appropriate gene
terminator sequences. One exemplary constitutive promoter is CaMV35S,
from the cauliflower mosaic virus, which generally results in a high
degree of expression in plants. Other promoters are more specific and
respond to cues in the plant's internal or external environment. An
exemplary light-inducible promoter is the promoter from the cab gene,
encoding the major chlorophyll a/b binding protein.
[0457] In one aspect, the nucleic acid is modified to achieve greater
expression in a plant cell. For example, a sequence of the invention is
likely to have a higher percentage of A-T nucleotide pairs compared to
that seen in a plant, some of which prefer G-C nucleotide pairs.
Therefore, A-T nucleotides in the coding sequence can be substituted with
G-C nucleotides without significantly changing the amino acid sequence to
enhance production of the gene product in plant cells.
[0458] Selectable marker gene can be added to the gene construct in order
to identify plant cells or tissues that have successfully integrated the
transgene. This may be necessary because achieving incorporation and
expression of genes in plant cells is a rare event, occurring in just a
few percent of the targeted tissues or cells. Selectable marker genes
encode proteins that provide resistance to agents that are normally toxic
to plants, such as antibiotics or herbicides. Only plant cells that have
integrated the selectable marker gene will survive when grown on a medium
containing the appropriate antibiotic or herbicide. As for other inserted
genes, marker genes also require promoter and termination sequences for
proper function.
[0459] In one aspect, making transgenic plants or seeds comprises
incorporating sequences of the invention and, optionally, marker genes
into a target expression construct (e.g., a plasmid), along with
positioning of the promoter and the terminator sequences. This can
involve transferring the modified gene into the plant through a suitable
method. For example, a construct may be introduced directly into the
genomic DNA of the plant cell using techniques such as electroporation
and microinjection of plant cell protoplasts, or the constructs can be
introduced directly to plant tissue using ballistic methods, such as DNA
particle bombardment. For example, see, e.g., Christou (1997) Plant Mol.
Biol. 35:197-203; Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987)
Nature 327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing
use of particle bombardment to introduce transgenes into wheat; and Adam
(1997) supra, for use of particle bombardment to introduce YACs into
plant cells. For example, Rinehart (1997) supra, used particle
bombardment to generate transgenic cotton plants. Apparatus for
accelerating particles is described U.S. Pat. No. 5,015,580; and, the
commercially available BioRad (Biolistics) PDS-2000 particle acceleration
instrument; see also, John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat.
No. 5,681,730, describing particle-mediated transformation of
gymnosperms.
[0460] In one aspect, protoplasts can be immobilized and injected with a
nucleic acids, e.g., an expression construct. Although plant regeneration
from protoplasts is not easy with cereals, plant regeneration is possible
in legumes using somatic embryogenesis from protoplast derived callus.
Organized tissues can be transformed with naked DNA using gene gun
technique, where DNA is coated on tungsten microprojectiles, shot 1/100th
the size of cells, which carry the DNA deep into cells and organelles.
Transformed tissue is then induced to regenerate, usually by somatic
embryogenesis. This technique has been successful in several cereal
species including maize and rice.
[0461] Nucleic acids, e.g., expression constructs, can also be introduced
in to plant cells using recombinant viruses. Plant cells can be
transformed using viral vectors, such as, e.g., tobacco mosaic virus
derived vectors (Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta
(1996) "Use of viral replicons for the expression of genes in plants,"
Mol. Biotechnol. 5:209-221.
[0462] Alternatively, nucleic acids, e.g., an expression construct, can be
combined with suitable T-DNA flanking regions and introduced into a
conventional Agrobacterium tumefaciens host vector. The virulence
functions of the Agrobacterium tumefaciens host will direct the insertion
of the construct and adjacent marker into the plant cell DNA when the
cell is infected by the bacteria. Agrobacterium tumefaciens-mediated
transformation techniques, including disarming and use of binary vectors,
are well described in the scientific literature. See, e.g., Horsch (1984)
Science 233:496-498; Fraley (1983) Proc. Natl. Acad. Sci. USA 80:4803
(1983); Gene Transfer to Plants, Potrykus, ed. (Springer-Verlag, Berlin
1995). The DNA in an A. tumefaciens cell is contained in the bacterial
chromosome as well as in another structure known as a Ti (tumor-inducing)
plasmid. The Ti plasmid contains a stretch of DNA termed T-DNA (.about.20
kb long) that is transferred to the plant cell in the infection process
and a series of vir (virulence) genes that direct the infection process.
A. tumefaciens can only infect a plant through wounds: when a plant root
or stem is wounded it gives off certain chemical signals, in response to
which, the vir genes of A. tumefaciens become activated and direct a
series of events necessary for the transfer of the T-DNA from the Ti
plasmid to the plant's chromosome. The T-DNA then enters the plant cell
through the wound. One speculation is that the T-DNA waits until the
plant DNA is being replicated or transcribed, then inserts itself into
the exposed plant DNA. In order to use A. tumefaciens as a transgene
vector, the tumor-inducing section of T-DNA have to be removed, while
retaining the T-DNA border regions and the vir genes. The transgene is
then inserted between the T-DNA border regions, where it is transferred
to the plant cell and becomes integrated into the plant's chromosomes.
[0463] The invention provides for the transformation of monocotyledonous
plants using the nucleic acids of the invention, including important
cereals, see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g.,
Horsch, Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci. USA
80:4803; Thykjaer (1997) supra; Park (1996) Plant Mol. Biol.
32:1135-1148, discussing T-DNA integration into genomic DNA. See also
D'Halluin, U.S. Pat. No. 5,712,135, describing a process for the stable
integration of a DNA comprising a gene that is functional in a cell of a
cereal, or other monocotyledonous plant.
[0464] In one aspect, the third step can involve selection and
regeneration of whole plants capable of transmitting the incorporated
target gene to the next generation. Such regeneration techniques rely on
manipulation of certain phytohormones in a tissue culture growth medium,
typically relying on a biocide and/or herbicide marker that has been
introduced together with the desired nucleotide sequences. Plant
regeneration from cultured protoplasts is described in Evans et al.,
Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp.
124-176, MacMillilan Publishing Company, New York, 1983; and Binding,
Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca
Raton, 1985. Regeneration can also be obtained from plant callus,
explants, organs, or parts thereof. Such regeneration techniques are
described generally in Klee (1987) Ann. Rev. of Plant Phys. 38:467-486.
To obtain whole plants from transgenic tissues such as immature embryos,
they can be grown under controlled environmental conditions in a series
of media containing nutrients and hormones, a process known as tissue
culture. Once whole plants are generated and produce seed, evaluation of
the progeny begins.
[0465] After the expression cassette is stably incorporated in transgenic
plants, it can be introduced into other plants by sexual crossing. Any of
a number of standard breeding techniques can be used, depending upon the
species to be crossed. Since transgenic expression of the nucleic acids
of the invention leads to phenotypic changes, plants comprising the
recombinant nucleic acids of the invention can be sexually crossed with a
second plant to obtain a final product. Thus, the seed of the invention
can be derived from a cross between two transgenic plants of the
invention, or a cross between a plant of the invention and another plant.
The desired effects (e.g., expression of the polypeptides of the
invention to produce a plant in which flowering behavior is altered) can
be enhanced when both parental plants express the polypeptides (e.g., a
glucanase, mannanase, or xylanase) of the invention. The desired effects
can be passed to future plant generations by standard propagation means.
[0466] The nucleic acids and polypeptides of the invention are expressed
in or inserted in any plant or seed. Transgenic plants of the invention
can be dicotyledonous or monocotyledonous. Examples of monocot transgenic
plants of the invention are grasses, such as meadow Brass (blue grass,
Poa), forage grass such as festuca, lolium, temperate grass, such as
Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and
maize (corn). Examples of dicot transgenic plants of the invention are
tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and
soybean, and cruciferous plants (family Brassicaceae), such as
cauliflower, rape seed, and the closely related model organism
Arabidopsis thaliana. Thus, the transgenic plants and seeds of the
invention include a broad range of plants, including, but not limited to,
species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena,
Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis,
Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus,
Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus,
Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza,
Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisuin, Pyrus, Prunus,
Raphanus, Ricinus, Secale, Senecio, Sinapis, Solaizum, Sorghum,
Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.
[0467] In alternative embodiments, the nucleic acids of the invention are
expressed in plants which contain fiber cells, including, e.g., cotton,
silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush,
winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca and
flax. In alternative embodiments, the transgenic plants of the invention
can be members of the genus Gossypium, including members of any Gossypium
species, such as G. arboreum; G. herbaceum, G. barbadense, and G.
hirsutum.
[0468] The invention also provides for transgenic plants to be used for
producing large amounts of the polypeptides (e.g., a glucanase,
mannanase, or xylanase or antibody) of the invention. For example, see
Palmgren (1997) Trends Genet. 13:348; Chong (1997) Transgenic Res.
6:289-296 (producing human milk protein beta-casein in transgenic potato
plants using an auxin-inducible, bidirectional mannopine synthase
(mas1',2') promoter with Agrobacterium tumefaciens-mediated leaf disc
transformation methods).
[0469] Using known procedures, one of skill can screen for plants of the
invention by detecting the increase or decrease of transgene mRNA or
protein in transgenic plants. Means for detecting and quantitation of
mRNAs or proteins are well known in the art.
Polypeptides and Peptides
[0470] In one aspect, the invention provides isolated, synthetic or
recombinant polypeptides having a sequence identity (e.g., at least about
50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,
64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)
sequence identity) to an exemplary sequence of the invention, e.g.,
proteins having a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID
NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID
NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID
NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID
NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID
NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID
NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID
NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID
NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ
ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,
SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132; SEQ ID
NO:134; SEQ ID NO:136; SEQ ID NO:138; SEQ ID NO:140; SEQ ID NO:142; SEQ
ID NO:144; NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID
NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ
ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172,
SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID
NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ
ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200,
SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID
NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ
ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228,
SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID
NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ
ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256,
SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID
NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ
ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284,
SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID
NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ
ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312,
SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID
NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ
ID NO:332, SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340,
SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:348, SEQ ID
NO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQ
ID NO:360, SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368,
SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:374, SEQ ID NO:376, SEQ ID
NO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ ID NO:384, SEQ ID NO:386, SEQ
ID NO:388, SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, SEQ ID NO:396,
SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402, SEQ ID NO:404, SEQ ID
NO:406, SEQ ID NO:408, SEQ ID NO:410, SEQ ID NO:412, SEQ ID NO:414, SEQ
ID NO:416, SEQ ID NO:418, SEQ ID NO:420, SEQ ID NO:422, SEQ ID NO:424,
SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, SEQ ID NO:432, SEQ ID
NO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:442, SEQ
ID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQ ID NO:450, SEQ ID NO:452,
SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458, SEQ ID NO:460, SEQ ID
NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ ID NO:470, SEQ
ID NO:472, SEQ ID NO:474, SEQ ID NO:476, SEQ ID NO:478, SEQ ID NO:480,
SEQ ID NO:482, SEQ ID NO:484, SEQ ID NO:486, SEQ ID NO:488, SEQ ID
NO:490, SEQ ID NO:492, SEQ ID NO:494, SEQ ID NO:496, SEQ ID NO:498, SEQ
ID NO:500, SEQ ID NO:502, SEQ ID NO:504, SEQ ID NO:506, SEQ ID NO:508,
SEQ ID NO:510, SEQ ID NO:512, SEQ ID NO:514, SEQ ID NO:516 or SEQ ID
NO:518. In one aspect, the polypeptide has a glucanase, mannanase, or
xylanase activity, e.g., can hydrolyze a glycosidic bond in a
polysaccharide, e.g., a glucan. In one aspect, the polypeptide has a
glucanase activity comprising catalyzing hydrolysis of
1,4-beta-D-glycosidic linkages or .beta.-1,3-glucosidic linkages. In one
aspect, the endoglucanase activity comprises an
endo-1,4-beta-endoglucanase activity. In one aspect, the endoglucanase
activity comprises hydrolyzing a glucan to produce a smaller molecular
weight glucan or glucan-oligomer. In one aspect, the glucan comprises a
beta-glucan, such as a water soluble beta-glucan.
[0471] Enzymes encoded by the polynucleotides of the invention include,
but are not limited to hydrolases such as glucanases, e.g.,
endoglucanases, mannanases, or xylanases. FIG. 5 is a table summarizing
the relative activities of several exemplary enzymes of the invention
under various conditions, e.g., varying pH and temperature. In FIG. 5:
ND=not determined; * pH or temperature optima not determined but enzyme
activities were measured at the indicated pH and/or temperature; 1
thermal stability, time that enzyme retained significant activity
(approx. >50%) or time where enzyme has lost 50% of its activity
(t1/2) at the indicated temperature; 2 RA=relative activity at pH's 2.6,
4.0, 5.5, 7.0, 8.0, 9.0 or at 25.degree. C., 37.degree. C., 50.degree.
C., 65.degree. C., 75.degree. C., 85.degree. C. relative to activity at
the pH and temperature optima respectively; 3 RA at pH 3.75, 5, 5.3,
6.25, 7 for pH opt and 40, 55, 70, 90 C for temp opt; 4
BBG=barley-beta-glucan, CMC=carboxymethyl cellulose. Family groupings of
glucanases are discussed below.
[0472] In one aspect, an enzyme of the invention can also have a mannanase
activity, e.g., it can degrade (or hydrolyze) mannans. Mannan containing
polysaccharides are a major component of the hemicellulose fraction in
both hardwoods and softwoods as well as in the endosperm in many
leguminous seeds and in some mature seeds of non-leguminous plants. In
one aspect, a mannanase of the invention hydrolyses beta-1,4 linkages in
mannans, glucomannans, galactomannans and galactoglucomannans (mannans
are polysaccharides having a backbone composed of beta-1,4 linked
mannose, glucomannans are polysaccharides having a backbone of more or
less regularly alternating beta-1,4 linked mannose and glucose). For
example, in one aspect, the polypeptide having a sequence as set forth in
SEQ ID NO:454, encoded by, e.g., SEQ ID NO:453, has a xylanase, a
glucanase, and a mannanase activity. Assays to determine mannanase
activity are well known in the art, see, e.g., U.S. Patent Application
Nos: 20030215812; 20030119093; U.S. Pat. Nos. 5,661,021; 5,795,764;
6,376,445; 6,420,331. Assays to determine xylanase activity are well
known in the art, see, e.g., U.S. Pat. Nos. 5,693,518; 5,885,819;
6,200,797; 6,586,209; 6,682,923.
[0473] The invention also provides chimeric polypeptides (and the nucleic
acids encoding them) comprising at least two enzymes of the invention or
subsequences thereof, e.g., active sites, or catalytic domains (CDs). A
chimeric protein of the invention (e.g., a fusion protein, or, other
heterodimer, e.g., two domains joined by other means, e.g., a linker, or,
electrostatically) can comprise one polypeptide (e.g., active site or
catalytic domain peptide) of the invention and another polypeptide (e.g.,
active site or catalytic domain peptide) of the invention or other
polypeptide. For example, a chimeric protein of the invention can have
mannanase and xylanase activity, mannanase and glycanase activity, etc.
In one aspect the chimeric protein of the invention comprises a fusion of
domains, e.g., a single domain can exhibit glucanase/xylanase/mannanase
or any combination of activities.
[0474] The invention provides glucanases having a common novelty in that
they were first derived from similar "glycosidase hydrolase" families.
Glycosidase hydrolases were first classified into families in 1991, see,
e.g., Henrissat (1991) Biochem, J. 280:309-316. Since then, the
classifications have been continually updated, see, e.g., Henrissat
(1993) Biochem. J. 293:781-788; Henrissat (1996) Biochem. J. 316:695-696;
Henrissat (2000) Plant Physiology 124:1515-1519. There are approximately
87 identified families of glycosidase hydrolases. In one aspect, the
glucanases of the invention are categorized as families, e.g., the
families 3, 5, 6, 8, 9, 12, and 16, as set forth below in Table 2.
TABLE-US-00003
TABLE 2
SEQ ID NO: Family
1, 2 5
101, 102 16
103, 104 5
105, 106 5
107, 108 5
109, 110 5
11, 12 8
111, 112 5
113, 114 16
115, 116 5
117, 118 5
119, 120 16
121, 122 12
123, 124 8
125, 126 16
127, 128 5
129, 130 5
13, 14 8
131, 132 9
133, 134 8
135, 136 5 + CBD
137, 138 5
139, 140 8
141, 142 9
143, 144 5
145, 146 5 + CBD + SLH
147, 148 5 + CBD + SLH
149, 150 5
15, 16 3
151, 152 16
153, 154 5
155, 156 9 + CBD
157, 158 16
159, 160 16
161, 162 16
163, 164 9
165, 166 5
167, 168 5
169, 170 5
17, 18 9 AND 1
171, 172 16
173, 174 16
175, 176 5
177, 178 16
179, 180 5 + CBD
181, 182 16
183, 184 8
185, 186 NA
187, 188 8 AND 1
189, 190 5
19, 20 5
191, 192 16
193, 194 5
195, 196 16
197, 198 16
199, 200 16
201, 202 5
203, 204 3
205, 206 5
207, 208 5
209, 210 16
21, 22 12
211, 212 8
213, 214 16
215, 216 6
217, 218 16
219, 220 5
221, 222 5
223, 224 5
225, 226 8
227, 228 5 + CBD
229, 230 5
23, 24 12
231, 232 5
233, 234 5
235, 236 5
237, 238 6
239, 240 NA + CBD
241, 242 5
245, 246 9
247, 248 8
249, 250 5
25, 26 8
251, 252 9 + CBD
253, 254 5
255, 256 5
257, 258 9
259, 260 5
261, 262 5
263, 264 1 + CBD
265, 266 NA
267, 268 5
269, 270 9
27, 28 8
271, 272 9
273, 274 48 + CBD
275, 276 8
277, 278 3
279, 280 5
281, 282 9
283, 284 5
285, 286 5
287, 288 6
289, 290 8
29, 30 9
291, 292 8
293, 294 6
295, 296 9 + DOCR
297, 298 9
299, 300 5
3, 4 8
301, 302 5
303, 304 9 + CBD
305, 306 5
307, 308 5
309, 310 10
31, 32 5
311, 312 5
313, 314 5
315, 316 5
317, 318 5
319, 320 5
321, 322 5
323, 324 5
325, 326 5
327, 328 5
329, 330 9 + CBD
33, 34 8
333, 334 5
335, 336 6
337, 338 5
339, 340 6
341, 342 5
343, 344 5
345, 346 6
347, 348 5
349, 350 5
35, 36 12
351, 352 5 + CBD
353, 354 12
355, 356 5 + CBD
357, 358 5
359, 360 5
361, 362 5
363, 364 CBD
365, 366 5
367, 368 5
369, 370 5
37, 38 5 and/or 6
371, 372 5
373, 374 5
375, 376 9
377, 378 5
379, 380 3
381, 382 9
383, 384 5
385, 386 8
387, 388 5
389, 390 9
39, 40 5
391, 392 9
395, 396 8
397, 398 3
399, 400 5
401, 402 5 or 6 + CDB
403, 404 5
405, 406 5
407, 408 5
409, 410 5
41, 42 12
411, 412 5
413, 414 6
415, 416 9 + CBD
417, 418 5
419, 420 5
421, 422 5
423, 424 9
425, 426 44
427, 428 5
429, 430 3
43, 44 16
431, 432 9
433, 434 6
435, 436 5
437, 438 5
439, 440 5
441, 442 9
443, 444 NA
445, 446 NA
447, 448 26
449, 450 5 + DOCR
45, 46 9
451, 452 5
453, 454 5 and 26
455, 456 1
457, 458 5
459, 460 9
461, 462 5
463, 464 5
465, 466 5
467, 468 10
469, 470 5
47, 48 8
471, 472 16
473, 474 5
475, 476 5
477, 478 11
481, 482 5
483, 484 16
485, 486 16
487, 488 12
489, 490 5
49, 50 5
491, 492 11
493, 494 16
495, 496 5
497, 498 16
499, 500 16
5, 6 5
501, 502 1
503, 504 5
505, 506 5
507, 508 1
509, 510 5
51, 52 5
511, 512 26
513, 514 26
515, 516 5
517, 518 3
53, 54 5
55, 56 5
57, 58 9
59, 60 16
61, 62 12
63, 64 16
65, 66 16
67, 68 9
69, 70 5
7, 8 9
71, 72 16
73, 74 5
75, 76 12
77, 78 5
79, 80 CBD
81, 82 16
83, 84 5
87, 88 16
89, 90 16
9, 10 5
91, 92 3
93, 94 6
95, 96 16
97, 98 5
99, 100 16
[0475] The polypeptides of the invention include glucanases, mannanases,
or xylanases in an active or inactive form. For example, the polypeptides
of the invention include proproteins before "maturation" or processing of
prepro sequences, e.g., by a proprotein-processing enzyme, such as a
proprotein convertase to generate an "active" mature protein. The
polypeptides of the invention include glucanases, mannanases, or
xylanases inactive for other reasons, e.g., before "activation" by a
post-translational processing event, e.g., an endo- or exo-peptidase or
proteinase action, a phosphorylation event, an amidation, a glycosylation
or a sulfation, a dimerization event, and the like. The polypeptides of
the invention include all active forms, including active subsequences,
e.g., catalytic domains or active sites, of the glucanase, mannanase, or
xylanases.
[0476] Methods for identifying "prepro" domain sequences and signal
sequences are well known in the art, see, e.g., Van de Ven (1993) Crit.
Rev. Oncog. 4(2):115-136. For example, to identify a prepro sequence, the
protein is purified from the extracellular space and the N-terminal
protein sequence is determined and compared to the unprocessed form.
[0477] The invention includes polypeptides with or without a signal
sequence and/or a prepro sequence. The invention includes polypeptides
with heterologous signal sequences and/or prepro sequences. The prepro
sequence (including a sequence of the invention used as a heterologous
prepro domain) can be located on the amino terminal or the carboxy
terminal end of the protein. The invention also includes isolated or
recombinant signal sequences, prepro sequences and catalytic domains
(e.g., "active sites") comprising sequences of the invention.
[0478] The percent sequence identity can be over the full length of the
polypeptide, or, the identity can be over a region of at least about 50,
60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,
650, 700 or more residues. Polypeptides of the invention can also be
shorter than the full length of exemplary polypeptides. In alternative
aspects, the invention provides polypeptides (peptides, fragments)
ranging in size between about 5 and the full length of a polypeptide,
e.g., an enzyme, such as a glucanase, mannanase, or xylanase; exemplary
sizes being of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450,
500, 550, 600, 650, 700, or more residues, e.g., contiguous residues of
an exemplary glucanase, mannanase, or xylanase of the invention.
[0479] Peptides of the invention (e.g., a subsequence of an exemplary
polypeptide of the invention) can be useful as, e.g., labeling probes,
antigens, toleragens, motifs, glucanase, mannanase, or xylanase active
sites (e.g., "catalytic domains"), signal sequences and/or prepro
domains.
[0480] Polypeptides and peptides of the invention can be isolated from
natural sources, be synthetic, or be recombinantly generated
polypeptides. Peptides and proteins can be recombinantly expressed in
vitro or in vivo. The peptides and polypeptides of the invention can be
made and isolated using any method known in the art. Polypeptide and
peptides of the invention can also be synthesized, whole or in part,
using chemical methods well known in the art. See e.g., Caruthers (1980)
Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res.
Symp. Ser. 225-232; Banga, A. K., Therapeutic Peptides and Proteins,
Formulation, Processing and Delivery Systems (1995) Technomic Publishing
Co., Lancaster, Pa. For example, peptide synthesis can be performed using
various solid-phase techniques (see e.g., Roberge (1995) Science 269:202;
Merrifield (1997) Methods Enzymol. 289:3-13) and automated synthesis may
be achieved, e.g., using the ABI 431A Peptide Synthesizer (Perlin Elmer)
in accordance with the instructions provided by the manufacturer.
[0481] The peptides and polypeptides of the invention can also be
glycosylated. The glycosylation can be added post-translationally either
chemically or by cellular biosynthetic mechanisms, wherein the later
incorporates the use of known glycosylation motifs, which can be native
to the sequence or can be added as a peptide or added in the nucleic acid
coding sequence. The glycosylation can be O-linked or N-linked.
[0482] The peptides and polypeptides of the invention, as defined above,
include all "mimetic" and "peptidomimetic" forms. The terms "mimetic" and
"peptidomimetic" refer to a synthetic chemical compound which has
substantially the same structural and/or functional characteristics of
the polypeptides of the invention. The mimetic can be either entirely
composed of synthetic, non-natural analogues of amino acids, or, is a
chimeric molecule of partly natural peptide amino acids and partly
non-natural analogs of amino acids. The mimetic can also incorporate any
amount of natural amino acid conservative substitutions as long as such
substitutions also do not substantially alter the mimetic's structure
and/or activity. As with polypeptides of the invention which are
conservative variants, routine experimentation will determine whether a
mimetic is within the scope of the invention, i.e., that its structure
and/or function is not substantially altered. Thus, in one aspect, a
mimetic composition is within the scope of the invention if it has a
glucanase, mannanase, or xylanase activity.
[0483] Polypeptide mimetic compositions of the invention can contain any
combination of non-natural structural components. In alternative aspect,
mimetic compositions of the invention include one or all of the following
three structural groups: a) residue linkage groups other than the natural
amide bond ("peptide bond") linkages; b) non-natural residues in place of
naturally occurring amino acid residues; or c) residues which induce
secondary structural mimicry, i.e., to induce or stabilize a secondary
structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix
conformation, and the like. For example, a polypeptide of the invention
can be characterized as a mimetic when all or some of its residues are
joined by chemical means other than natural peptide bonds. Individual
peptidomimetic residues can be joined by peptide bonds, other chemical
bonds or coupling means, such as, e.g., glutaraldehyde,
N-hydroxysuccinimide esters, bifunctional maleimides,
N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide
(DIC). Linking groups that can be an alternative to the traditional amide
bond ("peptide bond") linkages include, e.g., ketomethylene (e.g.,
--C(.dbd.O)--CH.sub.2-- for --C(.dbd.O)--NH--), aminomethylene
(CH.sub.2--NH), ethylene, olefin (CH.dbd.CH), ether (CH.sub.2--O),
thioether (CH.sub.2--S), tetrazole (CN.sub.4--), thiazole, retroamide,
thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and
Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357,
"Peptide Backbone Modifications," Marcell Dekker, NY).
[0484] A polypeptide of the invention can also be characterized as a
mimetic by containing all or some non-natural residues in place of
naturally occurring amino acid residues. Non-natural residues are well
described in the scientific and patent literature; a few exemplary
non-natural compositions useful as mimetics of natural amino acid
residues and guidelines are described below. Mimetics of aromatic amino
acids can be generated by replacing by, e.g., D- or L-naphylalanine; D-
or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2, 3-, or 4
pyreneylalanine; D- or L-3 thieneylalanine; D- or
L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or
L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;
D-trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;
D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- or
L-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,
D- or L-alkylamines, where alkyl can be substituted or unsubstituted
methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,
sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of a
non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,
benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
[0485] Mimetics of acidic amino acids can be generated by substitution by,
e.g., non-carboxylate amino acids while maintaining a negative charge;
(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,
aspartyl or glutamyl) can also be selectively modified by reaction with
carbodiimides (R'--N--C--N--R') such as, e.g.,
1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or
1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl or glutamyl
can also be converted to asparaginyl and glutaminyl residues by reaction
with ammonium ions. Mimetics of basic amino acids can be generated by
substitution with, e.g., (in addition to lysine and arginine) the amino
acids ornithine, citrulline, or (guanidino)-acetic acid, or
(guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrile
derivative (e.g., containing the CN-moiety in place of COOH) can be
substituted for asparagine or glutamine. Asparaginyl and glutaminyl
residues can be deaminated to the corresponding aspartyl or glutamyl
residues. Arginine residue mimetics can be generated by reacting arginyl
with, e.g., one or more conventional reagents, including, e.g.,
phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin, in
one aspect under alkaline conditions. Tyrosine residue mimetics can be
generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or
tetranitromethane. N-acetylimidizol and tetranitromethane can be used to
form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
Cysteine residue mimetics can be generated by reacting cysteinyl residues
with, e.g., alpha-haloacetates such as 2-chloroacetic acid or
chloroacetamide and corresponding amines; to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteine residue mimetics can also be
generated by reacting cysteinyl residues with, e.g.,
bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;
chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;
methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4
nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimetics can
be generated (and amino terminal residues can be altered) by reacting
lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine
and other alpha-amino-containing residue mimetics can also be generated
by reaction with imidoesters, such as methyl picolinimidate, pyridoxal
phosphate, pyridoxal, chloroborohydride, trinitro-benzenesulfonic acid,
O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions
with glyoxylate. Mimetics of methionine can be generated by reaction
with, e.g., methionine sulfoxide. Mimetics of proline include, e.g.,
pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy proline,
dehydroproline, 3- or 4-methylproline, or 3,3-dimethylproline. Histidine
residue mimetics can be generated by reacting histidyl with, e.g.,
diethylprocarbonate or para-bromophenacyl bromide. Other mimetics
include, e.g., those generated by hydroxylation of proline and lysine;
phosphorylation of the hydroxyl groups of seryl or threonyl residues;
methylation of the alpha-amino groups of lysine, arginine and histidine;
acetylation of the N-terminal amine; methylation of main chain amide
residues or substitution with N-methyl amino acids; or amidation of
C-terminal carboxyl groups.
[0486] A residue, e.g., an amino acid, of a polypeptide of the invention
can also be replaced by an amino acid (or peptidomimetic residue) of the
opposite chirality. Thus, any amino acid naturally occurring in the
L-configuration (which can also be referred to as the R or S, depending
upon the structure of the chemical entity) can be replaced with the amino
acid of the same chemical structural type or a peptidomimetic, but of the
opposite chirality, referred to as the D-amino acid, but also can be
referred to as the R- or S-form.
[0487] The invention also provides methods for modifying the polypeptides
of the invention by either natural processes, such as post-translational
processing (e.g., phosphorylation, acylation, etc), or by chemical
modification techniques, and the resulting modified polypeptides.
Modifications can occur anywhere in the polypeptide, including the
peptide backbone, the amino acid side-chains and the amino or carboxyl
termini. It will be appreciated that the same type of modification may be
present in the same or varying degrees at several sites in a given
polypeptide. Also a given polypeptide may have many types of
modifications. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin, covalent
attachment of a heme moiety, covalent attachment of a nucleotide or
nucleotide derivative, covalent attachment of a lipid or lipid
derivative, covalent attachment of a phosphatidylinositol, cross-linking
cyclization, disulfide bond formation, demethylation, formation of
covalent cross-links, formation of cysteine, formation of pyroglutamate,
formylation, gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristolyation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, and transfer-RNA mediated
addition of amino acids to protein such as arginylation. See, e.g.,
Creighton, T. E., Proteins--Structure and Molecular Properties 2nd Ed.,
W.H. Freeman and Company, New York (1993); Posttranslational Covalent
Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York,
pp. 1-12 (1983).
[0488] Solid-phase chemical peptide synthesis methods can also be used to
synthesize the polypeptide or fragments of the invention. Such method
have been known in the art since the early 1960's (Merrifield, R B., J.
Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,
J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,
Rockford, Ill., pp. 11-12)) and have recently been employed in
commercially available laboratory peptide design and synthesis kits
(Cambridge Research Biochemicals). Such commercially available laboratory
kits have generally utilized the teachings of H. M. Geysen et al, Proc.
Natl. Acad. Sci., USA, 81:3998 (1984) and provide for synthesizing
peptides upon the tips of a multitude of "rods" or "pins" all of which
are connected to a single plate. When such a system is utilized, a plate
of rods or pins is inverted and inserted into a second plate of
corresponding wells or reservoirs, which contain solutions for attaching
or anchoring an appropriate amino acid to the pin's or rod's tips. By
repeating such a process step, i.e., inverting and inserting the rod's
and pin's tips into appropriate solutions, amino acids are built into
desired peptides. In addition, a number of available FMOC peptide
synthesis systems are available. For example, assembly of a polypeptide
or fragment can be carried out on a solid support using an Applied
Biosystems, Inc. Model 431A.TM. automated peptide synthesizer. Such
equipment provides ready access to the peptides of the invention, either
by direct synthesis or by synthesis of a series of fragments that can be
coupled using other known techniques.
[0489] The invention includes glucanases, mannanases, or xylanases of the
invention with and without signal. The polypeptide comprising a signal
sequence of the invention can be a glucanase of the invention or another
glucanase or another enzyme or other polypeptide.
[0490] The invention includes immobilized glucanases, mannanases, or
xylanases, anti-glucanase, -mannanase, or -xylanase antibodies and
fragments thereof. The invention provides methods for inhibiting
glucanase, mannanase, or xylanase activity, e.g., using dominant negative
mutants or anti-glucanase, -mannanase, or -xylanase antibodies of the
invention. The invention includes heterocomplexes, e.g., fusion proteins,
heterodimers, etc., comprising the glucanases of the invention.
[0491] Polypeptides of the invention can have a glucanase, mannanase, or
xylanase activity under various conditions, e.g., extremes in pH and/or
temperature, oxidizing agents, and the like. The invention provides
methods leading to alternative glucanase, mannanase, or xylanase
preparations with different catalytic efficiencies and stabilities, e.g.,
towards temperature, oxidizing agents and changing wash conditions. In
one aspect, glucanase, mannanase, or xylanase variants can be produced
using techniques of site-directed mutagenesis and/or random mutagenesis.
In one aspect, directed evolution can be used to produce a great variety
of glucanase, mannanase, or xylanase variants with alternative
specificities and stability.
[0492] The proteins of the invention are also useful as research reagents
to identify glucanase, mannanase, or xylanase modulators, e.g.,
activators or inhibitors of glucanase, mannanase, or xylanase activity.
Briefly, test samples (compounds, broths, extracts, and the like) are
added to glucanase, mannanase, or xylanase assays to determine their
ability to inhibit substrate cleavage. Inhibitors identified in this way
can be used in industry and research to reduce or prevent undesired
proteolysis. Glucanase, mannanase, or xylanase inhibitors can be combined
to increase the spectrum of activity.
[0493] The enzymes of the invention are also useful as research reagents
to digest proteins or in protein sequencing. For example, a glucanase,
mannanase, or xylanase may be used to break polypeptides into smaller
fragments for sequencing using, e.g. an automated sequencer.
[0494] The invention also provides methods of discovering a new glucanase,
mannanase, or xylanase using the nucleic acids, polypeptides and
antibodies of the invention. In one aspect, phagemid libraries are
screened for expression-based discovery of a glucanase, mannanase, or
xylanase. In another aspect, lambda phage libraries are screened for
expression-based discovery of a glucanase, mannanase, or xylanase.
Screening of the phage or phagemid libraries can allow the detection of
toxic clones; improved access to substrate; reduced need for engineering
a host, by-passing the potential for any bias resulting from mass
excision of the library; and, faster growth at low clone densities.
Screening of phage or phagemid libraries can be in liquid phase or in
solid phase. In one aspect, the invention provides screening in liquid
phase. This gives a greater flexibility in assay conditions; additional
substrate flexibility; higher sensitivity for weak clones; and ease of
automation over solid phase screening.
[0495] The invention provides screening methods using the proteins and
nucleic acids of the invention and robotic automation to enable the
execution of many thousands of biocatalytic reactions and screening
assays in a short period of time, e.g., per day, as well as ensuring a
high level of accuracy and reproducibility (see discussion of arrays,
below). As a result, a library of derivative compounds can be produced in
a matter of weeks. For further teachings on modification of molecules,
including small molecules, see PCT/US94/09174.
[0496] Another aspect of the invention is an isolated or purified
polypeptide comprising the sequence of one of the invention, or fragments
comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or
150 consecutive amino acids thereof. As discussed above, such
polypeptides may be obtained by inserting a nucleic acid encoding the
polypeptide into a vector such that the coding sequence is operably
linked to a sequence capable of driving the expression of the encoded
polypeptide in a suitable host cell. For example, the expression vector
may comprise a promoter, a ribosome binding site for translation
initiation and a transcription terminator. The vector may also include
appropriate sequences for amplifying expression.
[0497] Another aspect of the invention is polypeptides or fragments
thereof which have at least about 50%, at least about 55%, at least about
60%, at least about 65%, at least about 70%, at least about 75%, at least
about 80%, at least about 85%, at least about 90%, at least about 95%, or
more than about 95% sequence identity (homology) to one of the
polypeptides of the invention, or a fragment comprising at least 5, 10,
15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more consecutive amino
acids thereof. Sequence identity (homology) may be determined using any
of the programs described above which aligns the polypeptides or
fragments being compared and determines the extent of amino acid identity
or similarity between them. It will be appreciated that amino acid
equivalence, or identity, or "homology," includes conservative amino acid
substitutions such as those described above.
[0498] The polypeptides or fragments having homology to one of the
polypeptides of the invention, or a fragment comprising at least about 5,
10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids
thereof may be obtained by isolating the nucleic acids encoding them
using the techniques described above.
[0499] Alternatively, the homologous polypeptides or fragments may be
obtained through biochemical enrichment or purification procedures. The
sequence of potentially homologous polypeptides or fragments may be
determined by glucan hydrolase digestion, gel electrophoresis and/or
microsequencing. The sequence of the prospective homologous polypeptide
or fragment can be compared to one of the polypeptides of the invention,
or a fragment comprising at least about 5, 10, 15, 20, 25, 30, 35, 40,
50, 75, 100, or 150 consecutive amino acids thereof using any of the
programs described above.
[0500] Another aspect of the invention is an assay for identifying
fragments or variants of the invention, which retain the enzymatic
function of the polypeptides of the invention. For example the fragments
or variants of said polypeptides, may be used to catalyze biochemical
reactions, which indicate that the fragment or variant retains the
enzymatic activity of a polypeptide of the invention.
[0501] The assay for determining if fragments of variants retain the
enzymatic activity of the polypeptides of the invention includes the
steps of: contacting the polypeptide fragment or variant with a substrate
molecule under conditions which allow the polypeptide fragment or variant
to function and detecting either a decrease in the level of substrate or
an increase in the level of the specific reaction product of the reaction
between the polypeptide and substrate.
[0502] The polypeptides of the invention or fragments comprising at least
5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino
acids thereof may be used in a variety of applications. For example, the
polypeptides or fragments thereof may be used to catalyze biochemical
reactions. In accordance with one aspect of the invention, there is
provided a process for utilizing the polypeptides of the invention or
polynucleotides encoding such polypeptides for hydrolyzing glycosidic
linkages. In such procedures, a substance containing a glycosidic linkage
(e.g., a starch) is contacted with one of the polypeptides of the
invention, or sequences substantially identical thereto under conditions
which facilitate the hydrolysis of the glycosidic linkage.
[0503] The present invention exploits the unique catalytic properties of
enzymes. Whereas the use of biocatalysts (i.e., purified or crude
enzymes, non-living or living cells) in chemical transformations normally
requires the identification of a particular biocatalyst that reacts with
a specific starting compound, the present invention uses selected
biocatalysts and reaction conditions that are specific for functional
groups that are present in many starting compounds, such as small
molecules. Each biocatalyst is specific for one functional group, or
several related functional groups and can react with many starting
compounds containing this functional group.
[0504] The biocatalytic reactions produce a population of derivatives from
a single starting compound. These derivatives can be subjected to another
round of biocatalytic reactions to produce a second population of
derivative compounds. Thousands of variations of the original small
molecule or compound can be produced with each iteration of biocatalytic
derivatization.
[0505] Enzymes react at specific sites of a starting compound without
affecting the rest of the molecule, a process which is very difficult to
achieve using traditional chemical methods. This high degree of
biocatalytic specificity provides the means to identify a single active
compound within the library. The library is characterized by the series
of biocatalytic reactions used to produce it, a so called "biosynthetic
history". Screening the library for biological activities and tracing the
biosynthetic history identifies the specific reaction sequence producing
the active compound. The reaction sequence is repeated and the structure
of the synthesized compound determined. This mode of identification,
unlike other synthesis and screening approaches, does not require
immobilization technologies and compounds can be synthesized and tested
free in solution using virtually any type of screening assay. It is
important to note, that the high degree of specificity of enzyme
reactions on functional groups allows for the "tracking" of specific
enzymatic reactions that make up the biocatalytically produced library.
[0506] Many of the procedural steps are performed using robotic automation
enabling the execution of many thousands of biocatalytic reactions and
screening assays per day as well as ensuring a high level of accuracy and
reproducibility. As a result, a library of derivative compounds can be
produced in a matter of weeks which would take years to produce using
current chemical methods.
[0507] In a particular aspect, the invention provides a method for
modifying small molecules, comprising contacting a polypeptide encoded by
a polynucleotide described herein or enzymatically active fragments
thereof with a small molecule to produce a modified small molecule. A
library of modified small molecules is tested to determine if a modified
small molecule is present within the library which exhibits a desired
activity. A specific biocatalytic reaction which produces the modified
small molecule of desired activity is identified by systematically
eliminating each of the biocatalytic reactions used to produce a portion
of the library and then testing the small molecules produced in the
portion of the library for the presence or absence of the modified small
molecule with the desired activity. The specific biocatalytic reactions
which produce the modified small molecule of desired activity is
optionally repeated. The biocatalytic reactions are conducted with a
group of biocatalysts that react with distinct structural moieties found
within the structure of a small molecule, each biocatalyst is specific
for one structural moiety or a group of related structural moieties; and
each biocatalyst reacts with many different small molecules which contain
the distinct structural moiety.
[0508] Signal Sequences, Prepro and Catalytic Domains
[0509] The invention provides glucanase, mannanase, or xylanase signal
sequences (e.g., signal peptides (SPs)), prepro domains and catalytic
domains (CDs) (e.g., active sites). The SPs, prepro domains and/or CDs of
the invention can be isolated or recombinant peptides or can be part of a
fusion protein, e.g., as a heterologous domain in a chimeric protein. The
invention provides nucleic acids encoding these catalytic domains (CDs),
prepro domains and signal sequences (SPs, e.g., a peptide having a
sequence comprising/consisting of amino terminal residues of a
polypeptide of the invention). In one aspect, the invention provides a
signal sequence comprising a peptide comprising/consisting of a sequence
as set forth in residues 1 to 1-5, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1
to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1
to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1
to 36, 1 to 37, 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1
to 44 of a polypeptide of the invention.
[0510] In one aspect, the invention also provides chimeric polypeptides
(and the nucleic acids encoding them) comprising at least two enzymes of
the invention or subsequences thereof, e.g., catalytic domains (CDs) or
active sites. For example, a chimeric protein of the invention can have
mannanase and xylanase activity, mannanase and glycanase activity, etc.
In one aspect the chimeric protein of the invention comprises a fusion of
domains, e.g., a single domain can exhibit glucanase/xylanase/mannanase
or any combination of activities (e.g.; as a recombinant chimeric
protein).
[0511] The invention also provides isolated, synthetic or recombinant
signal sequences comprising/consisting of a signal sequence of the
invention, e.g., exemplary signal sequences as set forth in Table 3,
below, and polypeptides comprising these signal sequences. The
polypeptide can be another glucanase, mannanase, or xylanase of the
invention, another glucanase, mannanase, or xylanase, another glycosidase
or hydrolase, or another type of enzyme or polypeptide. For example,
reading Table 3, the invention provides an isolated, synthetic or
recombinant signal sequence as set forth by residues 1 to 21 of SEQ ID
NO:102, which in one aspect is encoded by, e.g., a subsequence of SEQ ID
NO:101; or, the invention provides an isolated, synthetic or recombinant
signal sequence as set forth by residues 1 to 30 of SEQ ID NO:104, which
in one aspect is encoded by a subsequence of SEQ ID NO:103, etc.
TABLE-US-00004
TABLE 3
SEQ ID Signal
NO: (AA) Predicted Signal Sequence
101, 102 1-21
103, 104 1-30
105, 106 1-33
107, 108 1-18
109, 110 1-25
11, 12 1-25
111, 112 1-39
113, 114 1-24
115, 116 1-21
117, 118 1-29
121, 122 1-24
123, 124 1-30
125, 126 1-19
127, 128 1-22
129, 130 1-40
13, 14 1-22
133, 134 1-34
135, 136 1-53
137, 138 1-37
139, 140 1-22
141, 142 1-32
143, 144 1-29
145, 146 1-29
147, 148 1-29
149, 150 1-30
15, 16 1-44
151, 152 1-22
153, 154 1-24
155, 156 1-21
157, 158 1-38
159, 160 1-34
161, 162 1-19
163, 164 1-19
165, 166 1-51
169, 170 1-22
171, 172 1-22
173, 174 1-19
175, 176 1-19
177, 178 1-26
179, 180 1-71
181, 182 1-35
183, 184 1-22
185, 186 1-17
187, 188 1-22
19, 20 1-68
191, 192 1-20
193, 194 1-29
199, 200 1-22 MKTKLISTLVAGLIVISPATYA
201, 202 1-32
203, 204 1-27
205, 206 1-34
207, 208 1-28
209, 210 1-22
21, 22 1-34
211, 212 1-22
213, 214 1-22
215, 216 1-42
217, 218 1-23
219, 220 1-27
221, 222 1-29
223, 224 1-31
225, 226 1-29
227, 228 1-22 MTSKHFFKITLMSILLFTTTLA
229, 230 1-25 MKRRNWNYLLIILLVISAFTLISAQ
23, 24 1-22
235, 236 1-19 MKSVLALALIVSINLVLLA
237, 238 1-29 MTRRSIVRSSSNKWLVLAGAALLACTALG
241, 242 1-39 MSSFKASAINPRMAGALTRSLYAAGFSLAVSTLSTQ
AYA
243, 244 1-26 MKKLLKLSMLSTSVALGIMASSGAIA
247, 248 1-21 MNVLRSGIVTMLLLAAFSVQA
25, 26 1-21
251, 252 1-23 MLKKLALAAGIAAATLAASGSHG
255, 256 1-28 MKRTGWTLKLLLAALLLLPATLGLHNGA
257, 258 1-30 MYRLFFRSLKRSGILLPVLLYFFILPSATA
269, 270 1-22 MKFTLTPLLCGFALLLGCAVQA
27, 28 1-19
271, 272 1-18 MVSMLLLTVGAVSVSAVS
273, 274 1-37 MPRLRARTRPRRQLTALAAALSLPLGLTAVGATTAQ
A
275, 276 1-27 MQNLFKRVFFHLLLLALLAGCAGPSPV
277, 278 1-28 MSGRSAGRGPWARLWALAAVGALVAGA
279, 280 1-38 MQNKIINTKIKLRKFMSQLIKITYIFIIIIFCMQRT
YA
281, 282 1-18 MKKLILTLFSLWAISAYA
285, 286 1-27 MRKSIRSFSILLAITFIIALLSFPAMG
287, 288 1-30 MNPRSLRRRTTAALAALAACAALLATQAQA
289, 290 1-29 MFPRLSPSRFRQVTLTLLTLGLVSLTGCA
29, 30 1-19
291, 292 1-16 MKFFTVLLFFLSFVFS
293, 294 1-27 MRRRIRALVAALSALPLALVVAPSAHA
3, 4 1-25
301, 302 1-17 MAIGISATMLLAMPQQA
303, 304 1-30 MSCRTLMSRRVGWGLLLWGGLFLRTGSVTG
305, 306 1-26 MNPKYIYRITFLLISILSMTALQSFS
307, 308 1-52 MVWTPARSTLAGSSEIPLMTMMFPNRKDSRMSLWIK
LGILCMMAGTVMVHG
31, 32 1-39
311, 312 1-24 MKRREFMLGGAGVAALASTLGVSA
313, 314 1-20 MLIIGGLLVLLGFSSCGRQA
315, 316 1-32 MDKTITAKDSGKITALILIILLVLPYAGYVVA
319, 320 1-29 MREIILKSGALLMWILIVSILQILTVEA
323, 324 1-32 MFQSLKMRTLSFLLLMALLASFLALPTDVAHA
325, 326 1-29 MKKIILKSGILLLVVILIVSILQILPVFA
327, 328 1-46 MLVYRVSIQKHLASLTVLVSLLLILAGCSSSSDSIA
PVSSSSVSSA
33, 34 1-35
331, 332 1-28 MNNPTNGARRGRHRRRWSATALLLGVPA
333, 334 1-28 MNRTRVLSAATLLALVATLASVPVTAQA
337, 338 1-29 MRNHLNVPFYFIFFFLIASIFTVCSSSTA
339, 340 1-23 MNNPRILTYLLIGIWAVLIVFA
343, 344 1-31 MRKIVKQINYLTPSVLGLLVLSLFFQVPTQA
345, 346 1-33 MKRTRYGVRSPRSAPRFGVLFGAAAAGVLMTGA
349, 350 1-38 MNSSPVSVKKPCPVDRPNPLWAAGFSLALATLSTQT
QA
351, 352 1-28 MKKVSNARVLSFLLILVLIFGNLASVFA
357, 358 1-32 MEKQICSNVFSTMLIIGGLLVLLGFSSCGRQA
359, 360 1-22 MRRLITIILATAVAILSTTSCS
361, 362 1-20 MSRGILILVMLSVLSGAALA
363, 364 1-30 MRRTRSLLAGLALTAGLLTGAGAGAPPATA
365, 366 1-50 MLGAPSPHFPMRRGMTKSQRRTWLTAVGSAIAGIAG
LLLPVFATAGAAQA
367, 368 1-42 MPHPKLLTNGGSYVSSKQKTVAIFVLFWLAGVAGSI
PASYA
37, 38 1-33
373, 374 1-23 MNKILKLFSSLLLFAGICPALQA
377, 378 1-30 MKMLTTLKKPLLKKTALALLTSAMVAPAFA
381, 382 1-26 MRAIRLSLSIAAGAVLLLAGCTTKPA
383, 384 1-28 MTMHRKLHRSIAAGALSAIFFVGLQAGA
387, 388 1-25 MSIIKKVPLIFLCLLMFATSLFIFK
39, 40 1-24
393, 394 1-29 MSKFLSLSNFFSLLVVCVLLGACSGGSSS
401, 402 1-30 MGTSLMIKSTLTGMITAVAAAVFTTSAAFA
403, 404 1-32 MGKISKYFAMFLAFLMVFSSLFVNFQPRNVQA
407, 408 1-28 MRKNILMLAVAMIAAMCVTTSCGNKAQK
409, 410 1-24 MTRNWLGKILAALLLAGCAIPAPA
411, 412 1-24 MPYVVRLALVCAWTVLACTGAPIA
413, 414 1-28 MSRHLISLGLLVVVALGAMLWISSRDVA
419, 420 1-21 MLRKLILFCAVLLSMSWVALA
421, 422 1-26 MKTKSIYSIAILSIALFFFTTAQTFS
423, 424 1-25 MWSQDVRKVWLVGFLLLVAGMPALA
425, 426 1-20 MRIRLATLALCMLSPVTFA
427, 428 1-43 METPMTSARSARPRPRLRRYGIAGTALGALLLGLAT
LPPTATA
43, 44 1-48
431, 432 1-22 MKFTLMPLLCGFALLLGCAVQA
441, 442 1-26 MRAIRLSLSIAAGAVLLLAGCTTKPA
445, 446 1-29 MRRFRWFLGLFVFFGIVIASQYGQTAAA
449, 450 1-25 MKKIVSLVCVLVMLVSILGSFSWA
45, 46 1-16
457, 458 1-39 MKSKTSTAAPSAGPLRNYKKLTACIAVASTALLAGS
ASA
459, 460 1-18 MKKLILTLFSLWAISAYA
461, 462 1-28 MFKHLLHVLKIGFLPLLATLLLAGHAHG
465, 466 1-21 MLRKLIVSVFGFVMLTSAAAA
469, 470 1-25 MKYKAIFIYLIVLILFYSINIYANA
47, 48 1-23
471, 472 1-22 MKTKLISTLVAGLIVISPATYA
473, 474 1-28 MKRKRVFIHSLIVFFLMIGSFTSCGSVA
475, 476 1-25 MNLLAQYFSGLFLIFLISIFFVSSA
477, 478 1-27 MKSIRSRSLATAVLAGALGVAAAGAQA
481, 482 1-21 MKLLKLLIFLLITVIFSDVSA
483, 484 1-26 MYKRLLSSVLIIMLLLSAWSPISVQA
489, 490 1-23 MKYIFSYIIMMILIGFIPVYGFG
49, 50 1-42
491, 492 1-26 MSMFLSLKRVAALVCVAGFGISAANA
493, 494 1-28 MPYLKRVLLLLVTGLFMSLFAVTSTASA
495, 496 1-29 MSSKQKTVAIFVLFVALAGVAGSIPASYA
499, 500 1-17 MKKLVLVLLLFPVFILA
505, 506 1-24 MKSKVKMFFAAAIVWSACSSTGYA
509, 510 1-20 MPKKLLASFIALFFAANAAA
51, 52 1-38
511, 512 1-21 MKKLHILLLALTAMTAFASCS
[0512] The glucanase, mannanase, or xylanase signal sequences (SPs) and/or
prepro sequences of the invention can be isolated peptides, or, sequences
joined to another glucanase, mannanase, or xylanase or a non-glucanase,
mannanase, or xylanase polypeptide, e.g., as a fusion (chimeric) protein.
In one aspect, the invention provides polypeptides comprising glucanase,
mannanase, or xylanase signal sequences of the invention. In one aspect,
polypeptides comprising glucanase, mannanase, or xylanase signal
sequences SPs and/or prepro of the invention comprise sequences
heterologous to a glucanase, mannanase, or xylanase of the invention
(e.g., a fusion protein comprising an SP and/or prepro of the invention
and sequences from another glucanase or a non-glucanase protein). In one
aspect, the invention provides a glucanase, mannanase, or xylanase of the
invention with heterologous SPs and/or prepro sequences, e.g., sequences
with a yeast signal sequence. A glucanase, mannanase, or xylanase of the
invention can comprise a heterologous SP and/or prepro in a vector, e.g.,
a pPIC series vector (Invitrogen, Carlsbad, Calif.).
[0513] In one aspect, SPs and/or prepro sequences of the invention are
identified following identification of novel glucanase, mannanase, or
xylanase polypeptides. The pathways by which proteins are sorted and
transported to their proper cellular location are often referred to as
protein targeting pathways. One of the most important elements in all of
these targeting systems is a short amino acid sequence at the amino
terminus of a newly synthesized polypeptide called the signal sequence.
This signal sequence directs a protein to its appropriate location in the
cell and is removed during transport or when the protein reaches its
final destination. Most lysosomal, membrane, or secreted proteins have an
amino-terminal signal sequence that marks them for translocation into the
lumen of the endoplasmic reticulum. More than 100 signal sequences for
proteins in this group have been determined. The signal sequences can
vary in length from 13 to 36 amino acid residues. Various methods of
recognition of signal sequences are known to those of skill in the art.
For example, in one aspect, novel glucanase, mannanase, or xylanase
signal peptides are identified by a method referred to as SignalP.
SignalP uses a combined neural network which recognizes both signal
peptides and their cleavage sites. (Nielsen, et al., "Identification of
prokaryotic and eukaryotic signal peptides and prediction of their
cleavage sites." Protein Engineering, vol. 10, no. 1, p. 1-6 (1997).
[0514] It should be understood that in some aspects a glucanase,
mannanase, or xylanase of the invention may not have SPs and/or prepro
sequences, or "domains." In one aspect, the invention provides a
glucanase, mannanase, or xylanase of the invention lacking all or part of
an SP and/or a prepro domain. In one aspect, the invention provides a
nucleic acid sequence encoding a signal sequence (SP) and/or prepro from
one glucanase, mannanase, or xylanase operably linked to a nucleic acid
sequence of a different glucanase or, optionally, a signal sequence (SPs)
and/or prepro domain from a non-glucanase, mannanase, or xylanase protein
may be desired.
[0515] The invention also provides isolated or recombinant polypeptides
comprising signal sequences (SPs), prepro domain and/or catalytic domains
(CDs) of the invention and heterologous sequences. The heterologous
sequences are sequences not naturally associated (e.g., to a glucanase,
mannanase, or xylanase) with an SP, prepro domain and/or CD. The sequence
to which the SP, prepro domain and/or CD are not naturally associated can
be on the SP's, prepro domain and/or CD's amino terminal end, carboxy
terminal end, and/or on both ends of the SP and/or CD. In one aspect, the
invention provides an isolated or recombinant polypeptide comprising (or
consisting of) a polypeptide comprising a signal sequence (SP), prepro
domain and/or catalytic domain (CD) of the invention with the proviso
that it is not associated with any sequence to which it is naturally
associated (e.g., a glucanase, mannanase, or xylanase sequence).
Similarly in one aspect, the invention provides isolated or recombinant
nucleic acids encoding these polypeptides. Thus, in one aspect, the
isolated or recombinant nucleic acid of the invention comprises coding
sequence for a signal sequence (SP), prepro domain and/or catalytic
domain (CD) of the invention and a heterologous sequence (i.e., a
sequence not naturally associated with the a signal sequence (SP), prepro
domain and/or catalytic domain (CD) of the invention). The heterologous
sequence can be on the 3' terminal end, 5' terminal end, and/or on both
ends of the SP, prepro domain and/or CD coding sequence.
Hybrid (Chimeric) Glucanase, Mannanase, or Xylanase and Peptide Libraries
[0516] In one aspect, the invention provides hybrid glucanases,
mannanases, or xylanases and fusion proteins, including peptide
libraries, comprising sequences of the invention. The peptide libraries
of the invention can be used to isolate peptide modulators (e.g.,
activators or inhibitors) of targets, such as glucanase, mannanase, or
xylanase substrates, receptors, enzymes. The peptide libraries of the
invention can be used to identify formal binding partners of targets,
such as ligands, e.g., cytokines, hormones and the like. In one aspect,
the invention provides chimeric proteins, comprising a signal sequence
(SP), prepro domain and/or catalytic domain (CD) of the invention or a
combination thereof and a heterologous sequence (see above).
[0517] In one aspect, the fusion proteins of the invention (e.g., the
peptide moiety) are conformationally stabilized (relative to linear
peptides) to allow a higher binding affinity for targets. The invention
provides fusions of a glucanase, mannanase, or xylanase of the invention
and other peptides, including known and random peptides. They can be
fused in such a manner that the structure of a glucanase, mannanase, or
xylanase is not significantly perturbed and the peptide is metabolically
or structurally conformationally stabilized. This allows the creation of
a peptide library that is easily monitored both for its presence within
cells and its quantity.
[0518] Amino acid sequence variants of the invention can be characterized
by a predetermined nature of the variation, a feature that sets them
apart from a naturally occurring form, e.g., an allelic or interspecies
variation of a glucanase, mannanase, or xylanase sequence. In one aspect,
the variants of the invention exhibit the same qualitative biological
activity as the naturally occurring analogue. Alternatively, the variants
can be selected for having modified characteristics. In one aspect, while
the site or region for introducing an amino acid sequence variation is
predetermined, the mutation per se need not be predetermined. For
example, in order to optimize the performance of a mutation at a given
site, random mutagenesis may be conducted at the target codon or region
and the expressed glucanase, mannanase, or xylanase variants screened for
the optimal combination of desired activity. Techniques for making
substitution mutations at predetermined sites in DNA having a known
sequence are well known, as discussed herein for example, M13 primer
mutagenesis and PCR mutagenesis. Screening of the mutants can be done
using, e.g., assays of glucan hydrolysis. In alternative aspects, amino
acid substitutions can be single residues; insertions can be on the order
of from about 1 to 20 amino acids, although considerably larger
insertions can be done. Deletions can range from about 1 to about 20, 30,
40, 50, 60, 70 residues or more. To obtain a final derivative with the
optimal properties, substitutions, deletions, insertions or any
combination thereof may be used. Generally, these changes are done on a
few amino acids to minimize the alteration of the molecule. However,
larger changes may be tolerated in certain circumstances.
[0519] The invention provides a glucanase, mannanase, or xylanase where
the structure of the polypeptide backbone, the secondary or the tertiary
structure, e.g., an alpha-helical or beta-sheet structure, has been
modified. In one aspect, the charge or hydrophobicity has been modified.
In one aspect, the bulk of a side chain has been modified. Substantial
changes in function or immunological identity are made by selecting
substitutions that are less conservative. For example, substitutions can
be made which more significantly affect: the structure of the polypeptide
backbone in the area of the alteration, for example a alpha-helical or a
beta-sheet structure; a charge or a hydrophobic site of the molecule,
which can be at an active site; or a side chain. The invention provides
substitutions in polypeptide of the invention where (a) a hydrophilic
residues, e.g. seryl or threonyl, is substituted for (or by) a
hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or
alanyl; (b) a cysteine or proline is substituted for (or by) any other
residue; (c) a residue having an electropositive side chain, e.g. lysyl,
arginyl, or histidyl, is substituted for (or by) an electronegative
residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side
chain, e.g. phenylalanine, is substituted for (or by) one not having a
side chain, e.g. glycine. The variants can exhibit the same qualitative
biological activity (i.e. endoglucanase, mannanase, or xylanase activity)
although variants can be selected to modify the characteristics of the
glucanase, mannanase, or xylanase as needed.
[0520] In one aspect, glucanase, mannanase, or xylanase of the invention
comprise epitopes or purification tags, signal sequences or other fusion
sequences, etc. In one aspect, the glucanase, mannanase, or xylanase of
the invention can be fused to a random peptide to form a fusion
polypeptide. By "fused" or "operably linked" herein is meant that the
random peptide and the glucanase, mannanase, or xylanase are linked
together, in such a manner as to minimize the disruption to the stability
of the glucanase structure, e.g., it retains glucanase, mannanase, or
xylanase activity. The fusion polypeptide (or fusion polynucleotide
encoding the fusion polypeptide) can comprise further components as well,
including multiple peptides at multiple loops.
[0521] In one aspect, the peptides and nucleic acids encoding them are
randomized, either fully randomized or they are biased in their
randomization, e.g. in nucleotide/residue frequency generally or per
position. "Randomized" means that each nucleic acid and peptide consists
of essentially random nucleotides and amino acids, respectively. In one
aspect, the nucleic acids which give rise to the peptides can be
chemically synthesized, and thus may incorporate any nucleotide at any
position. Thus, when the nucleic acids are expressed to form peptides,
any amino acid residue may be incorporated at any position. The synthetic
process can be designed to generate randomized nucleic acids, to allow
the formation of all or most of the possible combinations over the length
of the nucleic acid, thus forming a library of randomized nucleic acids.
The library can provide a sufficiently structurally diverse population of
randomized expression products to affect a probabilistically sufficient
range of cellular responses to provide one or more cells exhibiting a
desired response. Thus, the invention provides an interaction library
large enough so that at least one of its members will have a structure
that gives it affinity for some molecule, protein, or other factor.
[0522] Endoglucanases are multidomain enzymes that consist optionally of a
signal peptide, a carbohydrate binding module, a glucanase catalytic
domain, a linker and/or another catalytic domain.
[0523] The invention provides a means for generating chimeric polypeptides
which may encode biologically active hybrid polypeptides (e.g., hybrid
glucanases, mannanases, or xylanases). In one aspect, the original
polynucleotides encode biologically active polypeptides. The method of
the invention produces new hybrid polypeptides by utilizing cellular
processes which integrate the sequence of the original polynucleotides
such that the resulting hybrid polynucleotide encodes a polypeptide
demonstrating activities derived from the original biologically active
polypeptides. For example, the original polynucleotides may encode a
particular enzyme from different microorganisms. An enzyme encoded by a
first polynucleotide from one organism or variant may, for example,
function effectively under a particular environmental condition, e.g.
high salinity. An enzyme encoded by a second polynucleotide from a
different organism or variant may function effectively under a different
environmental condition, such as extremely high temperatures. A hybrid
polynucleotide containing sequences from the first and second original
polynucleotides may encode an enzyme which exhibits characteristics of
both enzymes encoded by the original polynucleotides. Thus, the enzyme
encoded by the hybrid polynucleotide may function effectively under
environmental conditions shared by each of the enzymes encoded by the
first and second polynucleotides, e.g., high salinity and extreme
temperatures.
[0524] A hybrid polypeptide resulting from the method of the invention may
exhibit specialized enzyme activity not displayed in the original
enzymes. For example, following recombination and/or reductive
reassortment of polynucleotides encoding hydrolase activities, the
resulting hybrid polypeptide encoded by a hybrid polynucleotide can be
screened for specialized hydrolase activities obtained from each of the
original enzymes, i.e. the type of bond on which the hydrolase acts and
the temperature at which the hydrolase functions. Thus, for example, the
hydrolase may be screened to ascertain those chemical functionalities
which distinguish the hybrid hydrolase from the original hydrolases, such
as: (a) amide (peptide bonds), i.e., endoglucanases; (b) ester bonds,
i.e., esterases and lipases; (c) acetals, i.e., glycosidases and, for
example, the temperature, pH or salt concentration at which the hybrid
polypeptide functions.
[0525] Sources of the original polynucleotides may be isolated from
individual organisms ("isolates"), collections of organisms that have
been grown in defined media ("enrichment cultures"), or, uncultivated
organisms ("environmental samples"). The use of a culture-independent
approach to derive polynucleotides encoding novel bioactivities from
environmental samples is most preferable since it allows one to access
untapped resources of biodiversity.
[0526] "Environmental libraries" are generated from environmental samples
and represent the collective genomes of naturally occurring organisms
archived in cloning vectors that can be propagated in suitable
prokaryotic hosts. Because the cloned DNA is initially extracted directly
from environmental samples, the libraries are not limited to the small
fraction of prokaryotes that can be grown in pure culture. Additionally,
a normalization of the environmental DNA present in these samples could
allow more equal representation of the DNA from all of the species
present in the original sample. This can dramatically increase the
efficiency of finding interesting genes from minor constituents of the
sample which may be under-represented by several orders of magnitude
compared to the dominant species.
[0527] For example, gene libraries generated from one or more uncultivated
microorganisms are screened for an activity of interest. Potential
pathways encoding bioactive molecules of interest are first captured in
prokaryotic cells in the form of gene expression libraries.
Polynucleotides encoding activities of interest are isolated from such
libraries and introduced into a host cell. The host cell is grown under
conditions which promote recombination and/or reductive reassortment
creating potentially active biomolecules with novel or enhanced
activities.
[0528] Additionally, subcloning may be performed to further isolate
sequences of interest. In subcloning, a portion of DNA is amplified,
digested, generally by restriction enzymes, to cut out the desired
sequence, the desired sequence is ligated into a recipient vector and is
amplified. At each step in subcloning, the portion is examined for the
activity of interest, in order to ensure that DNA that encodes the
structural protein has not been excluded. The insert may be purified at
any step of the subcloning, for example, by gel electrophoresis prior to
ligation into a vector or where cells containing the recipient vector and
cells not containing the recipient vector are placed on selective media
containing, for example, an antibiotic, which will kill the cells not
containing the recipient vector. Specific methods of subcloning cDNA
inserts into vectors are well-known in the art (Sambrook et al.,
Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor
Laboratory Press (1989)). In another aspect, the enzymes of the invention
are subclones. Such subclones may differ from the parent clone by, for
example, length, a mutation, a tag or a label.
[0529] In one aspect, the signal sequences of the invention are identified
following identification of novel glucanase, mannanase, or xylanase
polypeptides. The pathways by which proteins are sorted and transported
to their proper cellular location are often referred to as protein
targeting pathways. One of the most important elements in all of these
targeting systems is a short amino acid sequence at the amino terminus of
a newly synthesized polypeptide called the signal sequence. This signal
sequence directs a protein to its appropriate location in the cell and is
removed during transport or when the protein reaches its final
destination. Most lysosomal, membrane, or secreted proteins have an
amino-terminal signal sequence that marks them for translocation into the
lumen of the endoplasmic reticulum. More than 100 signal sequences for
proteins in this group have been determined. The sequences vary in length
from 13 to 36 amino acid residues. Various methods of recognition of
signal sequences are known to those of skill in the art. In one aspect,
the peptides are identified by a method referred to as SignalP. SignalP
uses a combined neural network which recognizes both signal peptides and
their cleavage sites. See, e.g., Nielsen (1997) "Identification of
prokaryotic and eukaryotic signal peptides and prediction of their
cleavage sites." Protein Engineering, vol. 10, no. 1, p. 1-6. It should
be understood that some of the glucanases, mannanases, or xylanases of
the invention may or may not contain signal sequences. It may be
desirable to include a nucleic acid sequence encoding a signal sequence
from one glucanase, mannanase, or xylanase operably linked to a nucleic
acid sequence of a different glucanase, mannanase, or xylanase or,
optionally, a signal sequence from a non-glucanase, mannanase, or
xylanase protein may be desired.
[0530] The microorganisms from which the polynucleotide may be prepared
include prokaryotic microorganisms, such as Eubacteria and Archaebacteria
and lower eukaryotic microorganisms such as fungi, some algae and
protozoa. Polynucleotides may be isolated from environmental samples in
which case the nucleic acid may be recovered without culturing of an
organism or recovered from one or more cultured organisms. In one aspect,
such microorganisms may be extremophiles, such as hyperthermophiles,
psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles.
Polynucleotides encoding enzymes isolated from extremophilic
microorganisms can be used. Such enzymes may function at temperatures
above 100.degree. C. in terrestrial hot springs and deep sea thermal
vents, at temperatures below 0.degree. C. in arctic waters, in the
saturated salt environment of the Dead Sea, at pH values around 0 in coal
deposits and geothermal sulfur-rich springs, or at pH values greater than
11 in sewage sludge. For example, several esterases and lipases cloned
and expressed from extremophilic organisms show high activity throughout
a wide range of temperatures and pHs.
[0531] Polynucleotides selected and isolated as hereinabove described are
introduced into a suitable host cell. A suitable host cell is any cell
which is capable of promoting recombination and/or reductive
reassortment. The selected polynucleotides are in one aspect already in a
vector which includes appropriate control sequences. The host cell can be
a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic
cell, such as a yeast cell, or in one aspect, the host cell can be a
prokaryotic cell, such as a bacterial cell. Introduction of the construct
into the host cell can be effected by calcium phosphate transfection,
DEAE-Dextran mediated transfection, or electroporation (Davis et al.,
1986).
[0532] As representative examples of appropriate hosts, there may be
mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella
typhimurium; fungal cells, such as yeast; insect cells such as Drosophila
S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;
adenoviruses; and plant cells. The selection of an appropriate host is
deemed to be within the scope of those skilled in the art from the
teachings herein.
[0533] With particular references to various mammalian cell culture
systems that can be employed to express recombinant protein, examples of
mammalian expression systems include the COS-7 lines of monkey kidney
fibroblasts, described in "SV40-transformed simian cells support the
replication of early SV40 mutants" (Gluzman, 1981) and other cell lines
capable of expressing a compatible vector, for example, the C127, 3T3,
CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise
an origin of replication, a suitable promoter and enhancer and also any
necessary ribosome binding sites, polyadenylation site, splice donor and
acceptor sites, transcriptional termination sequences and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40 splice and
polyadenylation sites may be used to provide the required nontranscribed
genetic elements.
[0534] In another aspect, it is envisioned the method of the present
invention can be used to generate novel polynucleotides encoding
biochemical pathways from one or more operons or gene clusters or
portions thereof. For example, bacteria and many eukaryotes have a
coordinated mechanism for regulating genes whose products are involved in
related processes. The genes are clustered, in structures referred to as
"gene clusters," on a single chromosome and are transcribed together
under the control of a single regulatory sequence, including a single
promoter which initiates transcription of the entire cluster. Thus, a
gene cluster is a group of adjacent genes that are either identical or
related, usually as to their function. An example of a biochemical
pathway encoded by gene clusters are polyketides.
[0535] Gene cluster DNA can be isolated from different organisms and
ligated into vectors, particularly vectors containing expression
regulatory sequences which can control and regulate the production of a
detectable protein or protein-related array activity from the ligated
gene clusters. Use of vectors which have an exceptionally large capacity
for exogenous DNA introduction are particularly appropriate for use with
such gene clusters and are described by way of example herein to include
the f-factor (or fertility factor) of E. coli. This f-factor of E. coli
is a plasmid which affects high-frequency transfer of itself during
conjugation and is ideal to achieve and stably propagate large DNA
fragments, such as gene clusters from mixed microbial samples. One aspect
is to use cloning vectors, referred to as "fosmids" or bacterial
artificial chromosome (BAC) vectors. These are derived from E. coli
f-factor which is able to stably integrate large segments of genomic DNA.
When integrated with DNA from a mixed uncultured environmental sample,
this makes it possible to achieve large genomic fragments in the form of
a stable "environmental DNA library." Another type of vector for use in
the present invention is a cosmid vector. Cosmid vectors were originally
designed to clone and propagate large segments of genomic DNA. Cloning
into cosmid vectors is described in detail in Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press
(1989). Once ligated into an appropriate vector, two or more vectors
containing different polyketide synthase gene clusters can be introduced
into a suitable host cell. Regions of partial sequence homology shared by
the gene clusters will promote processes which result in sequence
reorganization resulting in a hybrid gene cluster. The novel hybrid gene
cluster can then be screened for enhanced activities not found in the
original gene clusters.
[0536] Therefore, in a one aspect, the invention relates to a method for
producing a biologically active hybrid polypeptide and screening such a
polypeptide for enhanced activity by: [0537] 1) introducing at least a
first polynucleotide in operable linkage and a second polynucleotide in
operable linkage, the at least first polynucleotide and second
polynucleotide sharing at least one region of partial sequence homology,
into a suitable host cell; [0538] 2) growing the host cell under
conditions which promote sequence reorganization resulting in a hybrid
polynucleotide in operable linkage; [0539] 3) expressing a hybrid
polypeptide encoded by the hybrid polynucleotide; [0540] 4) screening the
hybrid polypeptide under conditions which promote identification of
enhanced biological activity; and [0541] 5) isolating the a
polynucleotide encoding the hybrid polypeptide.
[0542] Methods for screening for various enzyme activities are known to
those of skill in the art and are discussed throughout the present
specification. Such methods may be employed when isolating the
polypeptides and polynucleotides of the invention.
Screening Methodologies and "On-Line" Monitoring Devices
[0543] In practicing the methods of the invention, a variety of apparatus
and methodologies can be used to in conjunction with the polypeptides and
nucleic acids of the invention, e.g., to screen polypeptides for
glucanase, mannanase, or xylanase activity (e.g., assays such as
hydrolysis of casein in zymograms, the release of fluorescence from
gelatin, or the release of p-nitroanilide from various small peptide
substrates), to screen compounds as potential modulators, e.g.,
activators or inhibitors, of a glucanase, mannanase, or xylanase
activity, for antibodies that bind to a polypeptide of the invention, for
nucleic acids that hybridize to a nucleic acid of the invention, to
screen for cells expressing a polypeptide of the invention and the like.
In addition to the array formats described in detail below for screening
samples, alternative formats can also be used to practice the methods of
the invention. Such formats include, for example, mass spectrometers,
chromatographs, e.g., high-throughput HPLC and other forms of liquid
chromatography, and smaller formats, such as 1536-well plates, 384-well
plates and so on. High throughput screening apparatus can be adapted and
used to practice the methods of the invention, see, e.g., U.S. Patent
Application No. 20020001809.
[0544] Capillary Arrays
[0545] Nucleic acids or polypeptides of the invention can be immobilized
to or applied to an array. Arrays can be used to screen for or monitor
libraries of compositions (e.g., small molecules, antibodies, nucleic
acids, etc.) for their ability to bind to or modulate the activity of a
nucleic acid or a polypeptide of the invention. Capillary arrays, such as
the GIGAMATRIX.TM., Diversa Corporation, San Diego, Calif.; and arrays
described in, e.g., U.S. Patent Application No. 20020080350 A1; WO
0231203 A, WO 0244336 A, provide an alternative apparatus for holding and
screening samples. In one aspect, the capillary array includes a
plurality of capillaries formed into an array of adjacent capillaries,
wherein each capillary comprises at least one wall defining a lumen for
retaining a sample. The lumen may be cylindrical, square, hexagonal or
any other geometric shape so long as the walls form a lumen for retention
of a liquid or sample. The capillaries of the capillary array can be held
together in close proximity to form a planar structure. The capillaries
can be bound together, by being fused (e.g., where the capillaries are
made of glass), glued, bonded, or clamped side-by-side. Additionally, the
capillary array can include interstitial material disposed between
adjacent capillaries in the array, thereby forming a solid planar device
containing a plurality of through-holes.
[0546] A capillary array can be formed of any number of individual
capillaries, for example, a range from 100 to 4,000,000 capillaries.
Further, a capillary array having about 100,000 or more individual
capillaries can be formed into the standard size and shape of a
Microtiter.RTM. plate for fitment into standard laboratory equipment. The
lumens are filled manually or automatically using either capillary action
or microinjection using a thin needle. Samples of interest may
subsequently be removed from individual capillaries for further analysis
or characterization. For example, a thin, needle-like probe is positioned
in fluid communication with a selected capillary to either add or
withdraw material from the lumen.
[0547] In a single-pot screening assay, the assay components are mixed
yielding a solution of interest, prior to insertion into the capillary
array. The lumen is filled by capillary action when at least a portion of
the array is immersed into a solution of interest. Chemical or biological
reactions and/or activity in each capillary are monitored for detectable
events. A detectable event is often referred to as a "hit", which can
usually be distinguished from "non-hit" producing capillaries by optical
detection. Thus, capillary arrays allow for massively parallel detection
of "hits".
[0548] In a multi-pot screening assay, a polypeptide or nucleic acid,
e.g., a ligand, can be introduced into a first component, which is
introduced into at least a portion of a capillary of a capillary array.
An air bubble can then be introduced into the capillary behind the first
component. A second component can then be introduced into the capillary,
wherein the second component is separated from the first component by the
air bubble. The first and second components can then be mixed by applying
hydrostatic pressure to both sides of the capillary array to collapse the
bubble. The capillary array is then monitored for a detectable event
resulting from reaction or non-reaction of the two components.
[0549] In a binding screening assay, a sample of interest can be
introduced as a first liquid labeled with a detectable particle into a
capillary of a capillary array, wherein the lumen of the capillary is
coated with a binding material for binding the detectable particle to the
lumen. The first liquid may then be removed from the capillary tube,
wherein the bound detectable particle is maintained within the capillary,
and a second liquid may be introduced into the capillary tube. The
capillary is then monitored for a detectable event resulting from
reaction or non-reaction of the particle with the second liquid.
[0550] Arrays, or "Biochips"
[0551] Nucleic acids or polypeptides of the invention can be immobilized
to or applied to an array. Arrays can be used to screen for or monitor
libraries of compositions (e.g., small molecules, antibodies, nucleic
acids, etc.) for their ability to bind to or modulate the activity of a
nucleic acid or a polypeptide of the invention. For example, in one
aspect of the invention, a monitored parameter is transcript expression
of a glucanase, mannanase, or xylanase gene. One or more, or, all the
transcripts of a cell can be measured by hybridization of a sample
comprising transcripts of the cell, or, nucleic acids representative of
or complementary to transcripts of a cell, by hybridization to
immobilized nucleic acids on an array, or "biochip." By using an "array"
of nucleic acids on a microchip, some or all of the transcripts of a cell
can be simultaneously quantified. Alternatively, arrays comprising
genomic nucleic acid can also be used to determine the genotype of a
newly engineered strain made by the methods of the invention. Polypeptide
arrays" can also be used to simultaneously quantify a plurality of
proteins. The present invention can be practiced with any known "array,"
also referred to as a "microarray" or "nucleic acid array" or
"polypeptide array" or "antibody array" or "biochip," or variation
thereof. Arrays are generically a plurality of "spots" or "target
elements," each target element comprising a defined amount of one or more
biological molecules, e.g., oligonucleotides, immobilized onto a defined
area of a substrate surface for specific binding to a sample molecule,
e.g., mRNA transcripts.
[0552] In practicing the methods of the invention, any known array and/or
method of making and using arrays can be incorporated in whole or in
part, or variations thereof, as described, for example, in U.S. Pat. Nos.
6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;
6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;
5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;
5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g., WO
99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g., Johnston
(1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques
23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)
Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature Genetics
Supp. 21:25-32. See also published U.S. patent applications Nos.
20010018642; 20010019827; 20010016322; 20010014449; 20010014448;
20010012537; 20010008765.
Antibodies and Antibody-Based Screening Methods
[0553] The invention provides isolated or recombinant antibodies that
specifically bind to a glucanase, mannanase, or xylanase of the
invention. These antibodies can be used to isolate, identify or quantify
a glucanase, mannanase, or xylanase of the invention or related
polypeptides. These antibodies can be used to isolate other polypeptides
within the scope the invention or other related glucanases, mannanases,
or xylanases. The antibodies can be designed to bind to an active site of
a glucanase, mannanase, or xylanase. Thus, the invention provides methods
of inhibiting glucanases, mannanases, or xylanases using the antibodies
of the invention (see discussion above regarding applications for
anti-glucanase, mannanase, or xylanase compositions of the invention).
[0554] The invention provides fragments of the enzymes of the invention,
including immunogenic fragments of a polypeptide of the invention. The
invention provides compositions comprising a polypeptide or peptide of
the invention and adjuvants or carriers and the like.
[0555] The antibodies can be used in immunoprecipitation, staining,
immunoaffinity columns, and the like. If desired, nucleic acid sequences
encoding for specific antigens can be generated by immunization followed
by isolation of polypeptide or nucleic acid, amplification or cloning and
immobilization of polypeptide onto an array of the invention.
Alternatively, the methods of the invention can be used to modify the
structure of an antibody produced by a cell to be modified, e.g., an
antibody's affinity can be increased or decreased. Furthermore, the
ability to make or modify antibodies can be a phenotype engineered into a
cell by the methods of the invention.
[0556] Methods of immunization, producing and isolating antibodies
(polyclonal and monoclonal) are known to those of skill in the art and
described in the scientific and patent literature, see, e.g., Coligan,
CURRENT PROTOCOLS IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.)
BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los
Altos, Calif. ("Stites"); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND
PRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)
Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, Cold
Spring Harbor Publications, New York. Antibodies also can be generated in
vitro, e.g., using recombinant antibody binding site expressing phage
display libraries, in addition to the traditional in vivo methods using
animals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz
(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.
[0557] The polypeptides of the invention or fragments comprising at least
5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino
acids thereof, may also be used to generate antibodies which bind
specifically to the polypeptides or fragments. The resulting antibodies
may be used in immunoaffinity chromatography procedures to isolate or
purify the polypeptide or to determine whether the polypeptide is present
in a biological sample. In such procedures, a protein preparation, such
as an extract, or a biological sample is contacted with an antibody
capable of specifically binding to one of the polypeptides of the
invention, or fragments comprising at least 5, 10, 15, 20, 25, 30, 35,
40, 50, 75, 100, or 150 consecutive amino acids thereof.
[0558] In immunoaffinity procedures, the antibody is attached to a solid
support, such as a bead or other column matrix. The protein preparation
is placed in contact with the antibody under conditions in which the
antibody specifically binds to one of the polypeptides of the invention,
or fragment thereof. After a wash to remove non-specifically bound
proteins, the specifically bound polypeptides are eluted.
[0559] The ability of proteins in a biological sample to bind to the
antibody may be determined using any of a variety of procedures familiar
to those skilled in the art. For example, binding may be determined by
labeling the antibody with a detectable label such as a fluorescent
agent, an enzymatic label, or a radioisotope. Alternatively, binding of
the antibody to the sample may be detected using a secondary antibody
having such a detectable label thereon. Particular assays include ELISA
assays, sandwich assays, radioimmunoassays and Western Blots.
[0560] Polyclonal antibodies generated against the polypeptides of the
invention, or fragments comprising at least 5, 10, 15, 20, 25, 30, 35,
40, 50, 75, 100, or 150 consecutive amino acids thereof can be obtained
by direct injection of the polypeptides into an animal or by
administering the polypeptides to an animal, for example, a nonhuman. The
antibody so obtained will then bind the polypeptide itself. In this
manner, even a sequence encoding only a fragment of the polypeptide can
be used to generate antibodies which may bind to the whole native
polypeptide. Such antibodies can then be used to isolate the polypeptide
from cells expressing that polypeptide.
[0561] For preparation of monoclonal antibodies, any technique which
provides antibodies produced by continuous cell line cultures can be
used. Examples include the hybridoma technique (Kohler and Milstein,
Nature, 256:495-497, 1975), the trioma technique, the human B-ell
hybridoma technique (Kozbor et al., Immunology Today 4:72, 1983) and the
EBV-hybridoma technique (Cole, et al., 1985, in Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0562] Techniques described for the production of single chain antibodies
(U.S. Pat. No. 4,946,778) can be adapted to produce single chain
antibodies to the polypeptides of the invention, or fragments comprising
at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive
amino acids thereof. Alternatively, transgenic mice may be used to
express humanized antibodies to these polypeptides or fragments thereof.
[0563] Antibodies generated against the polypeptides of the invention, or
fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,
or 150 consecutive amino acids thereof may be used in screening for
similar polypeptides from other organisms and samples. In such
techniques, polypeptides from the organism are contacted with the
antibody and those polypeptides which specifically bind the antibody are
detected. Any of the procedures described above may be used to detect
antibody binding. One such screening assay is described in "Methods for
Measuring Cellulase Activities", Methods in Enzymology, Vol 160, pp.
87-116.
Kits
[0564] The invention provides kits comprising the compositions, e.g.,
nucleic acids, expression cassettes, vectors, cells, transgenic seeds or
plants or plant parts, polypeptides (e.g., endoglucanases, mannanase, or
xylanase) and/or antibodies of the invention. The kits also can contain
instructional material teaching the methodologies and industrial uses of
the invention, as described herein.
Whole Cell Engineering and Measuring Metabolic Parameters
[0565] The methods of the invention provide whole cell evolution, or whole
cell engineering, of a cell to develop a new cell strain having a new
phenotype, e.g., a new or modified glucanase, mannanase, or xylanase
activity, by modifying the genetic composition of the cell. The genetic
composition can be modified by addition to the cell of a nucleic acid of
the invention, e.g., a coding sequence for an enzyme of the invention.
See, e.g., WO0229032; WO0196551.
[0566] To detect the new phenotype, at least one metabolic parameter of a
modified cell is monitored in the cell in a "real time" or "on-line" time
frame. In one aspect, a plurality of cells, such as a cell culture, is
monitored in "real time" or "on-line." In one aspect, a plurality of
metabolic parameters is monitored in "real time" or "on-line." Metabolic
parameters can be monitored using a glucanase, mannanase, or xylanase of
the invention.
[0567] Metabolic flux analysis (MFA) is based on a known biochemistry
framework. A linearly independent metabolic matrix is constructed based
on the law of mass conservation and on the pseudo-steady state hypothesis
(PSSH) on the intracellular metabolites. In practicing the methods of the
invention, metabolic networks are established, including the:
[0568] identity of all pathway substrates, products and intermediary
metabolites
[0569] identity of all the chemical reactions interconverting the pathway
metabolites, the stoichiometry of the pathway reactions,
[0570] identity of all the enzymes catalyzing the reactions, the enzyme
reaction kinetics,
[0571] the regulatory interactions between pathway components, e.g.
allosteric interactions, enzyme-enzyme interactions etc,
[0572] intracellular compartmentalization of enzymes or any other
supramolecular organization of the enzymes, and,
[0573] the presence of any concentration gradients of metabolites, enzymes
or effector molecules or diffusion barriers to their movement.
[0574] Once the metabolic network for a given strain is built, mathematic
presentation by matrix notion can be introduced to estimate the
intracellular metabolic fluxes if the on-line metabolome data is
available. Metabolic phenotype relies on the changes of the whole
metabolic network within a cell. Metabolic phenotype relies on the change
of pathway utilization with respect to environmental conditions, genetic
regulation, developmental state and the genotype, etc. In one aspect of
the methods of the invention, after the on-line MFA calculation, the
dynamic behavior of the cells, their phenotype and other properties are
analyzed by investigating the pathway utilization. For example, if the
glucose supply is increased and the oxygen decreased during the yeast
fermentation, the utilization of respiratory pathways will be reduced
and/or stopped, and the utilization of the fermentative pathways will
dominate. Control of physiological state of cell cultures will become
possible after the pathway analysis. The methods of the invention can
help determine how to manipulate the fermentation by determining how to
change the substrate supply, temperature, use of inducers, etc. to
control the physiological state of cells to move along desirable
direction. In practicing the methods of the invention, the MFA results
can also be compared with transcriptome and proteome data to design
experiments and protocols for metabolic engineering or gene shuffling,
etc.
[0575] In practicing the methods of the invention, any modified or new
phenotype can be conferred and detected, including new or improved
characteristics in the cell. Any aspect of metabolism or growth can be
monitored.
[0576] Monitoring Expression of an mRNA Transcript
[0577] In one aspect of the invention, the engineered phenotype comprises
increasing or decreasing the expression of an mRNA transcript (e.g., a
glucanase, mannanase, or xylanase message) or generating new (e.g.,
glucanase, mannanase, or xylanase) transcripts in a cell. This increased
or decreased expression can be traced by testing for the presence of a
glucanase, mannanase, or xylanase of the invention or by glucanase,
mannanase, or xylanase activity assays. mRNA transcripts, or messages,
also can be detected and quantified by any method known in the art,
including, e.g., Northern blots, quantitative amplification reactions,
hybridization to arrays, and the like. Quantitative amplification
reactions include, e.g., quantitative PCR, including, e.g., quantitative
reverse transcription polymerase chain reaction, or RT-PCR; quantitative
real time RT-PCR, or "real-time kinetic RT-PCR" (see, e.g., Kreuzer
(2001) Br. J. Haematol. 114:313-318; Xia (2001) Transplantation
72:907-914).
[0578] In one aspect of the invention, the engineered phenotype is
generated by knocking out expression of a homologous gene. The gene's
coding sequence or one or more transcriptional control elements can be
knocked out, e.g., promoters or enhancers. Thus, the expression of a
transcript can be completely ablated or only decreased.
[0579] In one aspect of the invention, the engineered phenotype comprises
increasing the expression of a homologous gene. This can be effected by
knocking out of a negative control element, including a transcriptional
regulatory element acting in cis- or trans-, or, mutagenizing a positive
control element. One or more, or, all the transcripts of a cell can be
measured by hybridization of a sample comprising transcripts of the cell,
or, nucleic acids representative of or complementary to transcripts of a
cell, by hybridization to immobilized nucleic acids on an array.
[0580] Monitoring Expression of a Polypeptides, Peptides and Amino Acids
[0581] In one aspect of the invention, the engineered phenotype comprises
increasing or decreasing the expression of a polypeptide (e.g., a
glucanase, mannanase, or xylanase) or generating new polypeptides in a
cell. This increased or decreased expression can be traced by determining
the amount of glucanase, mannanase, or xylanase present or by glucanase,
mannanase, or xylanase activity assays. Polypeptides, peptides and amino
acids also can be detected and quantified by any method known in the art,
including, e.g., nuclear magnetic resonance (NMR), spectrophotometry,
radiography (protein radiolabeling), electrophoresis, capillary
electrophoresis, high performance liquid chromatography (HPLC), thin
layer chromatography (TLC), hyperdiffusion chromatography, various
immunological methods, e.g. immunoprecipitation, immunodiffusion,
immuno-electrophoresis, radioimmunoassays (RIAs), enzyme-linked
immunosorbent assays (ELISAs), immuno-fluorescent assays, gel
electrophoresis (e.g., SDS-PAGE), staining with antibodies, fluorescent
activated cell sorter (FACS), pyrolysis mass spectrometry,
Fourier-Transform Infrared Spectrometry, Raman spectrometry, GC-MS, and
LC-Electrospray and cap-LC-tandem-electrospray mass spectrometries, and
the like. Novel bioactivities can also be screened using methods, or
variations thereof, described in U.S. Pat. No. 6,057,103. Furthermore, as
discussed below in detail, one or more, or, all the polypeptides of a
cell can be measured using a protein array.
INDUSTRIAL APPLICATIONS
[0582] The glucanase, mannanase, or xylanase enzymes of the invention can
be highly selective catalysts. They can catalyze reactions with exquisite
stereo-, regio- and chemo-selectivities that are unparalleled in
conventional synthetic chemistry. Moreover, enzymes are remarkably
versatile. The enzymes of the invention can be tailored to function in
organic solvents, operate at extreme pHs (for example, high pHs and low
pHs) extreme temperatures (for example, high temperatures and low
temperatures), extreme salinity levels (for example, high salinity and
low salinity) and catalyze reactions with compounds that are structurally
unrelated to their natural, physiological substrates.
[0583] Detergent Compositions
[0584] The invention provides detergent compositions comprising one or
more polypeptides (e.g., endoglucanases, mannanase, or xylanase) of the
invention, and methods of making and using these compositions. The
invention incorporates all methods of making and using detergent
compositions, see, e.g., U.S. Pat. Nos. 6,413,928; 6,399,561; 6,365,561;
6,380,147. The detergent compositions can be a one and two part aqueous
composition, a non-aqueous liquid composition, a cast solid, a granular
form, a particulate form, a compressed tablet, a gel and/or a paste and a
slurry form. The glucanases, mannanases, or xylanases of the invention
can also be used as a detergent additive product in a solid or a liquid
form. Such additive products are intended to supplement or boost the
performance of conventional detergent compositions and can be added at
any stage of the cleaning process.
[0585] The actual active enzyme content depends upon the method of
manufacture of a detergent composition and is not critical, assuming the
detergent solution has the desired enzymatic activity. In one aspect, the
amount of glucanase, mannanase, or xylanase present in the final solution
ranges from about 0.001 mg to 0.5 mg per gram of the detergent
composition. The particular enzyme chosen for use in the process and
products of this invention depends upon the conditions of final utility,
including the physical product form, use pH, use temperature, and soil
types to be degraded or altered. The enzyme can be chosen to provide
optimum activity and stability for any given set of utility conditions.
In one aspect, the glucanases, mannanases, or xylanases of the present
invention are active in the pH ranges of from about 4 to about 12 and in
the temperature range of from about 20.degree. C. to about 95.degree. C.
The detergents of the invention can comprise cationic, semi-polar
nonionic or zwitterionic surfactants; or, mixtures thereof.
[0586] Glucanases, mannanases, or xylanases of the invention can be
formulated into powdered and liquid detergents having pH between 4.0 and
12.0 at levels of about 0.01 to about 5% (in one aspect 0.1% to 0.5%) by
weight. These detergent compositions can also include other enzymes such
as other glucanases, mannanases, or xylanases, or cellulases,
endoglycosidases, endo-beta.-1,4-glucanases, beta-glucanases,
endo-beta-1,3(4)-glucanases, catalases, cutinases, peroxidases, laccases,
lipases, amylases, glucoamylases, pectinases, reductases, oxidases,
phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases,
mannanases, xyloglucanases, pectin acetyl esterases, rhamnogalacturonan
acetyl esterases, polygalacturonases, rhamnogalacturonases, galactanases,
proteases, pectate lyases, pectin methylesterases, cellobiohydrolases
and/or transglutaminases. These detergent compositions can also include
builders and stabilizers. These detergent compositions can also include
builders and stabilizers.
[0587] The addition of a glucanase, mannanase, or xylanase of the
invention to conventional cleaning compositions does not create any
special use limitation. In other words, any temperature and pH suitable
for the detergent is also suitable for the compositions of the invention
as long as the enzyme is active at or tolerant of the pH and/or
temperature of the intended use. In addition, a glucanase, mannanase, or
xylanase of the invention can be used in a cleaning composition without
detergents, again either alone or in combination with builders and
stabilizers.
[0588] The present invention provides cleaning compositions including
detergent compositions for cleaning hard surfaces, detergent compositions
for cleaning fabrics, dishwashing compositions, oral cleaning
compositions, denture cleaning compositions, and contact lens cleaning
solutions.
[0589] In one aspect, the invention provides a method for washing an
object comprising contacting the object with a polypeptide of the
invention under conditions sufficient for washing. A glucanase,
mannanase, or xylanase of the invention may be included as a detergent
additive. The detergent composition of the invention may, for example, be
formulated as a hand or machine laundry detergent composition comprising
a polypeptide of the invention. A laundry additive suitable for
pre-treatment of stained fabrics can comprise a polypeptide of the
invention. A fabric softener composition can comprise a glucanase,
mannanase, or xylanase of the invention. Alternatively, a glucanase,
mannanase, or xylanase of the invention can be formulated as a detergent
composition for use in general household hard surface cleaning
operations. In alternative aspects, detergent additives and detergent
compositions of the invention may comprise one or more other enzymes such
as another glucanase, mannanase, or xylanase, or, a xylanase, a lipase, a
cutinase, a carbohydrase, a cellulase, a pectinase, an arabinase, a
galactanase, an oxidase, e.g., a lactase, and/or a peroxidase (see also,
above). The properties of the enzyme(s) of the invention are chosen to be
compatible with the selected detergent (i.e. pH-optimum, compatibility
with other enzymatic and non-enzymatic ingredients, etc.) and the
enzyme(s) is present in effective amounts. In one aspect, enzymes of the
invention are used to remove malodorous materials from fabrics. Various
detergent compositions and methods for making them that can be used in
practicing the invention are described in, e.g., U.S. Pat. Nos.
6,387,690; 6,333,301; 6,329,333; 6,326,341; 6,297,038; 6,309,871;
6,204,232; 6,197,070; 5,856,164.
[0590] When formulated as compositions suitable for use in a laundry
machine washing method, the enzymes of the invention can comprise both a
surfactant and a builder compound. They can additionally comprise one or
more detergent components, e.g., organic polymeric compounds, bleaching
agents, additional enzymes, suds suppressors, dispersants, lime-soap
dispersants, soil suspension and anti-redeposition agents and corrosion
inhibitors. Laundry compositions of the invention can also contain
softening agents, as additional detergent components. Such compositions
containing carbohydrase can provide fabric cleaning, stain removal,
whiteness maintenance, softening, color appearance, dye transfer
inhibition and sanitization when formulated as laundry detergent
compositions.
[0591] The density of the laundry detergent compositions of the invention
can range from about 200 to 1500 g/liter, or, about 400 to 1200 g/liter,
or, about 500 to 950 g/liter, or, 600 to 800 g/liter, of composition;
this can be measured at about 20.degree. C.
[0592] The "compact" form of laundry detergent compositions of the
invention is best reflected by density and, in terms of composition, by
the amount of inorganic filler salt. Inorganic filler salts are
conventional ingredients of detergent compositions in powder form. In
conventional detergent compositions, the filler salts are present in
substantial amounts, typically 17% to 35% by weight of the total
composition. In one aspect of the compact compositions, the filler salt
is present in amounts not exceeding 15% of the total composition, or, not
exceeding 10%, or, not exceeding 5% by weight of the composition. The
inorganic filler salts can be selected from the alkali and
alkaline-earth-metal salts of sulphates and chlorides, e.g., sodium
sulphate.
[0593] Liquid detergent compositions of the invention can also be in a
"concentrated form." In one aspect, the liquid detergent compositions can
contain a lower amount of water, compared to conventional liquid
detergents. In alternative aspects, the water content of the concentrated
liquid detergent is less than 40%, or, less than 30%, or, less than 20%
by weight of the detergent composition. Detergent compounds of the
invention can comprise formulations as described in WO 97/01629.
[0594] Enzymes of the invention can be useful in formulating various
cleaning compositions. A number of known compounds are suitable
surfactants including nonionic, anionic, cationic, or zwitterionic
detergents, can be used, e.g., as disclosed in U.S. Pat. Nos. 4,404,128;
4,261,868; 5,204,015. In addition, glucanases, mannanases, or xylanases
of the invention can be used, for example, in bar or liquid soap
applications, dish care formulations, contact lens cleaning solutions or
products, peptide hydrolysis, waste treatment, textile applications, as
fusion-cleavage enzymes in protein production, and the like. Glucanases,
mannanases, or xylanases of the invention may provide enhanced
performance in a detergent composition as compared to another detergent
glucanase, that is, the enzyme group may increase cleaning of certain
enzyme sensitive stains such as grass or blood, as determined by usual
evaluation after a standard wash cycle. Glucanases, mannanases, or
xylanases of the invention can be formulated into known powdered and
liquid detergents having pH between 6.5 and 12.0 at levels of about 0.01
to about 5% (for example, about 0.1% to 0.5%) by weight. These detergent
cleaning compositions can also include other enzymes such as known
glucanases, mannanases, xylanases, amylases, cellulases, lipases or
endoglycosidases, as well as builders and stabilizers.
[0595] In one aspect, the invention provides detergent compositions having
glucanase, mannanase, xylanase activity (a glucanase, mannanase, or
xylanase of the invention) for use with fruit, vegetables and/or mud and
clay compounds (see, for example, U.S. Pat. No. 5,786,316).
[0596] Treating Fibers and Textiles
[0597] The invention provides methods of treating fibers and fabrics using
one or more glucanases, mannanases, or xylanases of the invention. The
enzymes of the invention can be used in any fiber- or fabric-treating
method, which are well known in the art, see, e.g., U.S. Pat. Nos.
6,387,690; 6,261,828; 6,077,316; 6,024,766; 6,021,536; 6,017,751;
5,980,581; US Patent Publication No. 20020142438 A1. For example, enzymes
of the invention can be used in fiber and/or fabric desizing. In one
aspect, the feel and appearance of a fabric is improved by a method
comprising contacting the fabric with an enzyme of the invention in a
solution. In one aspect, the fabric is treated with the solution under
pressure. For example, enzymes of the invention can be used in the
removal of stains.
[0598] In one aspect, enzymes of the invention are applied during or after
the weaving of textiles, or during the desizing stage, or during one or
more additional fabric processing steps. During the weaving of textiles,
the threads are exposed to considerable mechanical strain. Prior to
weaving on mechanical looms, warp yarns are often coated with sizing
starch or starch derivatives in order to increase their tensile strength
and to prevent breaking. After the textiles have been woven, a fabric can
proceed to a desizing stage. This can be followed by one or more
additional fabric processing steps. Desizing is the act of removing
"size" from textiles. After weaving, the size coating must be removed
before further processing the fabric in order to ensure a homogeneous and
wash-proof result.
[0599] The enzymes of the invention can be used to treat any cellulosic
material, including fibers (e.g., fibers from cotton, hemp, flax or
linen), sewn and unsewn fabrics, e.g., knits, wovens, denims, yarns, and
toweling, made from cotton, cotton blends or natural or manmade
cellulosics (e.g. originating from glucan-comprising cellulose fibers
such as from wood pulp) or blends thereof. Examples of blends are blends
of cotton or rayon/viscose with one or more companion material such as
wool, synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyester
fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,
polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers,
aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose,
ramie, hemp, flax/linen, jute, cellulose acetate fibers, lyocell).
[0600] The enzymes of the invention can be used to treat fabrics or any
glucan, mannanase, xylan or cellulose-comprising material, including
cotton-containing fabrics, as detergent additives, e.g., in aqueous
compositions. For the manufacture of clothes, the fabric can be cut and
sewn into clothes or garments. These can be finished before or after the
treatment. In particular, for the manufacture of denim jeans, different
enzymatic finishing methods have been developed. The finishing of denim
garment normally is initiated with an enzymatic desizing step, during
which garments are subjected to the action of amylolytic enzymes in order
to provide softness to the fabric and make the cotton more accessible to
the subsequent enzymatic finishing steps. The invention provides methods
of treating textiles, e.g., finishing denim garments, enzymatic desizing
and providing softness to fabrics by using any combination of enzymes,
such the mannanases, xylanases, or glucanases (e.g., endoglucanases) of
the invention. In one aspect, enzymes of the invention can be used in
treatments to prevent the graying of a textile.
[0601] In one aspect, an alkaline and/or thermostable mannanases,
xylanases, and glucanases (e.g., endoglucanases) of the invention are
combined in a single bath desizing and bioscouring. Among advantages of
combining desizing and scouring in one step are cost reduction and lower
environmental impact due to savings in energy and water usage and lower
waste production. Application conditions for desizing and bioscouring can
be between about pH 8.5 to pH 10.0 and temperatures at about 40.degree.
C. and up. Low enzyme dosages (e.g., about 5 g per a ton of cotton) and
short reaction times (e.g., about 15 minutes) can be used to obtain
efficient desizing and scouring with out added calcium.
[0602] The enzymes of the invention can be used in the treatment of
cellulose-containing fabrics for harshness reduction, for color
clarification, or to provide a localized variation in the color of such
fabrics. See, e.g., U.S. Pat. No. 6,423,524. For example, enzymes of the
invention can be used to reduce the harshness of cotton-containing
fabrics, e.g., as a harshness reducing detergent additive. The enzymes of
the invention can be used in the treatment of fabrics to give a
"stonewashed" look in a colored fabric while reducing the amount of
redeposition of colorant onto the fabric.
[0603] The textile treating processes of the invention (using enzymes of
the invention) can be used in conjunction with other textile treatments,
e.g., scouring and bleaching. Scouring is the removal of non-cellulosic
material from the cotton fiber, e.g., the cuticle (mainly consisting of
waxes) and primary cell wall (mainly consisting of pectin, protein and
xyloglucan). A proper wax removal is necessary for obtaining a high
wettability. This is needed for dyeing. Removal of the primary cell walls
by the processes of the invention improves wax removal and ensures a more
even dyeing. Treating textiles with the processes of the invention can
improve whiteness in the bleaching process. The main chemical used in
scouring is sodium, hydroxide in high concentrations and at high
temperatures. Bleaching comprises oxidizing the textile. Bleaching
typically involves use of hydrogen peroxide as the oxidizing agent in
order to obtain either a fully bleached (white) fabric or to ensure a
clean shade of the dye.
[0604] The invention also provides alkaline glucanases (e.g.,
endoglucanases active under alkaline conditions), mannanases, or
xylanases. These have wide-ranging applications in textile processing,
degumming of plant fibers (e.g., plant bast fibers), treatment of waste,
e.g., pectic wastewaters, paper-making, and coffee and tea fermentations.
See, e.g., Hoondal (2002) Applied Microbiology and Biotechnology
59:409-418.
[0605] The textile treating processes of the invention can also include
the use of any combination of other enzymes (including carbohydrate
degrading enzymes) such as catalases, other glucanases, cellulases,
lipases, endoglycosidases, endo-beta.-1,4-glucanases, beta-glucanases,
endo-beta-1,3(4)-glucanases, cutinases, peroxidases, laccases, amylases,
glucoamylases, pectinases, reductases, oxidases, phenoloxidases,
ligninases, pullulanases, arabinanases, hemicellulases, other mannanases,
xyloglucanases, other xylanases, pectin acetyl esterases,
rhamnogalacturonan acetyl esterases, proteases, polygalacturonases,
rhamnogalacturonases, galactanases, pectate lyases, pectin
methylesterases, cellobiohydrolases and/or transglutaminases. The enzymes
of the invention can be used in combination with other carbohydrate
degrading enzymes, e.g., cellulase, arabinanase, xyloglucanase,
pectinase, xylanase, and the like, for the preparation of fibers or for
cleaning of fibers. Proteases can also be used in a combination of
enzymes of the invention. These can be used in combination with
detergents.
[0606] Treating Foods and Food Processing
[0607] The glucanases, mannanases, or xylanases of the invention have
numerous applications in food processing industry. For example, in one
aspect, the enzymes of the invention are used to improve the extraction
of oil from oil-rich plant material, e.g., oil-rich seeds, for example,
soybean oil from soybeans, olive oil from olives, rapeseed oil from
rapeseed and/or sunflower oil from sunflower seeds.
[0608] The enzymes of the invention can be used for separation of
components of plant cell materials. For example, enzymes of the invention
can be used in the separation of glucan-rich material (e.g., plant cells)
into components. In one aspect, enzymes of the invention can be used to
separate glucan-rich or oil-rich crops into valuable protein and oil and
hull fractions. The separation process may be performed by use of methods
known in the art.
[0609] The enzymes of the invention can be used in the preparation of
fruit or vegetable juices, syrups, extracts and the like to increase
yield. The enzymes of the invention can be used in the enzymatic
treatment (e.g., hydrolysis of glucan-comprising plant materials) of
various plant cell wall-derived materials or waste materials, e.g. from
cereals, grains, wine or juice production, or agricultural residues such
as vegetable hulls, bean hulls, sugar beet pulp, olive pulp, potato pulp,
and the like. The enzymes of the invention can be used to modify the
consistency and appearance of processed fruit or vegetables. The enzymes
of the invention can be used to treat plant material to facilitate
processing of plant material, including foods, facilitate purification or
extraction of plant components. The enzymes of the invention can be used
to improve feed value, decrease the water binding capacity, improve the
degradability in waste water plants and/or improve the conversion of
plant material to ensilage, and the like. The enzymes of the invention
can also be used in the fruit and brewing industry for equipment cleaning
and maintenance.
[0610] In one aspect, enzymes, e.g., glucanases, mannanases, or xylanases
of the invention, are used in baking applications, e.g., cookies and
crackers, to hydrolyze glucans and reduce viscosity. The glucanases,
mannanases, or xylanases of the invention can also be used to create
non-sticky doughs that are not difficult to machine and to reduce biscuit
size. Use enzymes of the invention to hydrolyze glucans is used to
prevent rapid rehydration of the baked product resulting in loss of
crispiness and reduced shelf-life. In one aspect, enzymes of the
invention are used as additives in dough processing. In one aspect,
enzymes of the invention of the invention are used in dough conditioning,
wherein in one aspect the enzymes possess high activity over a
temperature range of about 25-35.degree. C. and at near neutral pH
(7.0-7.5). In one aspect, dough conditioning enzymes can be inactivated
at the extreme temperatures of baking (>500.degree. F.).
[0611] The food treatment processes of the invention can also include the
use of any combination of other enzymes such as catalases, glucanases,
cellulases, endoglycosidases, endo-beta.-1,4-glucanases,
amyloglucosidases, glucose isomerases, glycosyltransferases, lipases,
phospholipases, lipooxygenases, beta-glucanases,
endo-beta-1,3(4)-glucanases, cutinases, peroxidases, laccases, amylases,
glucoamylases, pectinases, reductases, oxidases, decarboxylases,
phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases,
mannanases, xyloglucanases, xylanases, pectin acetyl esterases,
rhamnogalacturonan acetyl esterases, proteases, peptidases, proteinases,
polygalacturonases, rhamnogalacturonases, galactanases, pectate lyases,
transglutaminases, pectin methylesterases, cellobiohydrolases and/or
transglutaminases.
[0612] Paper or Pulp Treatment
[0613] The glucanases, mannanases, or xylanases of the invention can be in
paper or pulp treatment or paper deinking. For example, in one aspect,
the invention provides a paper treatment process using a glucanase,
mannanase, or xylanase of the invention. In one aspect, an enzyme of the
invention is applicable both in reduction of the need for a chemical
bleaching agent, such as chlorine dioxide, and in high alkaline and high
temperature environments. In one aspect, an enzyme of the invention is a
thermostable alkaline glucanase which can effect a greater than 25%
reduction in the chlorine dioxide requirement of kraft pulp with a less
than 0.5% pulp yield loss. In one aspect, boundary parameters are pH 10,
65-85.degree. C. and treatment time of less than 60 minutes at an enzyme
loading of less than 0.001 wt %. A pool of endoglucanases may be tested
for the ability to hydrolyze dye-labeled glucan at, for example, pH 10
and 60.degree. C. The enzymes that test positive under these conditions
may then be evaluated at, for example pH 10 and 70.degree. C.
Alternatively, enzymes may be tested at pH 8 and pH 10 at 70.degree. C.
In discovery of endoglucanases desirable in the pulp and paper industry
libraries from high temperature or highly alkaline environments were
targeted. Specifically, these libraries were screened for enzymes
functioning at alkaline pH and a temperature of approximately 45.degree.
C. In another aspect, the glucanases of the invention are useful in the
pulp and paper industry in degradation of a lignin hemicellulose linkage,
in order to release the lignin.
[0614] Glucanases, mannanases, or xylanases of the invention can be used
in the paper and pulp industry as described in e.g., U.S. Pat. Nos.
5,661,021; 6,387,690; 6,083,733; 6,140,095 and 6,346,407. For example, as
in U.S. Pat. No. 6,140,095, an enzyme of the invention can be an
alkali-tolerant glucanase. An enzyme of the invention can be used in the
paper and pulp industry where the enzyme is active in the temperature
range of 65.degree. C. to 75.degree. C. and at a pH of approximately 10.
Additionally, an enzyme of the invention useful in the paper and pulp
industry would decrease the need for bleaching chemicals, such as
chlorine dioxide. An enzyme of the invention can have activity in
slightly acidic pH (5.5-6.0) in the 40.degree. C. to 70.degree. C.
temperature range with inactivation at 95.degree. C. In one aspect, an
enzyme of the invention has an optimal activity between 40-75.degree. C.,
and pH 5.5-6.0; stable at 70.degree. C. for at least 50 minutes, and
inactivated at 96-100.degree. C.
[0615] Additionally, glucanases, mannanases, or xylanases of the invention
can be useful in biobleaching and treatment of chemical pulps, as
described, e.g., in U.S. Pat. No. 5,202,249, biobleaching and treatment
of wood or paper pulps, as described, e.g., in U.S. Pat. Nos. 5,179,021,
5,116,746, 5,407,827, 5,405,769, 5,395,765, 5,369,024, 5,457,045,
5,434,071, 5,498,534, 5,591,304, 5,645,686, 5,725,732, 5,759,840,
5,834,301, 5,871,730 and 6,057,438, in reducing lignin in wood and
modifying wood, as described, e.g., in U.S. Pat. Nos. 5,486,468 and
5,770,012.
[0616] In one aspect, a mannanase or other enzyme of the invention is used
in the paper and pulp industry either alone or together with a xylanase
(e.g., a xylanase of the invention). In one aspect, the enzyme of the
invention is used in a bleaching process to enhance the brightness of
bleached pulps, e.g., fully or partially from softwood. Using an enzyme
of the invention, the amount of chlorine used in the bleaching stages may
be reduced. In one aspect, a mannanase of the invention is used to
increase the freeness of pulps in recycled paper process. In one aspect,
a mannanase of the invention is used alone or in combination with a
xylanase (e.g., a xylanase of the invention) in the treatment of
lignocellulosic pulp (e.g., fully or partially from softwood) to improve
the bleachability thereof. See, e.g., U.S. Pat. No. 5,795,764.
[0617] The pulp and paper processes of the invention can also include the
use of any combination of other enzymes such as catalases, glucanases,
cellulases, endoglycosidases, endo-beta.-1,4-glucanases,
amyloglucosidases, glucose isomerases, glycosyltransferases, lipases,
phospholipases, lipooxygenases, beta-glucanases,
endo-beta-1,3(4)-glucanases, cutinases, peroxidases, laccases, amylases,
glucoamylases, pectinases, reductases, oxidases, decarboxylases,
phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases,
mannanases, xyloglucanases, xylanases, pectin acetyl esterases,
rhamnogalacturonan acetyl esterases, proteases, peptidases, proteinases,
polygalacturonases, rhamnogalacturonases, galactanases, pectate lyases,
transglutaminases, pectin methylesterases, cellobiohydrolases and/or
transglutaminases.
[0618] Animal Feeds and Food or Feed Additives
[0619] The invention provides methods for treating animal feeds and foods
and food or feed additives using glucanases of the invention, animals
including mammals (e.g., humans), birds (e.g., chickens), reptiles, fish
and the like. The invention provides animal feeds, foods, and additives
comprising glucanases, mannanases, or xylanases of the invention. In one
aspect, treating animal feeds, foods and additives using glucanases,
mannanases, or xylanases of the invention can help in the availability of
nutrients, e.g., starch, protein, and the like, in the animal feed or
additive. By breaking down difficult to digest proteins or indirectly or
directly unmasking starch (or other nutrients), the enzyme of the
invention makes nutrients more accessible to other endogenous or
exogenous enzymes. The enzyme of the invention can also simply cause the
release of readily digestible and easily absorbed nutrients and sugars.
In another aspect, the enzymes of the invention are used in feed to
decrease the viscosity of glucans in a food or a feed, e.g., a
high-barley or a high-wheat diet, such as a poultry diet. In one aspect,
this can minimize wet droppings.
[0620] When added to animal feed, glucanases of the invention improve the
in vivo break-down of plant cell wall material partly due to a reduction
of the intestinal viscosity (see, e.g., Bedford et al., Proceedings of
the 1st Symposium on Enzymes in Animal Nutrition, 1993, pp. 73-77),
whereby a better utilization of the plant nutrients by the animal is
achieved. Thus, by using glucanases, mannanases, or xylanases of the
invention in feeds the growth rate and/or feed conversion ratio (i.e. the
weight of ingested feed relative to weight gain) of the animal is
improved.
[0621] The animal feed additive of the invention may be a granulated
enzyme product which may readily be-mixed with feed components.
Alternatively, feed additives of the invention can form a component of a
pre-mix. The granulated enzyme product of the invention may be coated or
uncoated. The particle size of the enzyme granulates can be compatible
with that of feed and pre-mix components. This provides a safe and
convenient mean of incorporating enzymes into feeds. Alternatively, the
animal feed additive of the invention may be a stabilized liquid
composition. This may be an aqueous or oil-based slurry. See, e.g., U.S.
Pat. No. 6,245,546.
[0622] Glucanases, mannanases, or xylanases of the present invention, in
the modification of animal feed or a food, can process the food or feed
either in vitro (by modifying components of the feed or food) or in vivo.
Glucanases, mannanases, or xylanases of the invention can be added to
animal feed or food compositions containing high amounts of glucans, e.g.
feed or food containing plant material from cereals, grains and the like.
When added to the feed or food the glucanase significantly improves the
in vivo break-down of glucan-containing material, e.g., plant cell walls,
whereby a better utilization of the plant nutrients by the animal (e.g.,
human) is achieved. In one aspect, the growth rate and/or feed conversion
ratio (i.e. the weight of ingested feed relative to weight gain) of the
animal is improved. For example a partially or indigestible
glucan-comprising protein is fully or partially degraded by glucanases,
mannanases, or xylanases of the invention, e.g. in combination with
another enzyme, e.g., beta-galactosidase, to peptides and galactose
and/or galactooligomers. These enzyme digestion products are more
digestible by the animal. Thus, glucanases of the invention can
contribute to the available energy of the feed or food. Also, by
contributing to the degradation of glucan-comprising proteins, a
glucanase of the invention can improve the digestibility and uptake of
carbohydrate and non-carbohydrate feed or food constituents such as
protein, fat and minerals.
[0623] In another aspect, glucanases, mannanases, or xylanases of the
invention can be supplied by expressing the enzymes directly in
transgenic feed crops (as, e.g., transgenic plants, seeds and the like),
such as grains, cereals, corn, soy bean, rape seed, lupin and the like.
As discussed above, the invention provides transgenic plants, plant parts
and plant cells comprising a nucleic acid sequence encoding a polypeptide
of the invention. In one aspect, the nucleic acid is expressed such that
the glucanase of the invention is produced in recoverable quantities. The
glucanases, mannanases, or xylanases of the invention can be recovered
from any plant or plant part. Alternatively, the plant or plant part
containing the recombinant polypeptide can be used as such for improving
the quality of a food or feed, e.g., improving nutritional value,
palatability, and rheological properties, or to destroy an antinutritive
factor.
[0624] In one aspect, the invention provides methods for removing
oligosaccharides from feed prior to consumption by an animal subject
using glucanase, mannanases, or xylanases of the invention. In this
process a feed is formed having an increased metabolizable energy value.
In addition to glucanases, mannanases, or xylanases of the invention,
galactosidases, cellulases and combinations thereof can be used. In one
aspect, the enzyme is added in an amount equal to between about 0.1% and
1% by weight of the feed material. In one aspect, the feed is a cereal, a
wheat, a grain, a soybean (e.g., a ground soybean) material. See, e.g.,
U.S. Pat. No. 6,399,123.
[0625] In another aspect, the invention provides methods for utilizing
glucanases, mannanases, or xylanases of the invention as a nutritional
supplement in the diets of animals by preparing a nutritional supplement
containing a recombinant enzyme of the invention, and administering the
nutritional supplement to an animal to increase the utilization of glucan
contained in food ingested by the animal.
[0626] In yet another aspect, the invention provides an edible pelletized
enzyme delivery matrix and method of use for delivery of glucanases,
mannanases, or xylanases of the invention to an animal, for example as a
nutritional supplement. The enzyme delivery matrix readily releases a
glucanase enzyme, such as one having an amino acid sequence of the
invention, or at least 30 contiguous amino acids thereof, in aqueous
media, such as, for example, the digestive fluid of an animal. The
invention enzyme delivery matrix is prepared from a granulate edible
carrier selected from such components as grain germ that is spent of oil,
hay, alfalfa, timothy, soy hull, sunflower seed meal, wheat midd, and the
like, that readily disperse the recombinant enzyme contained therein into
aqueous media. In use, the edible pelletized enzyme delivery matrix is
administered to an animal to delivery of glucanase to the animal.
Suitable grain-based substrates may comprise or be derived from any
suitable edible grain, such as wheat, corn, soy, sorghum, alfalfa,
barley, and the like. An exemplary grain-based substrate is a corn-based
substrate. The substrate may be derived from any suitable part of the
grain, but is in one aspect a grain germ approved for animal feed use,
such as corn germ that is obtained in a wet or dry milling process. The
grain germ in one aspect comprises spent germ, which is grain germ from
which oil has been expelled, such as by pressing or hexane or other
solvent extraction. Alternatively, the grain germ is expeller extracted,
that is, the oil has been removed by pressing.
[0627] The enzyme delivery matrix of the invention is in the form of
discrete plural particles, pellets or granules. By "granules" is meant
particles that are compressed or compacted, such as by a pelletizing,
extrusion, or similar compacting to remove water from the matrix. Such
compression or compacting of the particles also promotes intraparticle
cohesion of the particles. For example, the granules can be prepared by
pelletizing the grain-based substrate in a pellet mill. The pellets
prepared thereby are ground or crumbled to a granule size suitable for
use as an adjuvant in animal feed. Since the matrix is itself approved
for use in animal feed, it can be used as a diluent for delivery of
enzymes in animal feed.
[0628] In one aspect, the enzyme delivery matrix is in the form of
granules having a granule size ranging from about 4 to about 400 mesh
(USS); more in one aspect, about 8 to about 80 mesh; and most in one
aspect about 14 to about 20 mesh. If the grain germ is spent via solvent
extraction, use of a lubricity agent such as corn oil may be necessary in
the pelletizer, but such a lubricity agent ordinarily is not necessary if
the germ is expeller extracted. In other aspects of the invention, the
matrix is prepared by other compacting or compressing processes such as,
for example, by extrusion of the grain-based substrate through a die and
grinding of the extrudate to a suitable granule size.
[0629] The enzyme delivery matrix may further include a polysaccharide
component as a cohesiveness agent to enhance the cohesiveness of the
matrix granules. The cohesiveness agent is believed to provide additional
hydroxyl groups, which enhance the bonding between grain proteins within
the matrix granule. It is further believed that the additional hydroxyl
groups so function by enhancing the hydrogen bonding of proteins to
starch and to other proteins. The cohesiveness agent may be present in
any amount suitable to enhance the cohesiveness of the granules of the
enzyme delivery matrix. Suitable cohesiveness agents include one or more
of dextrins, maltodextrins, starches, such as corn starch, flours,
cellulosics, hemicellulosics, and the like. For example, the percentage
of grain germ and cohesiveness agent in the matrix (not including the
enzyme) is 78% corn germ meal and 20% by weight of corn starch.
[0630] Because the enzyme-releasing matrix of the invention is made from
biodegradable materials, the matrix may be subject to spoilage, such as
by molding. To prevent or inhibit such molding, the matrix may include a
mold inhibitor, such as a propionate salt, which may be present in any
amount sufficient to inhibit the molding of the enzyme-releasing matrix,
thus providing a delivery matrix in a stable formulation that does not
require refrigeration.
[0631] The glucanase enzyme contained in the invention enzyme delivery
matrix and methods is in one aspect a thermostable glucanase, as
described herein, so as to resist inactivation of the glucanase during
manufacture where elevated temperatures and/or steam may be employed to
prepare the palletized enzyme delivery matrix. During digestion of feed
containing the invention enzyme delivery matrix, aqueous digestive fluids
will cause release of the active enzyme. Other types of thermostable
enzymes and nutritional supplements that are thermostable can also be
incorporated in the delivery matrix for release under any type of aqueous
conditions.
[0632] A coating can be applied to the invention enzyme matrix particles
for many different purposes, such as to add a flavor or nutrition
supplement to animal feed, to delay release of animal feed supplements
and enzymes in gastric conditions, and the like. Or, the coating may be
applied to achieve a functional goal, for example, whenever it is
desirable to slow release of the enzyme from the matrix particles or to
control the conditions under which the enzyme will be released. The
composition of the coating material can be such that it is selectively
broken down by an agent to which it is susceptible (such as heat, acid or
base, enzymes or other chemicals). Alternatively, two or more coatings
susceptible to different such breakdown agents may be consecutively
applied to the matrix particles.
[0633] The invention is also directed towards a process for preparing an
enzyme-releasing matrix. In accordance with the invention, the process
comprises providing discrete plural particles of a grain-based substrate
in a particle size suitable for use as an enzyme-releasing matrix,
wherein the particles comprise a glucanase enzyme encoded by an amino
acid sequence of the invention. In one aspect, the process includes
compacting or compressing the particles of enzyme-releasing matrix into
granules, which most in one aspect is accomplished by pelletizing. The
mold inhibitor and cohesiveness agent, when used, can be added at any
suitable time, and in one aspect are mixed with the grain-based substrate
in the desired proportions prior to pelletizing of the grain-based
substrate. Moisture content in the pellet mill feed in one aspect is in
the ranges set forth above with respect to the moisture content in the
finished product, and in one aspect is about 14-15%. In one aspect,
moisture is added to the feedstock in the form of an aqueous preparation
of the enzyme to bring the feedstock to this moisture content. The
temperature in the pellet mill in one aspect is brought to about
82.degree. C. with steam. The pellet mill may be operated under any
conditions that impart sufficient work to the feedstock to provide
pellets. The pelleting process itself is a cost-effective process for
removing water from the enzyme-containing composition.
[0634] In one aspect, the pellet mill is operated with a 1/8 in. by 2 in.
die at 100 lb./min. pressure at 82.degree. C. to provide pellets, which
then are crumbled in a pellet mill crumbler to provide discrete plural
particles having a particle size capable of passing through an 8 mesh
screen but being retained on a 20 mesh screen.
[0635] The thermostable glucanases, mannanases, or xylanases of the
invention can be used in the pellets of the invention. They can have high
optimum temperatures and high heat resistance such that an enzyme
reaction at a temperature not hitherto carried out can be achieved. The
gene encoding the glucanase according to the present invention (e.g. as
set forth in any of the sequences of the invention) can be used in
preparation of glucanases, mannanases, or xylanases (e.g. using GSSM.TM.
as described herein) having characteristics different from those of the
glucanases, mannanases, or xylanase's of the invention (in terms of
optimum pH, optimum temperature, heat resistance, stability to solvents,
specific activity, affinity to substrate, secretion ability, translation
rate, transcription control and the like). Furthermore, a polynucleotide
of the invention may be employed for screening of variant glucanases,
mannanases, or xylanases prepared by the methods described herein to
determine those having a desired activity, such as improved or modified
thermostability or thermotolerance. For example, U.S. Pat. No. 5,830,732,
describes a screening assay for determining thermotolerance of a
glucanase.
[0636] In one aspect, glucanases, mannanases, or xylanases of the
invention in animal feeds are active in the animal's stomach. Thus, in
one aspect, an enzyme of the invention, e.g., in a feed, has an activity
at about 37.degree. C. and at low pH for monogastrics (pH 24) and near
neutral pH for ruminants (pH 6.5-7). The enzyme of the invention has
resistance to animal gut enzymes, e.g., proteases, and stability at the
higher temperatures involved in feed pelleting. In one aspect,
glucanases, mannanases, or xylanases of the invention are used in feed
additives, e.g., monogastric feeds, and can have a high specific
activity, e.g., activity at 35-40.degree. C. and pH 24, half life greater
than 30 minutes in SGF and a half-life >5 minutes at 85.degree. C. in
formulated state. For ruminant feed, glucanases, mannanases, or xylanases
of the invention in feed additives have a high specific activity, e.g.,
activity at 35-40.degree. C. and pH 6.5-7.0, half life greater than 30
minutes in SRF and stability as a concentrated dry powder.
[0637] The animal feed and animal feed production processes of the
invention can include any combination of other enzymes such as catalases,
other glucanases, cellulases, endoglycosidases,
endo-beta.-1,4-glucanases, amyloglucosidases, glucose isomerases,
glycosyltransferases, lipases, phospholipases, lipooxygenases,
beta-glucanases, endo-beta-1,3(4)-glucanases, cutinases, peroxidases,
laccases, amylases, glucoamylases, pectinases, reductases, oxidases,
decarboxylases, phenoloxidases, ligninases, pullulanases, phytases,
arabinanases, hemicellulases, other mannanases, xyloglucanases,
xylanases, pectin acetyl esterases, rhamnogalacturonan acetyl esterases,
polygalacturonases, rhamnogalacturonases, galactanases, pectate lyases,
transglutaminases, pectin methylesterases, cellobiohydrolases and/or
transglutaminases.
[0638] Waste Treatment
[0639] The glucanases, mannanases, or xylanases of the invention can be
used in a variety of other industrial applications, e.g., in waste
treatment (in addition to, e.g., biomass conversion to fuels). For
example, in one aspect, the invention provides a solid waste digestion
process using glucanases, mannanases, or xylanases of the invention. The
methods can comprise reducing the mass and volume of substantially
untreated solid waste. Solid waste can be treated with an enzymatic
digestive process in the presence of an enzymatic solution (including
glucanases, mannanases, or xylanases of the invention) at a controlled
temperature. This results in a reaction without appreciable bacterial
fermentation from added microorganisms. The solid waste is converted into
a liquefied waste and any residual solid waste. The resulting liquefied
waste can be separated from said any residual solidified waste. See e.g.,
U.S. Pat. No. 5,709,796.
[0640] The waste treatment processes of the invention can include the use
of any combination of other enzymes such as catalases, other glucanases,
cellulases, endoglycosidases, endo-beta.-1,4-glucanases,
amyloglucosidases, glucose isomerases, glycosyltransferases, lipases,
phospholipases, lipooxygenases, beta-glucanases,
endo-beta-1,3(4)-glucanases, cutinases, peroxidases, laccases, amylases,
glucoamylases, pectinases, reductases, oxidases, decarboxylases,
phenoloxidases, ligninases, pullulanases, phytases, arabinanases,
hemicellulases, other mannanases, xyloglucanases, xylanases, pectin
acetyl esterases, rhamnogalacturonan acetyl esterases, proteases,
peptidases, proteinases, polygalacturonases, rhamnogalacturonases,
galactanases, pectate lyases, transglutaminases, pectin methylesterases,
cellobiohydrolases and/or transglutaminases.
[0641] Oral Care Products
[0642] The invention provides oral care product comprising glucanases,
mannanases, or xylanases of the invention. Exemplary oral care products
include toothpastes, dental creams, gels or tooth powders, odontics,
mouth washes, pre- or post brushing rinse formulations, chewing gums,
lozenges, or candy. See, e.g., U.S. Pat. No. 6,264,925.
[0643] The oral products of the invention can include any combination of
other enzymes such as proteases, peptidases, proteinases, glucose
oxidases, peroxidases, glucanases, cellulases, endoglycosidases,
endo-beta-1,4-glucanases, amyloglucosidases, endo-beta-1,3(4)-glucanases,
amyloglucosidases and glucosidases.
[0644] Brewing and Fermenting
[0645] The invention provides methods of brewing (e.g., fermenting) beer
comprising glucanases, mannanases, or xylanases of the invention. In one
exemplary process, starch-containing raw materials are disintegrated and
processed to form a malt. An enzyme of the invention is used at any point
in the fermentation process. Glucanases, mannanases, or xylanases of the
invention can be used in the brewing industry for the degradation of
beta-glucans. In one aspect, glucanases, mannanases, or xylanases of the
invention are used in the brewing industry for the clarification of the
beverage.
[0646] In one aspect, glucanases, mannanases, or xylanases of the
invention can be used in the processing of barley malt. The major raw
material of beer brewing is barley malt. This can be a three stage
process. First, the barley grain can be steeped to increase water
content, e.g., to around about 40%. Second, the grain can be germinated
by incubation at 15 to 25.degree. C. for 3 to 6 days when enzyme
synthesis is stimulated under the control of gibberellins. In one aspect,
enzymes of the invention are added at this (or any other) stage of the
process.
[0647] In one aspect, enzymes of the invention are used in mashing and
conversion processes. In the brewing and fermentation industries, mashing
and conversion processes are performed at temperatures that are too low
to promote adequate degradation of water-soluble glucans and xylans.
These polymers form gummy substrates that can cause increased viscosity
in the mashing wort, resulting in longer mash run-off, residual haze and
precipitates in the final beer product due to inefficient filtration and
low extraction yield. For these reasons, enzymes are added during the
brewing processes to breakdown .beta.-1,4- and .beta.-1,3-linked glucan.
[0648] In one aspect, enzymes of the invention are used in malthouse
operations, e.g., glucanase is added to the process water, to shorten
germination times and/or to encourage conversion of poor quality barley
to acceptable malts. In one aspect, enzymes of the invention are used for
mashing, e.g., they are added to increase wort filterability and/or
improve lautering. In one aspect, enzymes of the invention are used in
the fermenter and/or settling tank to, e.g., assist in haze clearing
and/or to improve filtration. In one aspect, enzymes of the invention are
used in adjunct brewing, e.g., a glucanase of the invention is added to
breakdown glucans from barley, wheat, and/or other cereals, including
glycans in malt. In one aspect, enzymes of the invention are used in malt
brewing, e.g., a glucanase of the invention is added to modify poor malts
with high glucan content.
[0649] Glucanases, mannanases, or xylanases of the invention can be used
in any beer or alcoholic beverage producing process, as described, e.g.,
in U.S. Pat. Nos. 5,762,991; 5,536,650; 5,405,624; 5,021,246; 4,788,066.
[0650] The brewing processes of the invention can include the use of any
combination of other enzymes such as other xylanases, esterases,
cellulases, pectinases, pectate lyases, amylases, decarboxylases,
laccases, glucanases, proteases, peptidases, proteinases,
amyloglucosidases, glucose isomerases, glucoamylases, beta-glucanases,
endo-beta-1,3(4)-glucanases, hemicellulases, endoglycosidases,
endo-beta.-1,4-glucanases, glycosyltransferases, phospholipases,
lipooxygenases, reductases, oxidases, phenoloxidases, ligninases,
pullulanases, arabinanases, other mannanases, xyloglucanases, pectin
acetyl esterases, rhamnogalacturonan acetyl esterases,
polygalacturonases, rhamnogalacturonases, galactanases,
transglutaminases, pectin methylesterases, cellobiohydrolases and/or
transglutaminases.
[0651] Medical and Research Applications
[0652] Glucanases, mannanases, or xylanases of the invention can be used
as antimicrobial agents due to their bacteriolytic properties and
anti-fungal properties. Glucanases of the invention can be used to
eliminate or protect animals from salmonellae, e.g., as described in PCT
Application Nos. WO0049890 and WO9903497. Glucanases, mannanases, or
xylanases of the invention can be used in a method of use and composition
of a carbohydrase and/or a glucanase for the manufacture of an agent for
the treatments and/or prophylaxis of coccidiosis. The manufactured agent
can be in the form of a cereal-based animal feed. (see, for example, U.S.
Pat. No. 5,624,678).
[0653] Drilling Applications
[0654] Glucanases, mannanases, or xylanases of the invention can be used
in modifying the viscosity of plant derived material. In one aspect,
enzymes of the invention are used in the oil industry where guar gum and
modified guar are used in, e.g., fracturing fluids and drilling muds. The
enzymes of the invention can be used to clean oil wells, e.g. to break
the high viscosity or gel structure in fractural fluid after the
fracturation. In one aspect, the enzymes of the invention used in these
applications have a high thermostability. In one aspect, the enzymes of
the invention used in these applications are resistant to the elevated
temperatures in the ground or generated by drilling processes.
Glucanases, mannanases, or xylanases of the invention can be used to
treat drill mud (e.g., used mud).
OTHER INDUSTRIAL APPLICATIONS
[0655] Glucanases, mannanases, or xylanases of the invention can be used
in a wide variety of food, animal feed and beverage applications. New
glucanases, mannanases, or xylanases are discovered by screening existing
libraries and DNA libraries constructed from diverse mesophilic and
moderately thermophilic locations as well as from targeted sources
including digestive flora, microorganisms in animal waste, soil bacteria
and highly alkaline habitats. Biotrap and primary enrichment strategies
using glucan-comprising substrates and/or non-soluble polysaccharide
fractions of animal feed material are also useful.
[0656] Glucanases, mannanases, or xylanases of the invention can be used
in the conversion of biomass to fuels, and in the production of ethanol,
e.g., as described in PCT Application Nos. WO0043496 and WO8100857.
Glucanases of the invention can be used to produce fermentable sugars and
glucan-containing biomass that can be converted into fuel ethanol.
[0657] Glucanases, mannanases, or xylanases of the invention can be used
in combination with other enzymes involved in cellulose digestion like
cellobiohydrolases and beta-glucosidases.
[0658] Glucanases, mannanases, or xylanases of the invention can be used
in a number of other applications. For example, glucanases of the
invention can be used in improving the quality and quantity of milk
protein production in lactating cows (see, for example, Kung, L., et al,
J. Daily Science 2000 January 83:115-122), increasing the amount of
soluble saccharides in the stomach and small intestine of pigs (see, for
example, van der Meulen, J. et al, Arch. Tierernahr 2001 54:101-115),
improving late egg production efficiency and egg yields in hens (see, for
example, Jaroni, D., et al, Poult. Sci., 1999 June 78:841-847).
Additionally, glucanases, mannanases, or xylanases of the invention can
be used as flour, dough and bread improvers (see, for example, U.S. Pat.
Nos. 5,108,765 and 5,306,633) as feed additives and/or supplements, as
set forth above (see, for example, U.S. Pat. Nos. 5,432,074, 5,429,828,
5,612,055, 5,720,971, 5,981,233, 5,948,667, 6,099,844, 6,132,727 and
6,132,716), in manufacturing cellulose solutions (see, for example, U.S.
Pat. No. 5,760,211). Detergent compositions comprising glucanases,
mannanases, or xylanases of the invention can be used for fruit,
vegetables and/or mud and clay compounds (see, for example, U.S. Pat. No.
5,786,316).
[0659] Additional uses for glucanases, mannanases, or xylanases of the
invention include use in the production of water soluble dietary fiber
(see, for example, U.S. Pat. No. 5,622,738), in improving the
filterability, separation and production of starch (see, for example,
U.S. Pat. Nos. 4,960,705 and 5,023,176), in the beverage industry in
improving filterability of wort or beer (see, for example, U.S. Pat. No.
4,746,517), in an enzyme composition for promoting the secretion of milk
of livestock and improving the quality of the milk (see, for example,
U.S. Pat. No. 4,144,354), in reducing viscosity of plant material (see,
for example, U.S. Pat. No. 5,874,274), in increasing viscosity or gel
strength of food products such as jam, marmalade, jelly, juice, paste,
soup, salsa, etc. (see, for example, U.S. Pat. No. 6,036,981).
Glucanases, mannanases, or xylanases may also be used in hydrolysis of
hemicellulose for which it is selective, particularly in the presence of
cellulose. Additionally, the cellulase rich retentate is suitable for the
hydrolysis of cellulose (see, for example, U.S. Pat. No. 4,725,544).
[0660] Various uses of glucanases, mannanases, or xylanases of the
invention include transformation of a microbe that produces ethanol (see,
for example, PCT Application No. WO99/46362), in production of
oenological tannins and enzymatic composition (see, for example, PCT
Application No. WO0164830), in stimulating the natural defenses of plants
(see, for example, PCT Application No. WO0130161), in production of
sugars from hemicellulose substrates (see, for example, PCT Application
No. WO9203541), in the cleaning of fruit, vegetables, mud or clay
containing soils (see, for example, PCT Application No. WO9613568), in
cleaning beer filtration membranes (see, for example, PCT Application No.
WO9623579), in a method of killing or inhibiting microbial cells (see,
for example, PCT Application No. WO9732480) and in determining the
characteristics of process waters from wood pulp bleaching by using the
ratios of two UV absorption measurements and comparing the spectra (see,
for example, PCT Application No. WO9840721).
[0661] Any product or process of the invention can include any combination
of other enzymes such as catalases, glucanases, cellulases,
endoglycosidases, endo-beta.-1,4-glucanases, amyloglucosidases, glucose
isomerases, glycosyltransferases, lipases, esterase, phospholipases,
lipooxygenases, beta-glucanases, endo-beta-1,3(4)-glucanases, cutinases,
peroxidases, laccases, amylases, glucoamylases, pectinases, reductases,
oxidases, decarboxylases, phenoloxidases, ligninases, pullulanases,
phytases, arabinanases, hemicellulases, mannanases, xyloglucanases,
xylanases, pectin acetyl esterases, rhamnogalacturonan acetyl esterases,
polygalacturonases, rhamnogalacturonases, galactanases, pectate lyases,
transglutaminases, pectin methylesterases, cellobiohydrolases and/or
transglutaminases.
[0662] Two screening formats (activity-based and sequence-based) are used
in the discovery of novel glucanases, mannanases, or xylanases. The
activity-based approach is direct screening for glucanase activity in
agar plates using a substrate such as AZO-barley beta glucan (Megazyme).
Alternatively a sequence-based approach may be used, which relies on
bioinformatics and molecular biology to design probes for hybridization
and biopaning. See, for example, U.S. Pat. Nos. 6,054,267, 6,030,779,
6,368,798, 6,344,328. Hits from the screening are purified, sequenced,
characterized (for example, determination of specificity, temperature and
pH optima), analyzed using bioinformatics, subcloned and expressed for
basic biochemical characterization. These methods may be used in
screening for glucanases, mannanases, or xylanases useful in a myriad of
applications, including dough conditioning and as animal feed additive
enzymes.
[0663] In characterizing enzymes obtained from screening, the exemplary
utility in dough processing and baking applications may be assessed.
Characterization may include, for example, measurement of substrate
specificity (glucan, CMC, BBG), temperature and pH stability and specific
activity. A commercial enzyme may be used as a benchmark. In one aspect,
the enzymes of the invention have significant activity at pH=7 and
25-35.degree. C., are inactive on insoluble glucan, are stable and active
in 50-67% sucrose.
[0664] In another aspect, utility as feed additives may be assessed from
characterization of candidate enzymes. Characterization may include, for
example, measurement of substrate specificity (glucan, CMC, BBG),
temperature and pH stability, specific activity and gastric stability. In
one aspect the feed is designed for a monogastric animal and in another
aspect the feed is designed for a ruminant animal. In one aspect, the
enzymes of the invention have significant activity at pH 2-4 and
35-40.degree. C., a half-life greater than 30 minutes in gastric fluid,
formulation (in buffer or cells) half-life greater than 5 minutes at
85.degree. C. and are used as a monogastric animal feed additive. In
another aspect, the enzymes of the invention have one or more of the
following characteristics: significant activity at pH 6.5-7.0 and
35-40.degree. C., a half-life greater than 30 minutes in rumen fluid,
formulation stability as stable as dry powder and are used as a ruminant
animal feed additive.
[0665] Enzymes are reactive toward a wide range of natural and unnatural
substrates, thus enabling the modification of virtually any organic lead
compound. Moreover, unlike traditional chemical catalysts, enzymes are
highly enantio- and regio-selective. The high degree of functional group
specificity exhibited by enzymes enables one to keep track of each
reaction in a synthetic sequence leading to a new active compound.
Enzymes are also capable of catalyzing many diverse reactions unrelated
to their physiological function in nature. For example, peroxidases
catalyze the oxidation of phenols by hydrogen peroxide. Peroxidases can
also catalyze hydroxylation reactions that are not related to the native
function of the enzyme. Other examples are glucanases which catalyze the
breakdown of polypeptides. In organic solution some glucanases can also
acylate sugars, a function unrelated to the native function of these
enzymes.
[0666] The present invention exploits the unique catalytic properties of
enzymes. Whereas the use of biocatalysts (i.e., purified or crude
enzymes, non-living or living cells) in chemical transformations normally
requires the identification of a particular biocatalyst that reacts with
a specific starting compound, the present invention uses selected
biocatalysts and reaction conditions that are specific for functional
groups that are present in many starting compounds. Each biocatalyst is
specific for one functional group, or several related functional groups
and can react with many starting compounds containing this functional
group. The biocatalytic reactions produce a population of derivatives
from a single starting compound. These derivatives can be subjected to
another round of biocatalytic reactions to produce a second population of
derivative compounds. Thousands of variations of the original compound
can be produced with each iteration of biocatalytic derivatization.
[0667] Enzymes react at specific sites of a starting compound without
affecting the rest of the molecule, a process which is very difficult to
achieve using traditional chemical methods. This high degree of
biocatalytic specificity provides the means to identify a single active
compound within the library. The library is characterized by the series
of biocatalytic reactions used to produce it, a so-called "biosynthetic
history". Screening the library for biological activities and tracing the
biosynthetic history identifies the specific reaction sequence producing
the active compound. The reaction sequence is repeated and the structure
of the synthesized compound determined. This mode of identification,
unlike other synthesis and screening approaches, does not require
immobilization technologies and compounds can be synthesized and tested
free in solution using virtually any type of screening assay. It is
important to note, that the high degree of specificity of enzyme
reactions on functional groups allows for the "tracking" of specific
enzymatic reactions that make up the biocatalytically produced library.
[0668] Many of the procedural steps are performed using robotic automation
enabling the execution of many thousands of biocatalytic reactions and
screening assays per day as well as ensuring a high level of accuracy and
reproducibility. As a result, a library of derivative compounds can be
produced in a matter of weeks which would take years to produce using
current chemical methods. (For further teachings on modification of
molecules, including small molecules, see PCT/US94/09174).
[0669] The invention will be further described with reference to the
following examples; however, it is to be understood that the invention is
not limited to such examples.
EXAMPLES
Example 1
Plate Based Endoglycosidase Enzyme Discovery: Expression Screening
[0670] The following example demonstrates the isolation of and
confirmation of the enzymatic activity of exemplary enzymes and nucleic
acids of the invention. These assays can also be used to determine if a
polypeptide has the requisite enzyme (e.g., glucanase, mannanase, or
xylanase) activity to be within the scope of the invention.
Titer determination of Lambda Library: Add 1.0 .mu.L of Lambda Zap
Express amplified library stock to 600 .mu.L E. coli MRF' cells
(OD.sub.600=1.0). Dilute MRF' stock with 10 mM MgSO.sub.4. Incubate
mixture at 37.degree. C. for 15 minutes, then transfer suspension to 5-6
mL of NZY top agar at 50.degree. C. and gently mix. Immediately pour agar
solution onto large (150 mm) NZY media plate and allow top agar to
solidify completely (approximately 30 minutes). Invert the plate.
Incubate the plate at 39.degree. C. for 8-12 hours. (The number of
plaques is approximated. Phage titer determined to give 50,000 pfu/plate.
Dilute an aliquot of Library phage with SM buffer if needed.) Substrate
screening: Add Lambda Zap Express (50,000 pfu) from amplified library to
600 .mu.L of E. coli MRF' cells (OD.sub.600=1.0) and incubate at
37.degree. C. for 15 minutes. While phage/cell suspension is incubating,
add 11.0 mL of desired polysaccharide dye-labeled substrate (usually 1-2%
w/v) to 5.0 mL NZY top agar at 50.degree. C. and mix thoroughly.
(Solution kept at 50.degree. C. until needed.) Transfer the cell
suspension to substrate/top agar solution and gently mix. Immediately
pour solution onto large (150 mm) NZY media plate. Allow top agar to
solidify completely (approximately 30 minutes), then invert plate.
Incubate plate at 39.degree. C. for 8-12 hours. Observe plate for
clearing zones (halos) around plaques. Core plaques with halos out of
agar and transfer to a sterile micro tube. (A large bore 200 .mu.L
pipette tip works well to remove (core) the agar plug containing the
desired plaque.) Resuspend phage in 500 .mu.L SM buffer. Add 20 .mu.L
chloroform to inhibit any further cell growth. Isolation of pure clones:
Add 5 .mu.L of resuspended phage suspension to 500 .mu.L of E. coli MRF'
cells (OD.sub.600=1.0). Incubate at 37.degree. C. for 15 minutes. While
phage/cell suspension is incubating, add 600 .mu.L of desired
polysaccharide dye-labeled substrate (usually 1-2% w/v) to 3.0 mL NZY top
agar at 50.degree. C. and mix thoroughly. (Solution kept at 50.degree. C.
until needed.) Transfer cell suspension to substrate/top agar solution
and gently mix. Immediately pour solution onto small (90 mm) NZY media
plate and allow top agar to solidify completely (approximately 30
minutes), then invert plate. Incubate plate at 39.degree. C. for 8-12
hours. Plate observed for a clearing zone (halo) around a single plaque
(pure clone). (If a single plaque cannot be isolated, adjust titer and
replate phage suspension.) Phage are resuspended in 500 .mu.L SM buffer
and 20 .mu.L Chloroform is added to inhibit any further cell growth.
Excision of pure clone: Allow pure phage suspension to incubate at room
temperature for 2 to 3 hours or overnight at 4.degree. C. Add 100 .mu.L
of pure phage suspension to 200 .mu.L E. coli MRF' cells
(OD.sub.600=1.0). Add 1.0 .mu.L of ExAssist helper phage
(>1.times.10.sup.6 pfu/mL; Stratagene). Incubate suspension at
37.degree. C. for 15 minutes. Add 3.0 mL of 2.times.YT media to cell
suspension. Incubate at 37.degree. C. for 2-2.5 hours while shaking.
Transfer tube to 70.degree. C. for 20 minutes. Transfer 50-100 .mu.L of
phagemid suspension to a micro tube containing 200 .mu.L of E. coli Exp
505 cells (OD.sub.600=1.0). Incubate suspension at 37.degree. C. for 45
minutes. Plate 100 .mu.L of cell suspension on LB.sub.kan 50 media (LB
media with Kanamycin 50 .mu.g/mL). Incubate plate at 37.degree. C. for
8-12 hours. Observe plate for colonies. Any colonies that grow contain
the pure phagemid. Pick a colony and grow a small (3-10 mL) liquid
culture for 8-12 hours. Culture media is liquid LB.sub.kn 50. Activity
verification: Transfer 1.0 mL of liquid culture to a sterile micro tube.
Centrifuge at 13200 rpm (16000 g's) for 1 minute. Discard supernatant and
add 200 .mu.L of phosphate buffer pH 6.2. Sonicate for 5 to 10 seconds on
ice using a micro tip. Add 200 .mu.L of appropriate substrate, mix gently
and incubate at 37.degree. C. for 1.5-2 hours. A negative control should
also be run that contains only buffer and substrate. Add 1.0 mL absolute
ethanol (200 proof) to suspension and mixed. Centrifuge at 13200 rpm for
10 minutes. Observe supernatant for color. Amount of coloration may vary,
but any tubes with more coloration than control is considered positive
for activity. A spectrophotometer can be used for this step if so desired
or needed. (For Azo-barley beta glucan, Megazyme, read at 590 nm). RFLP
of pure clones from same Libraries: Transfer 11.0 mL of liquid culture to
a sterile micro tube. Centrifuge at 13200 rpm (16000 g's) for 1 minute.
Follow QIAprep spin mini kit (Qiagen) protocol for plasmid isolation and
use 40 .mu.L holy water as the elution buffer. Transfer 10 .mu.L plasmid
DNA to a sterile micro tube. Add 1.5 .mu.L Buffer 3 (New England
Biolabs), 1.5 .mu.L 100.times.BSA solution (New England Biolabs) and 2.0
.mu.L holy water. To this add 1.0 .mu.L Not 1 and 1.0 .mu.L Pst 1
restriction endonucleases (New England Biolabs). Incubate for 1.5 hours
at 37.degree. C. Add 3.0 .mu.L 6.times. Loading buffer (Invitrogen). Run
15 .mu.L of digested sample on a 1.0% agarose gel for 1-1.5 hours at 120
volts. View the gel with a gel imager. Perform sequence analysis on all
clones with a different digest pattern.
[0671] FIG. 5 is a table containing characterization of the enzymes of the
invention, including summarizing the relative activities of several
exemplary enzymes of the invention under various conditions, e.g.,
varying pH and temperature, as discussed above.
Example 2
Activity Assays
[0672] The following example demonstrates the enzymatic activity of
exemplary enzymes of the invention. These assays can also be used to
determine if a polypeptide has the requisite enzyme (e.g., glucanase,
mannanase, or xylanase) activity to be within the scope of the invention.
[0673] Polypeptides of the invention having sequences as set forth in the
SEQ ID NO:s listed below were demonstrated to have glucanase activity, as
described below. Specific activity was determined on barley .beta.-glucan
(BBG) or carboxymethylcellulose (CMC) using the BCA reducing sugar assay.
1 unit (U) of glucanase activity=1 .mu.mol/min.sup.-1 glucose reducing
equivalents released at 37.degree. C., pH 5.3.
TABLE-US-00005
Specific Activity
(U/mg)
6H 6H
Mw GH Native, tagged, tagged, T.sub.opt
Glucanase (kDa) pI Family BBG BBG CMC (.degree. C.) pH.sub.opt
SEQ ID NO: 6 (encoded 37.5 5.9 5 22 ND ND =90 5-7
by SEQ ID NO: 5)
SEQ ID NO: 400 (encoded 37.9 5.5 5 0.85 ND ND =90 5-7
by SEQ ID NO: 399)
SEQ ID NO: 162 (encoded 34.0 5.2 5 0.95 ND ND =85 ND
by SEQ ID NO: 161)
SEQ ID NO: 84 (encoded 36.9 6.3 5 >40 ND ND 80 4-6
by SEQ ID NO: 83)
SEQ ID NO: 172 (encoded 29.8 5.0 16 32 ND ND 50 5-6
by SEQ ID NO: 171)
SEQ ID NO: 104 (encoded 39.7 5.9 5 ND 3.2 2.8 85 5-6
by SEQ ID NO: 103)
SEQ ID NO: 10 (encoded 77.7 4.9 5 ND ND 0.5 85 5-6
by SEQ ID NO: 9)
SEQ ID NO: 222 (encoded 53.8 9.1 5 >40 16 24 85 5-6
by SEQ ID NO: 221)
SEQ ID NO: 108 (encoded 78.9 4.3 5 ND 3.8 4.0 75 ND
by SEQ ID NO: 107)
SEQ ID NO: 176 (encoded 37.2 6.0 5 ND 3.5 21 75 5-6
by SEQ ID NO: 175)
SEQ ID NO: 110 (encoded 39.9 6.2 5 ND 13 12 ND ND
by SEQ ID NO: 109)
SEQ ID NO: 268 (encoded 51.8 4.6 5 ND 3.6 2.8 50 ND
by SEQ ID NO: 267)
SEQ ID NO: 324 (encoded 49.3 6.1 5 ND ND ND ND ND
by SEQ ID NO: 323)
SEQ ID NO: 370 (encoded 42.1 5.8 5 ND ND ND ND ND
by SEQ ID NO: 369)
SEQ ID NO: 168 (encoded 37.3 5.7 5 ND ND ND ND ND
by SEQ ID NO: 167)
SEQ ID NO: 154 (encoded 35.6 5.4 5 ND ND ND ND ND
by SEQ ID NO: 153)
SEQ ID NO: 118 (encoded 34.5 6.1 5 ND ND ND ND ND
by SEQ ID NO: 117)
SEQ ID NO: 148 (encoded 74.1 5.3 5 ND ND ND ND ND
by SEQ ID NO: 147)
ND = Not determined
[0674] Exemplary polypeptides of the invention having a sequence as set
forth in the SEQ ID NO:s below were demonstrated to have alkaline
endoglucanase/cellulase activity, with pH and temperature optimums as set
forth, below. This activity was determined using a cellulase activity
assay (a BCA reducing ends assay), as described in detail in Example 3,
below.
TABLE-US-00006
SEQ ID Temperature
NO: Type pH optimum optimum
409, 410 Alkaline endoglucanase/cellulase 5 NA
343, 344 Alkaline endoglucanase/cellulase 6 60
319, 320 Alkaline endoglucanase/cellulase 6 70
383, 384 Alkaline endoglucanase/cellulase 7 60
301, 302 Alkaline endoglucanase/cellulase 7 60
257, 258 Alkaline endoglucanase/cellulase 8 42
419, 420 Alkaline endoglucanase/cellulase 8 70
421, 422 Alkaline endoglucanase/cellulase 9 60
405, 406 Alkaline endoglucanase/cellulase 9 50
329, 330 Alkaline endoglucanase/cellulase 9 50
325, 326 Alkaline endoglucanase/cellulase (5-7)* 70
415, 416 Alkaline endoglucanase/cellulase (6-10)* 70
303, 304 Alkaline endoglucanase/cellulase (6-10)* 60
271, 272 Alkaline endoglucanase/cellulase (6-7)* 60
175, 176 Alkaline endoglucanase/cellulase (6-7)* 70
9, 10 Alkaline endoglucanase/cellulase (6-7)* 70
297, 298 Alkaline endoglucanase/cellulase (6-8)* 50
109, 110 Alkaline endoglucanase/cellulase (6-8)* 60
267, 268 Alkaline endoglucanase/cellulase (6-8)* 70
107, 108 Alkaline endoglucanase/cellulase (6-8)* 70
305, 306 Alkaline endoglucanase/cellulase (7-10)* 60
417, 418 Alkaline endoglucanase/cellulase (7-8.5)* NA
227, 228 Alkaline endoglucanase/cellulase (7-9)* 60
375, 376 Alkaline endoglucanase/cellulase (7-9)* 70
335, 336 Alkaline endoglucanase/cellulase (7-9)* 50
155, 156 Alkaline endoglucanase/cellulase (7-9)* 60
445, 446 Alkaline endoglucanase/cellulase (7-9)* 60
259, 260 Alkaline endoglucanase/cellulase (8-10)* 50
423, 424 Alkaline endoglucanase/cellulase (8-10)* 50
345, 346 Alkaline endoglucanase/cellulase (8-9)* 25
285, 286 Alkaline endoglucanase/cellulase (8-9)* 50
351, 352 Alkaline endoglucanase/cellulase (9-10)* 80
Example 3
Cellulase Activity Assay: BCA Reducing Ends Assay
[0675] The following example describes an assay, a cellulase activity
assay (a BCA reducing ends assay) that can be used to determine if a
polypeptide has the requisite enzyme (e.g., glucanase, mannanase, or
xylanase) activity, e.g., an alkaline endoglucanase/cellulase activity
(see Example 2, above) to be within the scope of the invention.
[0676] This assay was designed to measure the amount of reducing ends
produced during the enzymatic degradation of carboxymethylcellulose (CMC)
in a high throughput multiple sample 96-well format.
Materials:
Substrate Solutions:
1% CMC
[0677] Dissolve 1 gm CMC in 100 ml 50 mM Britton-Robinson buffer at pH
.about.4, heat CMC solution in boiling water bath, while mixing, for
20-40 minutes until it dissolves (solution will still appear slightly
milky, but translucent). Adjust to desired pH with 1M NaOH or HCl.
Solution A:
[0678] 64 mg/ml sodium carbonate monohydrate 24 mg/ml sodium bicarbonate
1.95 mg/ml BCA (4,4'-dicarboxy-2,2'-biquinoline disodium salt (Sigma
Chemical cat #D-8284) Add above to dH2O, Might need to dissolve the BCA
by heating, don't heat more than .about.80.degree. C.
Solution B:
[0679] 1.24 mg/ml cupric sulfate pentahydrate 1.26 mg/ml L-serine Add
above to dH2O
Working Reagent:
[0680] 1:1 of solutions A & B, make fresh working reagent mixture every
day (usually only make enough for each assay), make fresh Solutions A & B
every week.
Glucose Stock Solution:
[0681] 10 mM Glucose in dH2O. 0.2 um filter, store at 4 C.
Glucose Standards:
[0682] Dilute the 10 mM Glucose stock in 1% CMC at desired pH; to a final
concentration of 0, 100, 200, 300, 400, 500 uM. Since the curve is
determined by adding 10 ul of the standards to the working reagent it
works out to 0-0.005 umole glucose/well. The standard curve needs to be
generated for each plate of sample time-points, as the heating cycle can
affect the amount of signal observed.
Method:
Set-Up:
[0683] Aliquot 1 ml of substrate solution (1% CMC) into deep-well plate
(if using ambient Temp) or Acme-tubes in hot-block, equilibrate to
desired temperature (.about.5 min) in heat block or heated water bath.
[0684] While solution is equilibrating, make 10 ml of the working reagent
and aliquot 100 ul into 96 well PCR-plate. Set plate on ice.
Reaction/Sampling:
[0685] After temperature equilibration is complete, add enzyme solution to
substrate solution. Mix immediately by pipetting up/down. Immediately
aliquot 10-ul into PCR-plate (this is t=0, zero time point). Aliquot
10-ul into PCR-plate at each desired time point (e.g. 0, 2, 5, 10, 15,
20, 30 minutes).
[0686] Save the last row on the plate for addition of 10 ul of glucose
standards (I.e. wells should only have the 100-ul working reagent in
them)
Assay Color Development:
[0687] When all time points are collected and standards are added, cover
plate and heat to 100 C for 10 min using PCR machine. Cool plate on ice
for 5-10 min (or set PCR machine to 1 C for 10 min).
[0688] Add 100 ul H2O to wells. Mix. Aliquot 100 ul of mixture into clear
flat bottomed 96-well plate and read absorbance at 560 nm.
Generate Standard Curve:
[0689] Plot the A560 vs. umole glucose from the wells containing the
glucose standards. Use linear regression to calculate the slope
(S.sub.std).
Generate Graph of Reaction Slope:
[0690] Plot A560 vs. time-points. Zero each sample's time points against
its own T=0 (i.e. subtract sample's T=0 absorbance value from all other
time-points of same sample). Generate the slope (S.sub.rxn) for each set
of sample time-points (A560/time).
Activity Determination:
[0691] Divide S.sub.rxn by the S.sub.std, and multiply by 100 (as the
umole product detected is the amount of reducing ends in the 10-ul used
in the assay, not the total amount generated in the 1 ml enzyme
reaction).
Specific Activity Determination:
[0692] Divide the Activity (in units of umole/min) by the total mg of
protein added in the 1-ml reaction. Determine the protein concentration
by Bradford or similar assay.
[0693] Divide the protein concentration by any dilutions used.
[0694] Multiply by the volume (in ml) used in the reaction.
[0695] All points should be done in duplicate with triplicate being
better.
[0696] The following chart sets forth an exemplary set of data ("sample
data") that is illustrated in graph form as a "standard curve" in FIG. 6.
TABLE-US-00007
SAMPLE DATA
date mg/ml Diln. ul/rxn 0 min 5 min 8 min 12 min 24 min 36 min 45 min
Enz x 06/09 20 500 20 0.1252 0.1654 0.1889 0.2315 0.3386 0.4036 0.4695
Slope of standard curve: 88.375 A560/umole glucose
Slope of reaction: 0.0076 A560/min
Activity (reaction slope/std slope): 8.70061E-05 umole/min
True activity/1 ml rxn (=Activity .times. 100): 0.0087 umole/min
Specific Activity: 10.87 umole/min, mg
Example 4
Codon Optimization
[0697] The following example demonstrates an exemplary codon optimization
of an exemplary enzyme-encoding sequence of the invention. Any codon
optimization protocol known in the art can be used to codon optimize any
nucleic acid of the invention.
[0698] An exemplary nucleic acid encoding the polypeptide having a
sequence as set forth in SEQ ID NO:6, i.e., SEQ ID NO:5, was subjected to
codon optimization for optimal expression in Pichia pastoris; the Pichia
pastoris codon-optimized enzyme-encoding nucleic acid is SEQ ID NO:463.
In addition to optimizing the codons of the enzyme-encoding nucleic acid,
one amino acid (A91V) was modified, and this new polypeptide sequence is
set forth as SEQ ID NO:464.
[0699] Glucanase activity assays (whose data are illustrated in FIGS. 7
and 8) demonstrated improved expression in Pichia pastoris of SEQ ID
NO:464 (encoded by, e.g., SEQ ID NO:463), which is the codon optimized
version of the polypeptide having a sequence a set forth in SEQ ID NO:6
(encoded, e.g., by SEQ ID NO:5). Expression level was improved by
changing the pH.
[0700] In FIG. 7, glucanase activity during the course of fermentation is
shown in U/mL of culture. 1 unit (U) of glucanase activity=1
.mu.mol/min.sup.-1 glucose reducing equivalents released at 37.degree.
C., pH 5.3. Codon-optimized glucanase SEQ ID NO:464 (encoded by SEQ ID
NO:463), expressed in Pichia pastoris was used. Fermentation was run at
5.0.
[0701] In FIG. 8, glucanase activity during the course of fermentation is
shown in U/mL of culture. 1 unit (U) of glucanase activity=1
.mu.mol/min.sup.-1 glucose reducing equivalents released at 37.degree.
C., pH 5.3. Codon-optimized glucanase SEQ ID NO:464 (encoded by SEQ ID
NO:463), expressed in Pichia pastoris was used. Fermentation was done at
pH 6.2.
Example 5
Enzyme Activity
[0702] The following example demonstrates confirmation of enzymatic
activity of exemplary enzymes of the invention. These assays can also be
used to determine if a polypeptide has the requisite enzyme (e.g.,
glucanase, mannanase, or xylanase) activity to be within the scope of the
invention.
[0703] Specific Activity of the Glucanase Encoded by SEQ ID NO:6
[0704] Specific activity of the exemplary enzyme of the invention having a
sequence as set forth in SEQ ID NO:6 (encoded by, e.g., SEQ ID NO:5) was
demonstrated using the following protocol:
[0705] The glucanase encoded by SEQ ID NO:6 was purified to homogeneity
using ion exchange chromatography. Specific activities were determined on
1% substrate in 50 mM sodium acetate buffer pH 5.3, at 37.degree. C.
using the BCA reducing sugar assay. 1 unit (U) of glucanase activity=1
mol/min.sup.1 glucose reducing equivalents released at 37.degree. C., pH
5.3.
[0706] Barley Beta Glucan (BBG): 30 U/mg
[0707] Oat Beta Glucan (OBG): 38 U/mg
[0708] Carboxymethylcellulose (CMC): 40 U/mg
[0709] Carob Galactomannan: 0.3 U/mg
[0710] Temperature Profile of the Glucanase Encoded by SEQ ID NO:6
[0711] Temperature profile was determined on three separate substrates
(BBG, OBG and CMC). The glucanase encoded by SEQ ID NO:6 had the highest
activity at higher temperatures. Specific activity of the glucanase
encoded by SEQ ID NO:6 on BBG and CMC at 80.degree. C. is 10.times.
better than the activity seen at 37.degree. C. In the presence of mannan,
the glucanase encoded by SEQ ID NO:6 showed the highest activity at
100.degree. C., as illustrated in FIG. 9.
[0712] Temperature profile was determined by incubating BD10 in the
presence of substrate (CMC, BBG or Mannan). Initial velocities were
determined using BCA reducing sugar assay and sodium acetate buffer pH
5.3. Initial velocities were normalized and plotted as % activity, as
illustrated in FIG. 9.
[0713] Half-Life Determination of the Glucanase Encoded by SEQ ID NO:6
[0714] The half-life of the glucanase encoded by SEQ ID NO:6 was
determined at 85.degree. C. and 90.degree. C. The glucanase encoded by
SEQ ID NO:6 was heat challenged for various times at 85 and 90 degrees
and the residual activity was measured at 37.degree. C. The glucanase
encoded by SEQ ID NO:6 retained more than 60% of its activity after 10
minutes of incubation at 85.degree. C. At 90.degree. C., there was no
residual activity left after 2 minutes, as illustrated in FIG. 10.
[0715] As illustrated in FIG. 10, half-life of BD10 was determined by heat
challenging the enzyme for 30 sec, 1 min, 2 min, 3 min, 4 min, 5 min, and
10 min at the indicated temperatures (85.degree. C. and 90.degree. C.)
and monitoring activity under standard conditions using the BCA reducing
sugar.
[0716] While the invention has been described in detail with reference to
certain Exemplary aspects thereof, it will be understood that
modifications and variations are within the spirit and scope of that
which is described and claimed.
Sequence CWU
0
SQTB
SEQUENCE LISTING
The patent application contains a lengthy "Sequence Listing" section. A
copy of the "Sequence Listing" is available in electronic form from the
USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20080233175A1).
An electronic copy of the "Sequence Listing" will also be available from
the USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
0
SQTB
SEQUENCE LISTING
The patent application contains a lengthy "Sequence Listing" section. A
copy of the "Sequence Listing" is available in electronic form from the
USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20080233175A1).
An electronic copy of the "Sequence Listing" will also be available from
the USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
* * * * *
