U.S. patent application number 15/218575 was filed with the patent office on 2017-05-11 for glucanases, nucleic acids encoding them and methods for making and using them.
The applicant listed for this patent is BP Corporation North America Inc., Syngenta Participations AG. Invention is credited to Walter Niles Callen, Shaun Healey, Derrick Pulliam, Brian Steer.
Application Number | 20170130215 15/218575 |
Document ID | / |
Family ID | 33564024 |
Filed Date | 2017-05-11 |
United States Patent
Application |
20170130215 |
Kind Code |
A1 |
Steer; Brian ; et
al. |
May 11, 2017 |
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 Niles; (San Diego, CA) ;
Healey; Shaun; (Carlsbad, CA) ; Pulliam; Derrick;
(Raleigh, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BP Corporation North America Inc.
Syngenta Participations AG |
Houston
Basel |
TX |
US
CH |
|
|
Family ID: |
33564024 |
Appl. No.: |
15/218575 |
Filed: |
July 25, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14019460 |
Sep 5, 2013 |
9422536 |
|
|
15218575 |
|
|
|
|
13156538 |
Jun 9, 2011 |
|
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|
14019460 |
|
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|
10560957 |
Apr 3, 2007 |
7960148 |
|
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PCT/US2014/021492 |
Jul 2, 2004 |
|
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|
13156538 |
|
|
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60484725 |
Jul 2, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23C 19/04 20130101;
C12N 9/2405 20130101; A23L 2/52 20130101; A61P 31/04 20180101; A23V
2002/00 20130101; C12P 19/18 20130101; D21C 1/00 20130101; C12N
11/14 20130101; G01N 33/573 20130101; D21C 5/005 20130101; A23K
20/189 20160501; C12Y 302/01004 20130101; A23K 50/10 20160501; A23L
29/10 20160801; C09K 8/62 20130101; C12N 11/16 20130101; C12N
9/2437 20130101; G01N 2333/924 20130101; A23K 10/14 20160501; G01N
33/53 20130101; G01N 2500/00 20130101; A61P 1/14 20180101; Y02E
50/10 20130101; Y02E 50/17 20130101; A23L 33/18 20160801; C12N
11/02 20130101; A61P 31/00 20180101; A23K 40/10 20160501; C12P
19/02 20130101; Y02E 50/16 20130101; C12P 7/10 20130101; C11D
3/38636 20130101; A61K 9/0056 20130101; A23C 9/1322 20130101; A61K
38/47 20130101; A61P 33/02 20180101 |
International
Class: |
C12N 9/24 20060101
C12N009/24; C12P 19/18 20060101 C12P019/18; C12P 19/02 20060101
C12P019/02; A61K 38/47 20060101 A61K038/47; A61K 9/00 20060101
A61K009/00; C11D 3/386 20060101 C11D003/386; D21C 5/00 20060101
D21C005/00; C09K 8/62 20060101 C09K008/62; A23C 9/13 20060101
A23C009/13; A23C 19/04 20060101 A23C019/04; A23K 10/14 20060101
A23K010/14; A23K 20/189 20060101 A23K020/189; A23K 40/10 20060101
A23K040/10; A23K 50/10 20060101 A23K050/10; A23L 2/52 20060101
A23L002/52; A23L 33/18 20060101 A23L033/18; G01N 33/573 20060101
G01N033/573 |
Claims
1. An isolated, synthetic, or recombinant polypeptide comprising:
(i) an amino acid sequence at least 70% or more or complete (100%)
sequence identity to SEQ ID NO: 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 an enzymatically active fragment
thereof, wherein the polypeptide or the enzymatically active
fragment has a glucanase activity; (ii) an amino acid sequence
encoded by a nucleic acid having at least 65% sequence identity to
SEQ ID NO.: 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 polypeptide has glucanase activity;
(iii) the amino acid sequence of (i), or (ii), lacking a signal
sequence and/or a carbohydrate binding module; or (iv) the amino
acid sequence of (i), (ii), or (iii), further comprising a
heterologous sequence.
2. The polypeptide of claim 1, 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.
3. A protein preparation comprising the polypeptide of claim 1,
wherein the protein preparation comprises a liquid, a solid or a
gel.
4. An immobilized polypeptide, wherein the polypeptide comprises:
(a) the sequence of claim 1; 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.
5. A method for identifying a polypeptide having a glucanase
activity comprising: (a) providing the polypeptide of claim 1; (b)
providing a glucanase substrate; and (c) contacting the polypeptide
with the substrate of (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 activity.
6. A method for identifying a glucanase substrate comprising: (a)
providing the polypeptide of claim 1; (b) providing a test
substrate; and (c) contacting the polypeptide of (a) with the test
substrate of (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 substrate.
7. A method of determining whether a test compound specifically
binds to a polypeptide comprising: (a) providing the polypeptide of
claim 1; (b) providing a test compound; (c) contacting the
polypeptide with the test compound; and (d) determining whether the
test compound of (b) specifically binds to the polypeptide.
8. A method for identifying a modulator of a glucanase activity
comprising: (A) (a) providing the polypeptide of claim 1; (b)
providing a test compound; (c) contacting the polypeptide of (a)
with the test compound of (b) and measuring an activity of the
glucanase, wherein a change in the glucanase 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 activity; or (B) the method of (A), wherein the
glucanase 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
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.
9. A method for hydrolyzing, breaking up or disrupting a
cellulose-, hemicellulose-, lignin-, or glucan-comprising
composition comprising: (A) (a) providing the polypeptide having a
glucanase activity of claim 1; (b) providing a composition
comprising a cellulose, hemicellulose, lignin, or glucan; and (c)
contacting the polypeptide of (a) with the composition of (b) under
conditions wherein the glucanase hydrolyzes, breaks up or disrupts
the cellulose-, hemicellulose-, lignin-, or glucan-comprising
composition; or (B) the method of (A), wherein the composition
comprises a biomass, a plant cell, a bacterial cell, a yeast cell,
an insect cell, or an animal cell.
10. A dough or a bread product comprising the polypeptide of claim
1.
11. A method of dough conditioning comprising contacting a dough or
a bread product with at least one polypeptide of claim 1 under
conditions sufficient for conditioning the dough.
12. A beverage comprising the polypeptide of claim 1.
13. A method of beverage production comprising (A) administration
of at least one polypeptide of claim 1 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.
14. A food, a feed or a nutritional supplement comprising the
polypeptide of claim 1.
15. A method for utilizing a glucanase nutritional supplement in an
animal diet, the method comprising: (A) preparing a nutritional
supplement containing a polypeptide as set forth in claim 1; and
administering the nutritional supplement to an animal to increase
utilization of a glucan contained in a feed or a food ingested by
the animal; (B) the method of (A), wherein the animal is a human;
or (C) the method of (A), wherein the animal is a ruminant or a
monogastric animal.
16. An edible enzyme delivery matrix comprising the polypeptide of
claim 1.
17. A method for delivering a glucanase 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 enzyme, wherein the pellets
readily disperse the glucanase 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 enzyme comprises the polypeptide of claim 1; (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 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; (g)
the method of (a), (b), (c), (d), (e) or (f), wherein the
pelletizing conditions include application of steam.
18. A cellulose- or cellulose derivative-composition comprising the
polypeptide of claim 1.
19. A wood, wood pulp, or wood product comprising the polypeptide
of claim 1.
20. A paper, paper pulp, or paper product comprising the
polypeptide of claim 1.
21. 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 1.
22. A detergent composition comprising a polypeptide as set forth
in claim 1.
23. A method for making a fuel comprising contacting a composition
comprising a cellulose, a hemicellulose, a lignin or a glucan with
the polypeptide of claim 1.
24. A dairy product comprising: (a) the polypeptide of claim 1; or
(b) the dairy product of (a), comprising a milk, an ice cream, a
cheese or a yogurt.
25. A method for improving texture and flavor of a dairy product
comprising: (a) providing the polypeptide of claim 1; (b) providing
a dairy product; and (c) contacting the polypeptide of (a) and the
dairy product of (b) under conditions wherein the glucanase can
improve the texture or flavor of the dairy product.
26. A composition comprising the polypeptide of claim 1.
27. A fracturing fluid composition comprising the polypeptide of
claim 1.
28. A method of treating a subterranean formation comprising use of
the polypeptide of claim 1.
29. An expression cassette, a vector or a cloning vehicle
comprising a nucleic acid comprising the sequence of claim 1,
wherein optionally the cloning vehicle comprises a viral vector, a
plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage
or an artificial chromosome, or wherein optionally the cloning
vehicle comprises a viral vector comprising an adenovirus vector, a
retroviral vector or an adeno-associated viral vector, or wherein
optionally the cloning vehicle comprises a bacterial artificial
chromosome (BAC), a plasmid, a bacteriophage P1-derived vector
(PAC), a yeast artificial chromosome (YAC), or a mammalian
artificial chromosome (MAC).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 14/019,460 filed Sep. 5, 2013, now pending;
which is a continuation application of U.S. application Ser. No.
13/156,538 filed Jun. 9, 2011, now abandoned; which is a divisional
application of U.S. application Ser. No. 10/560,957 filed Apr. 3,
2007, now issued as U.S. Pat. No. 7,960,148; which is a 35 USC
.sctn.371 National Stage application of International Application
No. PCT/US2004/021492 filed Jul. 2, 2004, now expired; which claims
the benefit under 35 USC .sctn.119 (e) to U.S. Application Ser. No.
60/484,725 filed Jul. 2, 2003, now expired. The disclosure of each
of the prior applications is considered part of and is incorporated
by reference in the disclosure of this application.
BACKGROUND OF THE INVENTION
[0002] Field of the Invention
[0003] 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.
[0004] Background Information
[0005] 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 1,413- 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.
[0006] 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.
[0007] 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.
[0008] 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
[0009] 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.
[0010] 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 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. 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.
[0011] 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: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, 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).
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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).
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] In one aspect, a polypeptide of the invention has at least
one glucanase, e.g., endoglucanase, a mannanase, or a xylanase
activity.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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).
[0055] 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).
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] The invention provides protein preparations comprising a
polypeptide of the invention, wherein the protein preparation
comprises a liquid, a solid or a gel.
[0061] 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.
[0062] 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.
[0063] The invention provides arrays comprising an immobilized
nucleic acid of the invention. The invention provides arrays
comprising an antibody of the invention.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[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 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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 yogurt. The
invention provides dairy products comprising a glucanase,
mannanase, or xylanase of the invention, or is encoded by a nucleic
acid of the invention.
[0098] 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.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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.
[0113] 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.
[0114] 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
gastro-intestinal 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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
[0120] 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.
[0121] FIG. 1 is a block diagram of a computer system.
[0122] 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.
[0123] FIG. 3 is a flow diagram illustrating one aspect of a
process in a computer for determining whether two sequences are
homologous.
[0124] FIG. 4 is a flow diagram illustrating one aspect of an
identifier process 300 for detecting the presence of a feature in a
sequence.
[0125] FIG. 5 is a table summarizing the relative activities of
several exemplary enzymes of the invention under various
conditions.
[0126] 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.
[0127] FIGS. 7 and 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.
[0128] 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.
[0129] 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.
[0130] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION OF THE INVENTION
[0131] 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
[0132] 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."
[0133] 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.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] 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.
[0139] 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.
[0140] "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.
[0141] 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.
[0142] "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.
[0143] "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.
[0144] "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.
[0145] 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.
[0146] 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.
[0147] "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.
[0148] 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.
[0149] "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.
[0150] "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.
[0151] 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.
[0152] 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.
[0153] "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.
[0154] "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.
[0155] 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.
[0156] 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.
[0157] The term "Saturation Mutagenesis" or "Gene Site Saturation
Mutagenesis.TM." or "GSSM.TM." includes a method that uses
degenerate oligonucleotide primers to introduce point mutations
into a polynucleotide, as described in detail, below.
[0158] 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.
[0159] 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
[0160] 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.
[0161] 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.
[0162] 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-00001 TABLE 1 SEQ ID NO: Source 291, 292 Aquifex 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
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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 o
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.
[0170] 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 III 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.
[0171] General Techniques
[0172] 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.
[0173] 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.
[0174] 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).
[0175] 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 (BAC); 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.
[0176] 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.
[0177] 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.
[0178] Transcriptional and Translational Control Sequences
[0179] 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.
[0180] 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.
[0181] Tissue-Specific Plant Promoters
[0182] 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.
[0183] 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.
[0184] 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 cassava 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).
[0185] 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).
[0186] 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 J12:367-77, 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.
[0187] 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).
[0188] 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 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). 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.
[0189] 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.
[0190] 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).
[0191] 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.
[0192] Expression Vectors and Cloning Vehicles
[0193] 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.
[0194] 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.
[0195] 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 TRP1 gene. Promoter regions can be selected from any
desired gene using chloramphenicol transferase (CAT) vectors or
other vectors with selectable markers.
[0196] 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 by 100 to 270,
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and the adenovirus
enhancers.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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 by 100 to 270,
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin and the adenovirus
enhancers.
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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).
[0211] Host Cells and Transformed Cells
[0212] 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.
[0213] 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)).
[0214] 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.
[0215] 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.
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] 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.
[0221] 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.
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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)).
[0226] 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.
[0227] 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.
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 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.
[0232] Amplification of Nucleic Acids
[0233] 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.
[0234] 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.
[0235] 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.
[0236] 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.
[0237] 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);
transcription 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
[0238] 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.
[0239] 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.
[0240] 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).
[0241] 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.
[0242] 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.
[0243] 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. 2:482, 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 (Fleischmann 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.
[0244] 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.
[0245] 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.
[0246] 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: [0247] (1) BLASTP and BLAST3 compare an amino acid
query sequence against a protein sequence database; [0248] (2)
BLASTN compares a nucleotide query sequence against a nucleotide
sequence database; [0249] (3) BLASTX compares the six-frame
conceptual translation products of a query nucleotide sequence
(both strands) against a protein sequence database; [0250] (4)
TBLASTN compares a query protein sequence against a nucleotide
sequence database translated in all six reading frames (both
strands); and [0251] (5) TBLASTX compares the six-frame
translations of a nucleotide query sequence against the six-frame
translations of a nucleotide sequence database.
[0252] 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 BLOSUM62 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.
[0253] 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
[0254] 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.
[0255] 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.
[0256] 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.
[0257] 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.
[0258] 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.
[0259] 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.
[0260] 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.
[0261] 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.
[0262] 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.
[0263] 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.
[0264] 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.
[0265] 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.
[0266] 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.
[0267] 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.
[0268] 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.
[0269] 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.
[0270] 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.
[0271] 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.
[0272] 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.
[0273] 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.
[0274] 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.
[0275] 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.
[0276] 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.
[0277] 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.
[0278] 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%.
[0279] 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.
[0280] 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.
[0281] 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.
[0282] 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.
[0283] 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.
[0284] 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.
[0285] 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.
[0286] 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.
[0287] 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.
[0288] 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 BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al,
J. Mol. Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc.
Natl. Acad. Sci. USA, 85: 2444, 1988), FASTDB (Brutlag 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.
[0289] 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
[0290] 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 be 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.
[0291] 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.
[0292] 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.
[0293] 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.
[0294] 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.
[0295] 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 Na2EDTA) 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.
[0296] All of the foregoing hybridizations would be considered to
be under conditions of high stringency.
[0297] 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.
[0298] Nucleic acids which have hybridized to the probe are
identified by autoradiography or other conventional techniques.
[0299] 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.quadrature.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.
[0300] 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.
[0301] 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.
[0302] 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.
[0303] 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
[0304] 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.
[0305] 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.
[0306] 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.
[0307] 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.
[0308] 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.
[0309] 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 sample 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.
[0310] 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.
[0311] 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.
[0312] 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. for a particular probe. The melting temperature of the
probe may be calculated using the following formulas:
[0313] For probes between 14 and 70 nucleotides in length the
melting temperature (Tm) 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.
[0314] 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.
[0315] Prehybridization may be carried out in 6.times.SSC,
5.times.Denhardt's reagent, 0.5% SDS, 100.quadrature.g denatured
fragmented salmon sperm DNA or 6.times.SSC, 5.times.Denhardt's
reagent, 0.5% SDS, 100 g denatured fragmented salmon sperm DNA, 50%
formamide. The formulas for SSC and Denhardt's solutions are listed
in Sambrook et al., supra.
[0316] 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)
[0317] 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.
[0318] 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).
[0319] 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.
[0320] Antisense Oligonucleotides
[0321] 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. J. Pharm. Sci. 11:191-198.
[0322] 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, phosphoramidate, alkyl
phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino),
3'-N-carbamate, and morpholino carbamate nucleic acids, as
described above.
[0323] 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).
[0324] Inhibitory Ribozymes
[0325] 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.
[0326] In some circumstances, the enzymatic nature of a ribozyme
can be advantageous over other technologies, such as anti sense
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.
[0327] 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.
[0328] RNA interference (RNAi)
[0329] 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
[0330] 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.
[0331] 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., nitrosoguanidine,
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.
[0332] 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.
[0333] 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.
[0334] 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).
[0335] 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.
[0336] 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."
[0337] 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 CHARACTERISTICS" 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.
[0338] 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.
[0339] Saturation Mutagenesis, or, GSSM.TM.
[0340] 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.
[0341] 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.
[0342] 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 X 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.
[0343] 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.
[0344] 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.
[0345] 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.
[0346] 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.
[0347] 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.
[0348] 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) sequence.
[0349] 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.
[0350] 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.
[0351] 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.
[0352] 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.
[0353] 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.
[0354] 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.
[0355] 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).
[0356] 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.
[0357] Defined sequences to be mutagenized include a whole gene,
pathway, cDNA, an entire open reading frame (ORF) 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.
[0358] 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.
[0359] Synthetic Ligation Reassembly (SLR)
[0360] 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.
[0361] 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.
[0362] 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.
[0363] 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.
[0364] 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.
[0365] 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.
[0366] 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.
[0367] 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.
[0368] 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.
[0369] 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.
[0370] 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.
[0371] 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.
[0372] 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.
[0373] 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.
[0374] 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.
[0375] 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.
[0376] 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.
[0377] 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.
[0378] 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).
[0379] 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.
[0380] 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).
[0381] 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.
[0382] 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.
[0383] 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.
[0384] 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.
[0385] 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.
[0386] 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).
[0387] 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.
[0388] 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.
[0389] 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.
[0390] 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.
[0391] 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.
[0392] Optimized Directed Evolution System
[0393] 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.
[0394] 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.
[0395] 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.
[0396] 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.
[0397] 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.
[0398] 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.
[0399] 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 1013 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.
[0400] 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.
[0401] Determining Crossover Events
[0402] 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.
[0403] Iterative Processes
[0404] 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.
[0405] 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.
[0406] In vivo Shuffling
[0407] 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.
[0408] 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.
[0409] 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.
[0410] 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.
[0411] 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.
[0412] 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.
[0413] Sequences can be assembled in a head to tail orientation
using any of a variety of methods, including the following: [0414]
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. [0415] 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. [0416] c) The inner
few bases of the primer could be thiolated and an exonuclease used
to produce properly tailed molecules.
[0417] 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:
[0418] 1) The use of vectors only stably maintained when the
construct is reduced in complexity. [0419] 2) The physical recovery
of shortened vectors by physical procedures. [0420] 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. [0421] 3) The recovery of vectors containing
interrupted genes which can be selected when insert size decreases.
[0422] 4) The use of direct selection techniques with an expression
vector and the appropriate selection.
[0423] 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.
[0424] 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).
[0425] 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.
[0426] 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.
[0427] 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.
[0428] 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-(N3-Adenine (See Sun and
Hurley (1992); an N-acetylated or deacetylated
4'-fluro-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-J]-quinoline ("N-hydroxy-IQ")
and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-J]-pyridine
("N-hydroxy-PhIP"). Exemplary means for slowing or halting PCR
amplification consist of UV light (+)-CC-1065 and
(+)-CC-1065-(N3-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.
[0429] 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.
[0430] Producing Sequence Variants
[0431] 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.
[0432] 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.
[0433] 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 MgCl2, 0.5 mM MnC12, 5 units of Taq polymerase, 0.2
mM dGTP, 0.2 mM dATP, 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.
[0434] 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.
[0435] 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.
[0436] 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/.mu.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 (30-45 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.
[0437] 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".
[0438] 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.
[0439] 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 Arkin,
A. P. and Youvan, D.C., PNAS, USA, 89:7811-7815, 1992.
[0440] 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.
[0441] 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.
[0442] 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.
[0443] 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.
[0444] 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 bearing 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.
[0445] Other variants are those in which one or more of the amino
acid residues of a polypeptide of the invention includes a
substituent group.
[0446] 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).
[0447] 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.
[0448] 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.
[0449] Optimizing Codons to Achieve High Levels of Protein
Expression in Host Cells
[0450] 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.
[0451] 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.
[0452] 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.
[0453] 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
[0454] 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.
[0455] 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).
[0456] "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
[0457] 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.
[0458] 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.
[0459] 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.
[0460] 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.
[0461] 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.
[0462] 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.
[0463] 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.
[0464] 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.
[0465] 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.
[0466] 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.
[0467] 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.
[0468] 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.
[0469] 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.
[0470] 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 grass (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, Malta, Manihot, Majorana, Medicago,
Nicotiana, Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus,
Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale,
Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella,
Triticum, Vicia, Vitis, Vigna, and Zea.
[0471] 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.
arboretum, G. herbaceum, G. barbadense, and G. hirsutum.
[0472] 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).
[0473] 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
[0474] 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.
[0475] 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.
[0476] 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.
[0477] 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.
[0478] 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-00002 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.sup. 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
[0479] 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.
[0480] 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.
[0481] 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.
[0482] 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.
[0483] 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.
[0484] 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 (Perkin Elmer) in accordance with the instructions
provided by the manufacturer.
[0485] 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.
[0486] 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.
[0487] 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).
[0488] 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-biphenylphenyl alanine; D- or
L-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines;
and, D- or L-alkylainines, 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.
[0489] 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 0-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.
[0490] 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.
[0491] 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).
[0492] 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.
[0493] 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.
[0494] 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.
[0495] 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.
[0496] 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.
[0497] 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.
[0498] 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.
[0499] 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.
[0500] 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.
[0501] 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.
[0502] 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.
[0503] 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.
[0504] 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.
[0505] 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.
[0506] 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.
[0507] 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.
[0508] 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.
[0509] 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.
[0510] 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.
[0511] 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.
[0512] Signal Sequences, Prepro and Catalytic Domains
[0513] 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
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 39, 1 to 40, 1 to 41, 1 to 42, 1 to 43, 1 to 44 of a
polypeptide of the invention.
[0514] 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).
[0515] 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-00003 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 MSSFKASAINPRMAGALTRSLYAAGFSLAVSTL STQAYA 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
MPRLRARTRPRRQLTALAAALSLPLGLTAVGAT TAQA 275, 276 1-27
MQNLFKRVFFHLLLLALLAGCAGPSPV 277, 278 1-28
MSGRSAGRGPWARLVVALAAVGALVAGA 279, 280 1-38
MQNKIINTKIKLRKFMSQLIKITYIFIIIIFCM QRTYA 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
MVWTPARSTLAGSSEIPLMTMNIFPNRKDSRMS LWIKLGILCMMAGTVMVHG 31, 32 1-39
311, 312 1-24 MKRREFMLGGAGVAALASTLGVSA 313, 314 1-20
MLIIGGLLVLLGFSSCGRQA 315, 316 1-32 MDKTITAKDSGKITALILIILLVLPYAGYVVA
319, 320 1-29 MREIILKSGALLMVVILIVSILQILTVFA 323, 324 1-32
MFQSLKMRTLSFLLLMALLASFLALPTDVAHA 325, 326 1-29
MKKIILKSGILLLVVILIVSILQILPVFA 327, 328 1-46
MLVYRVSIQKHLASLTVLVSLLLILAGCSSSSD SIAPVSSSSVSSA 33, 34 1-35 331,
332 1-28 MNNPTNGARRGRHRRRWSATALLLGVPA 333, 334 1-28
MNRTRVLSAATLLALVATLASVPVTAQA 337, 338 1-29
MRNHLNVPFYFIFFFLIASIFTVCSSSTA 339, 340 1-23 MNNPRILTYLLIGIVVAVLIVFA
343, 344 1-31 MRKIVKQINYLTPSVLGLLVLSLFFQVPTQA 345, 346 1-33
MKRTRYGVRSPRSAPRFGVLFGAAAAGVLMTGA 349, 350 1-38
MNSSPVSVKKPCPVDRPNPLWAAGFSLALATLS TQTQA 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
MLGAPSPHFPMRRGMTKSQRRTWLTAVGSAIAG IAGLLLPVFATAGAAQA 367, 368 1-42
MPHPKLLTNGGSYVSSKQKTVAIFVLFVVLAGV AGSIPASYA 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 MRIRLATLALCAALSPVTFA 427,
428 1-43 METPMTSARSARPRPRLRRYGIAGTALGALLLG LATLPPTATA 43, 44 1-48
431, 432 1-22 MKFTLMPLLCGFALLLGCAVQA 441, 442 1-26
MRAIRLSLSIAAGAVLLLAGCTTKPA 445, 446 1-29
MRRFRVVFLGLFVFFGIVIASQYGQTAAA 449, 450 1-25
MKKIVSLVCVLVMLVSILGSFSVVA 45, 46 1-16 457, 458 1-39
MKSKTSTAAPSAGPLRNYKKLTACIAVASTALL AGSASA 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
[0516] 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.).
[0517] 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).
[0518] 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.
[0519] 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
[0520] 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).
[0521] 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.
[0522] 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.
[0523] 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.
[0524] 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.
[0525] 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.
[0526] 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.
[0527] 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.
[0528] 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.
[0529] 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.
[0530] "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.
[0531] 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.
[0532] 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.
[0533] 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.
[0534] 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.
[0535] 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).
[0536] 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.
[0537] 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.
[0538] 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.
[0539] 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.
[0540] 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: [0541] 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;
[0542] 2) growing the host cell under conditions which promote
sequence reorganization resulting in a hybrid polynucleotide in
operable linkage; [0543] 3) expressing a hybrid polypeptide encoded
by the hybrid polynucleotide; [0544] 4) screening the hybrid
polypeptide under conditions which promote identification of
enhanced biological activity; and [0545] 5) isolating the a
polynucleotide encoding the hybrid polypeptide.
[0546] 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
[0547] 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-nitroanalide 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.
[0548] Capillary Arrays
[0549] 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.
[0550] 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.
[0551] 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".
[0552] 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.
[0553] 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.
[0554] Arrays or "Biochips"
[0555] 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.
[0556] 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
[0557] 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).
[0558] 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.
[0559] 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.
[0560] 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 IN IMMUNOLOGY, Wiley/Greene, N Y (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.
[0561] 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.
[0562] 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.
[0563] 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.
[0564] 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.
[0565] 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-cell 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).
[0566] 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.
[0567] 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
[0568] 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
[0569] 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.
[0570] 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.
[0571] 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: [0572] identity of all
pathway substrates, products and intermediary metabolites [0573]
identity of all the chemical reactions interconverting the pathway
metabolites, the stoichiometry of the pathway reactions, [0574]
identity of all the enzymes catalyzing the reactions, the enzyme
reaction kinetics, [0575] the regulatory interactions between
pathway components, e.g. allosteric interactions, enzyme-enzyme
interactions etc, [0576] intracellular compartmentalization of
enzymes or any other supramolecular organization of the enzymes,
and, [0577] the presence of any concentration gradients of
metabolites, enzymes or effector molecules or diffusion barriers to
their movement.
[0578] 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.
[0579] 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.
[0580] Monitoring Expression of an mRNA Transcript
[0581] 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).
[0582] 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.
[0583] 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.
[0584] Monitoring Expression of a Polypeptides, Peptides and Amino
Acids
[0585] 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
[0586] 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.
[0587] Detergent Compositions
[0588] 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.
[0589] 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.
[0590] 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.
[0591] 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.
[0592] 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.
[0593] 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.
[0594] 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.
[0595] 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.
[0596] 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.
[0597] 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.
[0598] 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.
[0599] 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).
[0600] Treating Fibers and Textiles
[0601] 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.
[0602] 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.
[0603] 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).
[0604] The enzymes of the invention can be used to treat fabrics or
any glucan, mannanan, 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.
[0605] 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.
[0606] 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.
[0607] 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.
[0608] 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.
[0609] 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.
[0610] Treating Foods and Food Processing
[0611] 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.
[0612] 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.
[0613] 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.
[0614] 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.).
[0615] 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.
[0616] Paper or Pulp Treatment
[0617] 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.
[0618] 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.
[0619] 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.
[0620] 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.
[0621] 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.
[0622] Animal Feeds and Food or Feed Additives
[0623] 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.
[0624] 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.
[0625] 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.
[0626] 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.
[0627] 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.
[0628] 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.
[0629] 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.
[0630] 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.
[0631] 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.
[0632] 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.
[0633] 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.
[0634] 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.
[0635] 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.
[0636] 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.
[0637] 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.
[0638] 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.
[0639] 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 xylanases 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.
[0640] 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
2-4) 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 2-4, 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.
[0641] 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.
[0642] Waste Treatment
[0643] 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.
[0644] 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.
[0645] Oral Care Products
[0646] 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.
[0647] 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.
[0648] Brewing and Fermenting
[0649] 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.
[0650] 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.
[0651] 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.
[0652] 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.
[0653] 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.
[0654] 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.
[0655] Medical and Research Applications
[0656] 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).
[0657] Drilling Applications
[0658] 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).
[0659] Other Industrial Applications
[0660] 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.
[0661] 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.
[0662] Glucanases, mannanases, or xylanases of the invention can be
used in combination with other enzymes involved in cellulose
digestion like cellobiohydrolases and beta-glucosidases.
[0663] 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. Dairy 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).
[0664] 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).
[0665] 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).
[0666] 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.
[0667] 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 biopanning. 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.
[0668] 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, B.beta.G), 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.gtoreq.7 and 25-35.degree. C., are
inactive on insoluble glucan, are stable and active in 50-67%
sucrose.
[0669] 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, B.beta.G), 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.
[0670] 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.
[0671] 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.
[0672] 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.
[0673] 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).
[0674] 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
[0675] 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.
[0676] Titer Determination of Lambda Library:
[0677] 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 MgS0.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.)
[0678] Substrate Screening:
[0679] 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 1.0 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.
[0680] Isolation of Pure Clones:
[0681] 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.
[0682] Excision of Pure Clone:
[0683] 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.kan 50.
[0684] Activity Verification:
[0685] 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 pt 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).
[0686] RFLP of Pure Clones from Same Libraries:
[0687] Transfer 1.0 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.
[0688] 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
[0689] 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.
[0690] 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.mnol/min.sup.-1 glucose reducing equivalents released at
37.degree. C., pH 5.3.
TABLE-US-00004 Specific Activity (U/mg) Mw GH Native, 6H tagged, 6H
tagged, T.sub.opt Glucanase (kDa) pI Family BBG BBG CMC (.degree.
C.) pH.sub.opt SEQ ID NO: 6 37.5 5.9 5 22 ND ND .gtoreq.90 5-7
(encoded by SEQ ID NO: 5) SEQ ID NO: 400 37.9 5.5 5 0.85 ND ND
.gtoreq.90 5-7 (encoded by SEQ ID NO: 399) SEQ ID NO: 162 34.0 5.2
5 0.95 ND ND .gtoreq.85 ND (encoded by SEQ ID NO: 161) SEQ ID NO:
84 36.9 6.3 5 >40 ND ND 80 4-6 (encoded by SEQ ID NO: 83) SEQ ID
NO: 172 29.8 5.0 16 32 ND ND 50 5-6 (encoded by SEQ ID NO: 171) SEQ
ID NO: 104 39.7 5.9 5 ND 3.2 2.8 85 5-6 (encoded by SEQ ID NO: 103)
SEQ ID NO: 10 77.7 4.9 5 ND ND 0.5 85 5-6 (encoded by SEQ ID NO: 9)
SEQ ID NO: 222 53.8 9.1 5 >40 16 24 85 5-6 (encoded by SEQ ID
NO: 221) SEQ ID NO: 108 78.9 4.3 5 ND 3.8 4.0 75 ND (encoded by SEQ
ID NO: 107) SEQ ID NO: 176 37.2 6.0 5 ND 3.5 21 75 5-6 (encoded by
SEQ ID NO: 175) SEQ ID NO: 110 39.9 6.2 5 ND 13 12 ND ND (encoded
by SEQ ID NO: 109) SEQ ID NO: 268 51.8 4.6 5 ND 3.6 2.8 50 ND
(encoded by SEQ ID NO: 267) SEQ ID NO: 324 49.3 6.1 5 ND ND ND ND
ND (encoded by SEQ ID NO: 323) SEQ ID NO: 370 42.1 5.8 5 ND ND ND
ND ND (encoded by SEQ ID NO: 369) SEQ ID NO: 168 37.3 5.7 5 ND ND
ND ND ND (encoded by SEQ ID NO: 167) SEQ ID NO: 154 35.6 5.4 5 ND
ND ND ND ND (encoded by SEQ ID NO: 153) SEQ ID NO: 118 34.5 6.1 5
ND ND ND ND ND (encoded by SEQ ID NO: 117) SEQ ID NO: 148 74.1 5.3
5 ND ND ND ND ND (encoded by SEQ ID NO: 147) ND = Not
determined
[0691] 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-00005 pH Temperature SEQ ID NO: Type 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 .sup. (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
[0692] 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.
[0693] 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:
[0694] 1% CMC
[0695] Dissolve 1 gm CMC in 100 ml 50 mM Britton-Robinson buffer at
pH 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:
[0696] 64 mg/ml sodium carbonate monohydrate
[0697] 24 mg/ml sodium bicarbonate
[0698] 1.95 mg/ml BCA (4,4'-dicarboxy-2,2'-biquinoline disodium
salt (Sigma Chemical cat # D-8284)
[0699] Add above to dH2O,
[0700] Might need to dissolve the BCA by heating, don't heat more
than .about.80 C.
Solution B:
[0701] 1.24 mg/ml cupric sulfate pentahydrate
[0702] 1.26 mg/ml L-serine
[0703] Add above to dH2O
Working Reagent:
[0704] 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:
[0705] 10 mM Glucose in dH2O. 0.2 um filter, store at 4 C.
Glucose Standards:
[0706] 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:
[0707] 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.
[0708] 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:
[0709] 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).
[0710] 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:
[0711] 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).
[0712] Add 100 ul H2O 2O to wells. Mix. Aliquot 100 ul of mixture
into clear flat bottomed 96-well plate and read absorbance at 560
nm.
Generate Standard Curve:
[0713] 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:
[0714] 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).
[0715] Generate the slope (S.sub.r.times.n) for each set of sample
time-points (A560/time).
Activity Determination:
[0716] Divide S.sub.r.times.n 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:
[0717] 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.
[0718] Divide the protein concentration by any dilutions used.
[0719] Multiply by the volume (in ml) used in the reaction.
[0720] All points should be done in duplicate with triplicate being
better.
[0721] 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.
Sample Data
TABLE-US-00006 [0722] date mg/ml Diln. ul/rxn 0 min 5 min 8 min 12
min 24 min 36 min 45 min Enz x June 2009 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 r.times.n
(=Activity.times.100): 0.0087 umole/min Specific Activity: 10.87
umole/min,mg
Example 4
Codon Optimization
[0723] 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.
[0724] 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.
[0725] 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.
[0726] 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.
[0727] 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
[0728] 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.
[0729] Specific Activity of the Glucanase Encoded by SEQ ID
NO:6
[0730] 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:
[0731] 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-1 glucose reducing equivalents
released at 37.degree. C., pH 5.3. [0732] Barley Beta Glucan (BBG):
30 U/mg [0733] Oat Beta Glucan (OBG): 38 U/mg [0734]
Carboxymethylcellulose (CMC): 40 U/mg [0735] Carob Galactomannan:
0.3 U/mg
[0736] Temperature Profile of the Glucanase Encoded by SEQ ID
NO:6
[0737] 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.
[0738] 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.
[0739] Half-Life Determination of the Glucanase Encoded by SEQ ID
NO:6
[0740] 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.
[0741] 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.
[0742] 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=US20170130215A1).
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=US20170130215A1).
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).
* * * * *
References