U.S. patent application number 16/292057 was filed with the patent office on 2020-03-19 for alpha-amylase combinatorial variants.
The applicant listed for this patent is DANISCO US INC. Invention is credited to Pieter Augustinus, Richard R Bott, Luis G. Cascao-Pereira, Dina Finan, Roel Hermant, Marc Kolkman, Monica Ocha Ruiz, Dewy Van Tol, David Edward Wildes.
Application Number | 20200087644 16/292057 |
Document ID | / |
Family ID | 50680117 |
Filed Date | 2020-03-19 |
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United States Patent
Application |
20200087644 |
Kind Code |
A1 |
Cascao-Pereira; Luis G. ; et
al. |
March 19, 2020 |
ALPHA-AMYLASE COMBINATORIAL VARIANTS
Abstract
Disclosed are compositions and methods relating to variant
alpha-amylases. The variant alpha-amylases are useful, for example,
for starch liquefaction and saccharification, for cleaning starchy
stains in laundry, dishwashing, and other applications, for textile
processing (e.g., desizing), in animal feed for improving
digestibility, and for baking and brewing.
Inventors: |
Cascao-Pereira; Luis G.;
(Redwood City, CA) ; Finan; Dina; (Palo Alto,
CA) ; Wildes; David Edward; (Palo Alto, CA) ;
Augustinus; Pieter; (Palo Alto, CA) ; Hermant;
Roel; (Palo Alto, CA) ; Ruiz; Monica Ocha;
(Palo Alto, CA) ; Van Tol; Dewy; (Palo Alto,
CA) ; Bott; Richard R; (Palo Alto, CA) ;
Kolkman; Marc; (Palo Alto, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DANISCO US INC |
Palo Alto |
CA |
US |
|
|
Family ID: |
50680117 |
Appl. No.: |
16/292057 |
Filed: |
March 4, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14775595 |
Sep 11, 2015 |
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PCT/US14/23458 |
Mar 11, 2014 |
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16292057 |
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61776699 |
Mar 11, 2013 |
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61906617 |
Nov 20, 2013 |
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61907131 |
Nov 21, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/2414 20130101;
C12P 19/14 20130101; A23L 2/382 20130101; C12N 9/2417 20130101;
C11D 3/386 20130101; C11D 3/38681 20130101; A23V 2002/00 20130101;
C12P 19/02 20130101 |
International
Class: |
C12N 9/26 20060101
C12N009/26; C12N 9/28 20060101 C12N009/28; C11D 3/386 20060101
C11D003/386; A23L 2/38 20060101 A23L002/38; C12P 19/02 20060101
C12P019/02; C12P 19/14 20060101 C12P019/14 |
Claims
1. A recombinant variant of a parent .alpha.-amylase comprising: a
mutation at an amino acid residue corresponding to E187 or S241;
and at least one mutation at an amino acid residue corresponding to
an amino acid residue selected from the group consisting of N126,
Y150, F153, L171, T180, and, I203; wherein the variant
.alpha.-amylase or the parent .alpha.-amylase has at least 60%
amino acid sequence identity relative to SEQ ID NO: 1, which is
used for numbering; and wherein the variant has increased
thermostability, detergent stability, starch liquifaction activity,
and/or cleaning performance compared to the parent .alpha.-amylase
or a reference .alpha.-amylase differing from the variant
.alpha.-amylase only by the absence of the mutations.
2. The variant .alpha.-amylase of claim 1, comprising at least two
mutations at amino acid residues corresponding N126, Y150, F153,
L171, and, I203, using SEQ ID NO: 1 for numbering.
3. The variant .alpha.-amylase of claim 1, further comprising a
deletion of at least one amino acid residue corresponding to R178,
G179, T180, and G181, using SEQ ID NO: 1 for numbering.
4. The variant .alpha.-amylase of claim 1, further comprising
deletions of amino acid residues corresponding to R178 and G179, or
T180 and G181.
5. The variant .alpha.-amylase of claim 1, further comprising a
mutation at an amino acid residue corresponding to G476 and/or
G477, using SEQ ID NO: 1 for numbering.
6. The variant .alpha.-amylase of claim 1, further comprising a
mutation in an amino acid residue corresponding to an amino acid
residue selected from the group consisting of E132, Q167, T180, and
A277, using SEQ ID NO: 1 for numbering.
7. The variant .alpha.-amylase of claim 1, further comprising a
mutation in an amino acid residue corresponding to an amino acid
residue selected from the group consisting of R458, T459, and D460,
using SEQ ID NO: 1 for numbering.
8. The variant .alpha.-amylase of claim 1, further comprising a
mutation in an amino acid residue corresponding to T180, using SEQ
ID NO: 1 for numbering.
9. The variant .alpha.-amylase of claim 1, further comprising a
mutation in an amino acid residue corresponding to N205, using SEQ
ID NO: 3 for numbering.
10. The variant .alpha.-amylase of claim 1, further comprising a
mutation in an amino acid residue corresponding to an amino acid
residue selected from the group consisting of T333G, A335S, and
Q337E, using SEQ ID NO: 3 for numbering.
11. The variant .alpha.-amylase of claim 1, further comprising a
mutation in an amino acid residue corresponding to an amino acid
residue position selected from the group consisting of 6, 7, 8, 11,
14, 15, 20, 21, 23, 26, 27, 28, 37, 38, 39, 40, 42, 45, 46, 48, 49,
50, 51, 52, 53, 54, 58, 61, 62, 68, 70, 71, 72, 73, 79, 80, 81, 82,
84, 85, 87, 88, 89, 92, 93, 94, 95, 96, 97, 98, 101, 108, 111, 112,
113, 114, 115, 116, 117, 118, 120, 122, 123, 124, 126, 127, 129,
130, 131, 132, 133, 134, 136, 137, 138, 140, 142, 143, 144, 147,
148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 165, 167,
168, 170, 171, 172, 175, 176, 177, 180, 181, 182, 187, 190, 191,
193, 199, 200, 201, 203, 206, 208, 210, 211, 212, 214, 215, 216,
219, 221, 223, 225, 226, 227, 235, 238, 239, 240, 241, 242, 243,
245, 246, 247, 248, 249, 250, 252, 253, 254, 256, 257, 258, 260,
261, 262, 266, 267, 268, 269, 270, 271, 273, 276, 277, 279, 280,
282, 284, 285, 286, 288, 296, 299, 300, 301, 302, 303, 304, 307,
308, 310, 311, 312, 313, 316, 317, 318, 320, 321, 325, 327, 335,
338, 342, 348, 349, 352, 356, 357, 360, 362, 363, 368, 369, 377,
381, 382, 383, 384, 385, 388, 390, 392, 394, 395, 396, 397, 398,
400, 401, 402, 403, 404, 405, 407, 408, 410, 414, 415, 416, 418,
419, 420, 421, 422, 423, 424, 426, 428, 429, 430, 431, 434, 435,
436, 439, 441, 442, 444, 445, 446, 447, 448, 449, 450, 451, 454,
455, 457, 460, 461, 462, 463, 464, 465, 466, 467, 469, 470, 471,
473, 474, 475, 476, 477, 479, 480, 481, 482, 483, and 484, using
SEQ ID NO: 1 for numbering.
12. The variant .alpha.-amylase of claim 1, comprising a
combinations of mutations corresponding to mutations selected from
the group consisting of: E187P+I203Y+G476K,
E187P+I203Y+G476K+R458N+T459S+D460T, T180D+E187P+I203Y+G476K,
N126Y+T180D+E187P+I203Y+G476K,
N126Y+T180D+E187P+I203Y+Y303D+G476T+G477E,
N126Y+T180D+E187P+I203Y+Y303D+N475E+G477Q,
N126Y+T180D+E187P+I203Y+Y303R+N475E+G476T+G477R,
T038N+N088H+N126Y+T129I+N134M+F153W+L171R+T180D+E187P+I203Y+G476K+G477E,
N126Y+E132H+T180D+E187P+I203Y+Y303D+G476T+G477E, N126Y+E187P+G476K,
N126Y+F153W+E187P+G476K, N126Y+F153W+E187P+G4726+G477R,
N126Y+E187P+I203Y, N126Y+I203Y+S241Q, N126Y+T180H+E187P+I203Y,
N126Y+T180H+I203Y+S241Q, N126Y+F153W+T180H+E187P+I203Y,
N126Y+F153W+T180H+I203Y+S241Q, N126Y+Y150H+F153W+L171N+E187P+I203Y,
N126Y+Y150H+F153W+L171N+I203Y+S241Q,
N126Y+Y150H+F153W+L171N+T180H+E187P+I203Y,
N126Y+Y150H+F153W+L171N+T180H+I203Y+S241Q, and
N126Y+F153W+T180D+I203Y+S241Q; wherein the variant has increased
thermostability, detergent stability, stability starch liquifaction
activity, or cleaning performance compared to the parent; wherein
the variant or the parent has at least 60% amino acid sequence
identity relative to SEQ ID NO: 1, which is used for numbering.
13. The variant amylase of claim 1, comprising the combinations of
mutations corresponding to N126Y+F153W+T180D+I203Y+S241Q and one or
more mutations corresponding to mutations selected from the group
consisting of E132H, Q167E, A277F, and T400K.
14. The variant amylase of claim 13, comprising the combinations of
mutations corresponding to mutations selected from the group
consisting of: N126Y+E132H+F153W+T180D+I203Y+S241Q+A277F,
N126Y+E132H+F153W+Q167E+T180D+I203Y+S241Q+A277F, and
N126Y+E132H+F153W+Q167E+T180D+I203Y+S241Q+A277F+T400K.
15. The variant amylase of claim 1, wherein the parental
.alpha.-amylase is from a Cytophaga species.
16. The variant amylase of claim 1, wherein the parental
.alpha.-amylase is from a Paenibacillus species.
17. The variant amylase of claim 1, wherein the parental
.alpha.-amylase is not from a Bacillus species.
18. The variant amylase of claim 1, wherein the parental
.alpha.-amylase or the variant .alpha.-amylase has at least 70%
amino acid sequence identity to the amino acid sequence of SEQ ID
NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.
19. The variant amylase of claim 1, wherein the parental
.alpha.-amylase or the variant .alpha.-amylase has at least 70%
amino acid sequence identity to the amino acid sequence of SEQ ID
NO: 1 or SEQ ID NO: 3.
20. The variant amylase of claim 1, wherein the parental
.alpha.-amylase or the variant .alpha.-amylase has at least 80%
amino acid sequence identity to the amino acid sequence of SEQ ID
NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.
21. The variant amylase of claim 1, wherein the parental
.alpha.-amylase or the variant .alpha.-amylase has at least 90%
amino acid sequence identity to the amino acid sequence of SEQ ID
NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.
22. A composition comprising the variant .alpha.-amylase of any of
the preceding claims.
23. The composition of claim 22, wherein the composition is
effective for removing starchy stains from laundry, dishes, or
textiles.
24. The composition of claim 22, further comprising a
surfactant.
25. The composition of claim 22, wherein the composition is a
detergent composition.
26. The composition of claim 22, wherein the composition is a
laundry detergent or a laundry detergent additive.
27. The composition of claim 22, wherein the composition is a
manual or automatic dishwashing detergent.
28. The composition of claim 22, further comprising one or more
additional enzymes selected from the group consisting of protease,
hemicellulase, cellulase, peroxidase, lipolytic enzyme,
metallolipolytic enzyme, xylanase, lipase, phospholipase, esterase,
perhydrolase, cutinase, pectinase, pectate lyase, mannanase,
keratinase, reductase, oxidase, phenoloxidase, lipoxygenase,
ligninase, pullulanase, tannase, pentosanase, malanase,
.beta.-glucanase, arabinosidase, hyaluronidase, chondroitinase,
laccase, metalloproteinase, amadoriase, glucoamylase,
arabinofuranosidase, phytase, isomerase, transferase, and an
amylase other than the amylase of claim 1.
29. The composition of claim 21, wherein the composition is for
liquifying starch.
30. The composition of claim 21, wherein the composition is for
saccharifying a composition comprising starch, for SSF post
liquefaction, or for direct SSF without prior liquefaction.
31. The composition of claim 21, wherein the composition is for
producing a fermented beverage.
32. The composition of claim 21, wherein the composition is for
producing a baked food product.
33. The composition of claim 21, wherein the composition is for
textile desizing.
34-56. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority from US
provisional applications U.S. Ser. No. 61/776,699, filed 11 Mar.
2013, U.S. Ser. No. 61/906,617, filed 20 Nov. 2013, and U.S. Ser.
No. 61/907,131, filed 21 Nov. 2013, and are incorporated herein by
reference in their entirety.
FIELD OF THE INVENTION
[0002] Disclosed are compositions and methods relating to variant
.alpha.-amylases containing a plurality of combinable mutations.
The variant .alpha.-amylases are useful, for example, for starch
liquefaction and saccharification, cleaning starchy stains, textile
desizing, baking, and brewing.
BACKGROUND
[0003] Starch consists of a mixture of amylose (15-30% w/w) and
amylopectin (70-85% w/w). Amylose consists of linear chains of
.alpha.-1,4-linked glucose units having a molecular weight (MW)
from about 60,000 to about 800,000. Amylopectin is a branched
polymer containing .alpha.-1,6 branch points every 24-30 glucose
units; its MW may be as high as 100 million.
[0004] Sugars from starch, in the form of concentrated dextrose
syrups, are currently produced by an enzyme catalyzed process
involving: (1) liquefaction (or viscosity reduction) of solid
starch with an .alpha.-amylase into dextrins having an average
degree of polymerization of about 7-10, and (2) saccharification of
the resulting liquefied starch (i.e. starch hydrolysate) with
amyloglucosidase (also called glucoamylase or GA). The resulting
syrup has a high glucose content. Much of the glucose syrup that is
commercially produced is subsequently enzymatically isomerized to a
dextrose/fructose mixture known as isosyrup. The resulting syrup
also may be fermented with microorganisms, such as yeast, to
produce commercial products including ethanol, citric acid, lactic
acid, succinic acid, itaconic acid, monosodium glutamate,
gluconates, lysine, other organic acids, other amino acids, and
other biochemicals, for example. Fermentation and saccharification
can be conducted simultaneously (i.e., an SSF process) to achieve
greater economy and efficiency.
[0005] .alpha.-amylases hydrolyze starch, glycogen, and related
polysaccharides by cleaving internal .alpha.-1,4-glucosidic bonds
at random. .alpha.-amylases, particularly from Bacilli, have been
used for a variety of different purposes, including starch
liquefaction and saccharification, textile desizing, starch
modification in the paper and pulp industry, brewing, baking,
production of syrups for the food industry, production of
feedstocks for fermentation processes, and in animal feed to
increase digestability. These enzymes can also be used to remove
starchy soils and stains during dishwashing and laundry
washing.
[0006] Numerous publications have described mutations in
.alpha.-amylases. However, not all mutations produce the same
effect in different molecules and not all mutation can be combined.
In addition, many mutations produce molecules that have certain
desirable qualities at the expense of other properties. The need
exists for robust engineered .alpha.-amylases molecules.
SUMMARY
[0007] The present compositions and methods relate to variant
amylase polypeptides, and methods of use, thereof. Aspects and
embodiments of the present compositions and methods are summarized
in the following separately-numbered paragraphs:
[0008] 1. In one aspect, a recombinant variant of a parent
.alpha.-amylase is provided, comprising: a mutation at an amino
acid residue corresponding to E187 or S241; and at least one
mutation at an amino acid residue corresponding to an amino acid
residue selected from the group consisting of N126, Y150, F153,
L171, T180, and, I203; wherein the variant .alpha.-amylase or the
parent .alpha.-amylase has at least 60% amino acid sequence
identity relative to SEQ ID NO: 1, which is used for numbering; and
wherein the variant has increased thermostability, detergent
stability, starch liquifaction activity, and/or cleaning
performance compared to the parent .alpha.-amylase or a reference
.alpha.-amylase differing from the variant .alpha.-amylase only by
the absence of the mutations.
[0009] 2. In some embodiments, the variant .alpha.-amylase of
paragraph 1 comprises at least two mutations at amino acid residues
corresponding N126, Y150, F153, L171, and, I203, using SEQ ID NO: 1
for numbering.
[0010] 3. In some embodiments, the variant .alpha.-amylase of any
of the preceding paragraphs further comprises a deletion of at
least one amino acid residue corresponding to R178, G179, T180, and
G181, using SEQ ID NO: 1 for numbering.
[0011] 4. In some embodiments, the variant .alpha.-amylase of any
of the preceding paragraphs further comprise deletions of amino
acid residues corresponding to R178 and G179, or T180 and G181.
[0012] 5. In some embodiments, the variant .alpha.-amylase of any
of the preceding paragraphs further comprises a mutation at an
amino acid residue corresponding to G476 and/or G477, using SEQ ID
NO: 1 for numbering.
[0013] 6. In some embodiments, the variant .alpha.-amylase of any
of the preceding paragraphs further comprises a mutation in an
amino acid residue corresponding to an amino acid residue selected
from the group consisting of E132, Q167, T180, and A277, using SEQ
ID NO: 1 for numbering.
[0014] 7. In some embodiments, the variant .alpha.-amylase of any
of the preceding paragraphs further comprises a mutation in an
amino acid residue corresponding to an amino acid residue selected
from the group consisting of R458, T459, and D460, using SEQ ID NO:
1 for numbering.
[0015] 8. In some embodiments, the variant .alpha.-amylase of any
of the preceding paragraphs further comprises a mutation in an
amino acid residue corresponding to T180, using SEQ ID NO: 1 for
numbering.
[0016] 9. In some embodiments, the variant .alpha.-amylase of any
of the preceding paragraphs further comprises a mutation in an
amino acid residue corresponding to N205, using SEQ ID NO: 3 for
numbering.
[0017] 10. In some embodiments, the variant .alpha.-amylase of any
of the preceding paragraphs further comprises a mutation in an
amino acid residue corresponding to an amino acid residue selected
from the group consisting of T333G, A335S, and Q337E, using SEQ ID
NO: 3 for numbering.
[0018] 11. In some embodiments, the variant .alpha.-amylase of any
of the preceding paragraphs further comprises a mutation in an
amino acid residue corresponding to an amino acid residue position
selected from the group consisting of 6, 7, 8, 11, 14, 15, 20, 21,
23, 26, 27, 28, 37, 38, 39, 40, 42, 45, 46, 48, 49, 50, 51, 52, 53,
54, 58, 61, 62, 68, 70, 71, 72, 73, 79, 80, 81, 82, 84, 85, 87, 88,
89, 92, 93, 94, 95, 96, 97, 98, 101, 108, 111, 112, 113, 114, 115,
116, 117, 118, 120, 122, 123, 124, 126, 127, 129, 130, 131, 132,
133, 134, 136, 137, 138, 140, 142, 143, 144, 147, 148, 149, 150,
151, 152, 153, 154, 155, 156, 158, 159, 165, 167, 168, 170, 171,
172, 175, 176, 177, 180, 181, 182, 187, 190, 191, 193, 199, 200,
201, 203, 206, 208, 210, 211, 212, 214, 215, 216, 219, 221, 223,
225, 226, 227, 235, 238, 239, 240, 241, 242, 243, 245, 246, 247,
248, 249, 250, 252, 253, 254, 256, 257, 258, 260, 261, 262, 266,
267, 268, 269, 270, 271, 273, 276, 277, 279, 280, 282, 284, 285,
286, 288, 296, 299, 300, 301, 302, 303, 304, 307, 308, 310, 311,
312, 313, 316, 317, 318, 320, 321, 325, 327, 335, 338, 342, 348,
349, 352, 356, 357, 360, 362, 363, 368, 369, 377, 381, 382, 383,
384, 385, 388, 390, 392, 394, 395, 396, 397, 398, 400, 401, 402,
403, 404, 405, 407, 408, 410, 414, 415, 416, 418, 419, 420, 421,
422, 423, 424, 426, 428, 429, 430, 431, 434, 435, 436, 439, 441,
442, 444, 445, 446, 447, 448, 449, 450, 451, 454, 455, 457, 460,
461, 462, 463, 464, 465, 466, 467, 469, 470, 471, 473, 474, 475,
476, 477, 479, 480, 481, 482, 483, and 484, using SEQ ID NO: 1 for
numbering.
[0019] 12. In some embodiments, the variant .alpha.-amylase of any
of the preceding paragraphs comprises a combinations of mutations
corresponding to mutations selected from the group consisting of:
[0020] E187P+I203Y+G476K, [0021]
E187P+I203Y+G476K+R458N+T459S+D460T, [0022]
T180D+E187P+I203Y+G476K, [0023] N126Y+T180D+E187P+I203Y+G476K,
[0024] N126Y+T180D+E187P+I203Y+Y303D+G476T+G477E, [0025]
N126Y+T180D+E187P+I203Y+Y303D+N475E+G477Q, [0026]
N126Y+T180D+E187P+I203Y+Y303R+N475E+G476T+G477R, [0027]
T038N+N088H+N126Y+T129I+N134M+F153W+L171R+T180D+E187P+I203Y+G476K+G477E,
[0028] N126Y+E132H+T180D+E187P+I203Y+Y303D+G476T+G477E, [0029]
N126Y+E187P+G476K, [0030] N126Y+F153W+E187P+G476K, [0031]
N126Y+F153W+E187P+G4726+G477R, [0032] N126Y+E187P+I203Y, [0033]
N126Y+I203Y+S241Q, [0034] N126Y+T180H+E187P+I203Y, [0035]
N126Y+T180H+I203Y+S241Q, [0036] N126Y+F153W+T180H+E187P+I203Y,
[0037] N126Y+F153W+T180H+I203Y+S241Q, [0038]
N126Y+Y150H+F153W+L171N+E187P+I203Y, [0039]
N126Y+Y150H+F153W+L171N+I203Y+S241Q, [0040]
N126Y+Y150H+F153W+L171N+T180H+E187P+I203Y, [0041]
N126Y+Y150H+F153W+L171N+T180H+I203Y+S241Q, and [0042]
N126Y+F153W+T180D+I203Y+S241Q;
[0043] wherein the variant has increased thermostability, detergent
stability, stability starch liquifaction activity, or cleaning
performance compared to the parent; and wherein the variant or the
parent has at least 60% amino acid sequence identity relative to
SEQ ID NO: 1, which is used for numbering.
[0044] 13. In some embodiments, the variant amylase of any of
paragraphs 1-12 comprises the combinations of mutations
corresponding to N126Y+F153W+T180D+I203Y+S241Q and one or more
mutations corresponding to mutations selected from the group
consisting of E132H, Q167E, A277F, and T400K.
[0045] 14. In some embodiments, the variant amylase of paragraph 13
comprises the combinations of mutations corresponding to mutations
selected from the group consisting of: [0046]
N126Y+E132H+F153W+T180D+I203Y+S241Q+A277F, [0047]
N126Y+E132H+F153W+Q167E+T180D+I203Y+S241Q+A277F, and [0048]
N126Y+E132H+F153W+Q167E+T180D+I203Y+S241Q+A277F+T400K.
[0049] 15. In some embodiments, the variant amylase of any of the
preceding paragraphs is from a Cytophaga species.
[0050] 16. In some embodiments, the variant amylase of any of the
preceding paragraphs is from a Paenibacillus species.
[0051] 17. In some embodiments, the variant amylase of any of the
preceding paragraphs is not from a Bacillus species.
[0052] 18. In some embodiments, the variant amylase of any of the
preceding paragraphs has at least 70% amino acid sequence identity
to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID
NO: 5.
[0053] 19. In some embodiments, the variant amylase of any of the
preceding paragraphs has at least 70% amino acid sequence identity
to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
[0054] 20. In some embodiments, the variant amylase of any of
paragraphs 1-18 has at least 80% amino acid sequence identity to
the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID
NO: 5.
[0055] 21 In some embodiments, the variant amylase of any of
paragraphs 1-18 has at least 90% amino acid sequence identity to
the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID
NO: 5.
[0056] 22. In another aspect, a composition comprising the variant
.alpha.-amylase of any of the preceding paragraphs is provided.
[0057] 23. In some embodiments, the composition of paragraph 22 is
effective for removing starchy stains from laundry, dishes, or
textiles.
[0058] 24. In some embodiments, the composition of paragraph 22 or
23 further comprises a surfactant.
[0059] 25. In some embodiments, the composition of any of
paragraphs 22-24 is a detergent composition.
[0060] 26. In some embodiments, the composition of any of
paragraphs 22-24 is a laundry detergent or a laundry detergent
additive.
[0061] 27. In some embodiments, the composition of any of
paragraphs 22-24 is a manual or automatic dishwashing
detergent.
[0062] 28. In some embodiments, the composition of any of
paragraphs 22-24 further comprises one or more additional enzymes
selected from the group consisting of protease, hemicellulase,
cellulase, peroxidase, lipolytic enzyme, metallolipolytic enzyme,
xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase,
pectinase, pectate lyase, mannanase, keratinase, reductase,
oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase,
tannase, pentosanase, malanase, .beta.-glucanase, arabinosidase,
hyaluronidase, chondroitinase, laccase, metalloproteinase,
amadoriase, glucoamylase, arabinofuranosidase, phytase, isomerase,
transferase, and an amylase other than the amylase of any one of
paragraphs 1-21.
[0063] 29. In some embodiments, the composition of paragraph 21 is
for liquifying starch.
[0064] 30. In some embodiments, the composition of paragraph 21 is
for saccharifying a composition comprising starch, for SSF post
liquefaction, or for direct SSF without prior liquefaction.
[0065] 31. In some embodiments, the composition of paragraph 21 is
for producing a fermented beverage.
[0066] 32. In some embodiments, the composition of paragraph 21 is
for producing a baked food product.
[0067] 33. In some embodiments, the composition of paragraph 21 is
for textile desizing.
[0068] 34. In another aspect, a method for removing a starchy stain
or soil from a surface is provided, comprising: contacting the
surface in the presence of a composition comprising an effective
amount of the variant amylase of any of the paragraphs 1-21, and
allowing the polypeptide to hydrolyze starch components present in
the starchy stain to produce smaller starch-derived molecules that
dissolve in the aqueous composition, thereby removing the starchy
stain from the surface.
[0069] 35. In some embodiments of the method of paragraph 34 the
aqueous composition further comprises a surfactant.
[0070] 36. In some embodiments of the method of any of paragraphs
34 or 35 the surface is a textile surface or a surface on
dishes.
[0071] 37. In some embodiments of the method of any of paragraphs
34-36 the composition further comprises at least one additional
enzymes selected from the group consisting of protease,
hemicellulase, cellulase, peroxidase, lipolytic enzyme,
metallolipolytic enzyme, xylanase, lipase, phospholipase, esterase,
perhydrolase, cutinase, pectinase, pectate lyase, mannanase,
keratinase, reductase, oxidase, phenoloxidase, lipoxygenase,
ligninase, pullulanase, tannase, pentosanase, malanase,
.beta.-glucanase, arabinosidase, hyaluronidase, chondroitinase,
laccase, metalloproteinase, amadoriase, glucoamylase,
arabinofuranosidase, phytase, isomerase, transferase, and an
amylase other than the amylase of any one of paragraphs 1-21.
[0072] 38. In another aspect, a method for saccharifying a
composition comprising starch to produce a composition comprising
glucose is provided, wherein the method comprises: [0073] (i)
contacting the solution comprising starch with effective amount of
the variant amylase of any of the paragraphs 1-21; and [0074] (ii)
saccharifying the solution comprising starch to produce the
composition comprising glucose; wherein the variant amylase
catalyzes the saccharification of the starch solution to glucose or
other enriched carbohydrate syrups.
[0075] 39. In some embodiments of the method of paragraph 38 the
composition comprising starch comprises liquefied starch,
gelatinized starch, granular starch, or starch heat-treated below
its gelatinization temperature.
[0076] 40. In some embodiments of the method of paragraph 38 or 39
the fermentation is a simultaneous saccharification and
fermentation (SSF) reaction.
[0077] 41. In some embodiments of the method of any of paragraphs
38-40 the method further comprises contacting a mash and/or a wort
with an amylase.
[0078] 42. In some embodiments, the method of any one of paragraphs
38-41 further comprises adding glucoamylase, hexokinase, xylanase,
glucose isomerase, xylose isomerase, phosphatase, phytase,
pullulanase, .beta.-amylase, .alpha.-amylase that is not the
variant .alpha.-amylase, protease, cellulase, hemicellulase,
lipase, cutinase, isoamylase, redox enzyme, esterase, transferase,
pectinase, alpha-glucosidase, beta-glucosidase, or a combination
thereof, to the starch solution.
[0079] 43. In some embodiments of the method of any one of
paragraphs 38-42 the amylase is expressed and secreted by a host
cell.
[0080] 44. In some embodiments of the method of paragraph 43 the
composition comprising starch is contacted with the host cell.
[0081] 45. In some embodiments of the method of paragraph 43 or 44
the host cell further expresses and secretes one or more enzymes
selected from the group consisting of glucoamylase, hexokinase,
xylanase, glucose isomerase, xylose isomerase, phosphatase,
phytase, pullulanase, .beta.-amylase, .alpha.-amylase that is not
the variant .alpha.-amylase, protease, cellulase, hemicellulase,
lipase, cutinase, isoamylase, redox enzyme, esterase, transferase,
pectinase, alpha-glucosidase, and beta-glucosidase.
[0082] 46. In some embodiments of the method of any one of
paragraphs 43-45 the host cell further expresses and secretes a
glucoamylase.
[0083] 47. In some embodiments of the method of any one of
paragraphs 43-46 the host cell is capable of fermenting the
composition.
[0084] 48. In another aspect, a composition comprising glucose
produced by the method of any one of paragraphs 38-47 is
provided.
[0085] 49. In another aspect, a liquefied starch produced by the
method of any one of paragraphs 38-47 is provided.
[0086] 50. In another aspect, a fermented beverage produced by the
method of any one of paragraphs 38-47 is provided.
[0087] 51. In another aspect, use of an amylase of any of
paragraphs 1-21 in the production of a composition comprising
glucose, in the production of a liquefied starch, in the production
of a fermented beverage, in cleaning starchy stains, or in textile
desizing, is provided.
[0088] 52. In another aspect, a method of desizing a textile is
provided, comprising contacting a desizing composition with a sized
textile for a time sufficient to desize the textile, wherein the
desizing composition comprises a variant .alpha.-amylase of any one
of paragraphs 1-21.
[0089] 53. In another aspect, an isolated polynucleotide encoding a
polypeptide of any of paragraphs 1-21 is provided.
[0090] 54. In another aspect, an expression vector comprising the
polynucleotide of paragraph 53 is provided.
[0091] 55. In another aspect, a host cell comprising the expression
vector of paragraph 54 is provided
[0092] 56. In another aspect, a polypeptide according to any one of
paragraphs 1-21 encoded by a polynucleotide that hybridizes under
stringent conditions to a polynucleotide complementary to the
full-length of the polynucleotide of SEQ ID NO: 7, SEQ ID NO: 33,
or SEQ ID NO: 38 is provided.
[0093] These and other aspects and embodiments of the compositions
and methods will be apparent from the present description and
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0094] FIG. 1 shows an amino acid sequence alignment of CspAmy2
.alpha.-amylase (SEQ ID NO: 1), PcuAmy1 .alpha.-amylase (SEQ ID NO:
3), and BASE .alpha.-amylase, using Clustal W with default
parameters.
[0095] FIG. 2 is a graph showing the cleaning benefit of different
doses of CspAmy2-v5 and CspAmy2-v6 on CS-28 rice starch at pH
8.
[0096] FIG. 3 is a graph showing the thermal stability of
CspAmy2-v5 and CspAmy2-v6 in buffer.
[0097] FIG. 4 is a graph showing the thermal stability of
CspAmy2-v5 and CspAmy2-v6 in buffer with calcium.
[0098] FIG. 5 is a graph showing the detergent stability of
CspAmy2-v5 and CspAmy2-v6 in OMO.TM. Color detergent.
[0099] FIG. 6 is a graph showing the detergent stability of
CspAmy2-v5 and CspAmy2-v6 in EPSIL.TM. Perfect detergent.
[0100] FIG. 7 is a table showing the relative half-lives and
performance indexes of the C16 variants and reference molecules
CspAmy2-v1-E187P and CspAmy2-v1-S241Q.
[0101] FIG. 8 is a graph showing the thermal stability of the C16
variants and reference molecules CspAmy2-v1-E187P and
CspAmy2-v1-S241Q at pH 4.5 and 65.degree. C.
[0102] FIG. 9 is a graph showing the thermal stability of the C16
variants and reference molecules CspAmy2-v1-E187P and
CspAmy2-v1-S241Q at pH 5.0 and 70.degree. C.
[0103] FIG. 10 is a graph showing the thermal stability of the C16
variants and reference molecules CspAmy2-v1-E187P and
CspAmy2-v1-S241Q at pH 5.7 and 85.degree. C.
[0104] FIG. 11 is a graph showing the detergent stability of
CspAmy2-v5, CspAmy2-v171, CspAmy2-v172, and ACE-QK.
[0105] FIG. 12 is a graph showing the relative cleaning performance
of CspAmy2-v5 and STAINZYME.RTM. in a hand dishwashing
application.
[0106] FIG. 13 FIGS. 13A and 13B, includes are tables showing the
compositions of WfK B citrate-based detergent (FIG. 13A) and WfK C
phospate-based detergent (FIG. 13B).
[0107] FIGS. 14 and 15 show the cleaning performance of CspAmy2-v6
(squares) compared to POWERASE.RTM. (diamonds), dosed at 0, 1, 2,
4, or 8 ppm in WfK B detergent against the mixed starch stain (FIG.
14) and the pasta stain (FIG. 15).
[0108] FIGS. 16 and 17 show the cleaning performance of CspAmy2-v6
(squares) compared to STAINZYME.RTM. (circles), dosed at 0, 1, 2,
4, or 8 ppm WfK B detergent against the mixed starch stain (FIG.
16) and the pasta stain (FIG. 17).
[0109] FIGS. 18 and 19 show the cleaning performance of CspAmy2-v6
(squares) compared to POWERASE.RTM. (diamonds), dosed at 0, 1, 2,
4, or 8 ppm in WfK C detergent against the mixed starch stain (FIG.
18) and the pasta stain (FIG. 19). CspAmy2-v6 clearly outperformed
POWERASE.RTM. against both stains.
[0110] FIG. 20 is a graph showing examples of C18P variants
demonstrating improved hydrolysis of corn starch at high
temperatures. CspAmy2-C18P (N126Y+F153W+T180D+I203Y+S241Q) is shown
as a reference.
[0111] FIG. 21 is a graph showing examples of C18P variants
demonstrating improved hydrolysis of amylopectin from corn. C18P is
shown as a reference.
[0112] FIG. 22 is a graph showing examples of variants
demonstrating improved generation of reducing sugars from starch.
C18P is shown as a reference.
[0113] FIG. 23 is a graph showing examples of C18P variants
demonstrating improved release of iodine staining material from
starch. C18P is shown as a reference.
[0114] FIG. 24 is a graph showing the viscosity reduction of corn
flour slurry produced by three C18P variants reported as fluidity
(1/viscosity) versus dose of the variants (in .mu.g). C18P and C16F
are shown as references.
[0115] FIG. 25 is a table showing the PI values for C16F variants
having different pairwise combinations of mutations at positions
G476 and G477, relative to a C16F control, in a corn starch
microswatch assay. PI values for revertants (i.e., G476G and G477G)
are empirically determined.
[0116] FIG. 26 is a table showing the PI values for C16F variants
having different pairwise combinations of mutations at positions
G476 and G477, relative to a C16F control, in a corn amylose
hydrolysis assay. PI values for revertants (i.e., G476G and G477G)
are empirically determined.
[0117] FIG. 27 is a graph showing the relative liquefaction
performance of CspAmy2-C25F, B, and A compared to C16F.
[0118] FIG. 28 is a graph showing the results of cleaning assays
performed at 0.015 ppm with CspAmy2-v179, v186, and v191 compared
to STAINZYME.RTM. and ACE-QK.
[0119] FIG. 29 is a graph showing the relative thermostability of
CspAmy2 variants v5, v179, v186, and v191 compared to
STAINZYME.RTM. and ACE-QK at temperatures ranging from 77.degree.
C. to 97.degree. C.
[0120] FIG. 30 is a graph showing the relative in-detergent storage
stability of CspAmy2 variants v5 and v179 compared to
STAINZYME.RTM. and ACE-QK in TIDE.RTM. regular HDL.
[0121] FIG. 31 is a graph showing the relative in-detergent storage
stability of CspAmy2 variants v5 and v179 compared to
STAINZYME.RTM. and ACE-QK in US TIDE.RTM. PODS.TM..
[0122] FIG. 32 is a graph showing the relative in-detergent storage
stability of CspAmy2 variants v5 and v179 compared to
STAINZYME.RTM. and ACE-QK in European ARIEL.TM. HDL.
[0123] FIG. 33 is a graph showing the relative in-detergent storage
stability of CspAmy2 variants v5 and v179 compared to
STAINZYME.RTM. and ACE-QK in European OMO.TM. Color HDL.
[0124] FIG. 34 is a graph showing the relative in-detergent storage
stability of CspAmy2 variants v5 and v179 compared to
STAINZYME.RTM. and ACE-QK in Chinese OMO.TM. Color HDL.
[0125] FIG. 35 is a graph showing the relative in-detergent storage
stability of CspAmy2 variants v5 and v179 compared to
STAINZYME.RTM. and ACE-QK in Chinese LIBY.TM. HDL.
[0126] FIG. 36 is a graph showing the relative cleaning performance
of PcuAmy1 variants v1, v6, v8, and v16 compared to STAINZYME.RTM.
and ACE-QK in buffer at pH 8.0. Enzyme doses are noted on the
x-axis.
[0127] FIG. 37 is a graph showing the relative thermal stability of
PcuAmy1 variants v1, v6, v8, and v16 compared to STAINZYME.RTM. in
buffer at the temperatures indicated 5 ppm of PcuAmy1 variants and
10 ppm of STAINZYME.RTM. were used.
[0128] FIG. 38 is a graph showing the relative thermal stability of
.DELTA.RG BASE variants incubated for the indicated amounts of time
at 95.degree. C.
[0129] FIG. 39 shows a portion of the three-dimensional structure
of CspAmy2-v1 highlighting the potential for interaction between a
glutamate at position 132 and a threonine at position 180.
[0130] FIG. 40 shows a portion of the three-dimensional structure
of CspAmy2-v1 highlighting the potential for interaction between a
glutamate at position 132 and a histidine at position 180.
[0131] FIG. 41 shows a portion of the three-dimensional structure
of CspAmy2-v1 highlighting the potential for interaction between a
glutamate at position 132 and an aspartate at position 180.
[0132] FIG. 42 shows a portion of the three-dimensional structure
of CspAmy2-v1 highlighting the potential for interaction between a
histidine at position 132 and an aspartate at position 180.
[0133] FIG. 43 is an image of an SDS/PAGE gel showing the cleavage
of PcuAmy1-v1 in the presence of increasing amounts of GG36
protease. The letters on the right side of the gel indicate (A)
intact full-length PcuAmy1-v1, (B) a first cleavage product of
PcuAmy1-v1, (C) GG36 protease, (D) a contaminant in the GG36
protein preparation, and (E) a second cleavage product of
PcuAmy1-v1.
[0134] FIG. 44 is a graph showing the residual .alpha.-amylase
activity of PcuAmy1 and several engineered variants following
incubation with GG36 protease.
[0135] FIG. 45 is an image of an SDS/PAGE gel showing the
proteolytic cleavage of PcuAmy1 and several engineered variants
following incubation with GG36 protease.
[0136] FIG. 46 is a graph showing the stability of PcuAmy1-v1 and
several engineered variants following incubation with GG36 protease
in MIFA Total detergent for up to 14 days at 37.degree. C.
[0137] FIG. 47 is a graph showing the stability of PcuAmy1-v1 and
several engineered variants following incubation with GG36 protease
in MIFA Total detergent for 3 or 14 days at 37.degree. C.
[0138] FIG. 48 is a graph showing the stability of PcuAmy1-v1 and
several engineered variants following incubation with GG36 protease
in Unilever Omo detergent for up to 14 days at 37.degree. C.
[0139] FIG. 49 is a graph showing the stability of PcuAmy1-v1 and
several engineered variants following incubation with GG36 protease
in Unilever Omo detergent for 3 or 14 days at 37.degree. C.
[0140] FIG. 50 is a graph showing the dose-dependent cleaning
performance of PcuAmy1-3B and PcuAmy1-3L in buffer at pH 8.0
compared to two commercial benchmarks.
[0141] FIG. 51 is a graph showing the stability of PcuAmy1-3B and
PcuAmy1-3L in Persil Universal Gel Gold detergent compared to two
commercial benchmarks.
[0142] FIG. 52 is a graph showing the stability of PcuAmy1-v1 and
several engineered variants following incubation with GG36 protease
in MIFA Total detergent for 3 or 14 days at 37.degree. C.
DETAILED DESCRIPTION
[0143] Described are compositions and methods relating to variant
amylase enzymes. The variants were discovered by a combination of
experimental approaches, as detailed in the appended Examples. The
approaches include the use of site evaluation libraries (SELs) and
structure-based analysis. Exemplary applications for the variant
amylase enzymes are for starch liquefaction and saccharification,
for cleaning starchy stains in laundry, dishwashing, and other
applications, for textile processing (e.g., desizing), in animal
feed for improving digestibility, and for baking and brewing. These
and other aspects of the compositions and methods are described in
detail, below.
[0144] Prior to describing the various aspects and embodiments of
the present compositions and methods, the following definitions and
abbreviations are described.
1. Definitions and Abbreviations
[0145] In accordance with this detailed description, the following
abbreviations and definitions apply. Note that the singular forms
"a," "an," and "the" include plural referents unless the context
clearly dictates otherwise. Thus, for example, reference to "an
enzyme" includes a plurality of such enzymes, and reference to "the
dosage" includes reference to one or more dosages and equivalents
thereof known to those skilled in the art, and so forth.
[0146] The present document is organized into a number of sections
for ease of reading; however, the reader will appreciate that
statements made in one section may apply to other sections. In this
manner, the headings used for different sections of the disclosure
should not be construed as limiting.
[0147] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. The following terms are provided
below.
1.1. Abbreviations and Acronyms
[0148] The following abbreviations/acronyms have the following
meanings unless otherwise specified:
[0149] ABTS 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic
acid
[0150] AE or AEO alcohol ethoxylate
[0151] AES or AEOS alcohol ethoxysulfate
[0152] AkAA Aspergillus kawachii .alpha.-amylase
[0153] AnGA Aspergillus niger glucoamylase
[0154] AOS .alpha.-olefinsulfonate
[0155] AS alkyl sulfate
[0156] cDNA complementary DNA
[0157] ct/kg cents/kg (US currency)
[0158] CMC carboxymethylcellulose
[0159] DE dextrose equivalent
[0160] DNA deoxyribonucleic acid
[0161] DPn degree of saccharide polymerization having n
subunits
[0162] ds or DS dry solids
[0163] DTMPA diethylenetriaminepentaacetic acid
[0164] EC Enzyme Commission
[0165] EDTA ethylenediaminetetraacetic acid
[0166] EO ethylene oxide (polymer fragment)
[0167] EOF end of fermentation
[0168] FH French hardness
[0169] GA glucoamylase
[0170] GAU/g ds glucoamylase activity unit/gram dry solids
[0171] GH general hardness
[0172] HDL high density liquid detergent
[0173] HDD heavy duty powder detergent
[0174] HSG high suds granular detergent
[0175] HFCS high fructose corn syrup
[0176] HgGA Humicola grisea glucoamylase
[0177] IPTG isopropyl .beta.-D-thiogalactoside
[0178] IRS insoluble residual starch
[0179] kDa kiloDalton
[0180] LAS linear alkylbenzenesulfonate
[0181] LAT, BLA B. licheniformis amylase
[0182] MW molecular weight
[0183] MWU modified Wohlgemuth unit; 1.6.times.10.sup.-5
mg/MWU=unit of activity
[0184] NCBI National Center for Biotechnology Information
[0185] NOBS nonanoyloxybenzenesulfonate
[0186] NTA nitriloacetic acid
[0187] OxAm Purastar HPAM 5000L (Danisco US Inc.)
[0188] PAHBAH p-hydroxybenzoic acid hydrazide
[0189] PEG polyethyleneglycol
[0190] pI isoelectric point
[0191] PI performance index
[0192] ppm parts per million, e.g., .mu.g protein per gram dry
solid
[0193] PVA poly(vinyl alcohol)
[0194] PVP poly(vinylpyrrolidone)
[0195] RCF relative centrifugal/centripetal force (i.e., x
gravity)
[0196] RNA ribonucleic acid
[0197] SAS alkanesulfonate
[0198] SDS-PAGE sodium dodecyl sulfate polyacrylamide gel
electrophoresis
[0199] SSF simultaneous saccharification and fermentation
[0200] SSU/g solid soluble starch unit/gram dry solids
[0201] sp. species
[0202] TAED tetraacetylethylenediamine
[0203] Tm melting temperature
[0204] TrGA Trichoderma reesei glucoamylase
[0205] w/v weight/volume
[0206] w/w weight/weight
[0207] v/v volume/volume
[0208] wt % weight percent
[0209] .degree. C. degrees Centigrade
[0210] H.sub.2O water
[0211] dH.sub.2O or DI deionized water
[0212] dIH.sub.2O deionized water, Milli-Q filtration
[0213] g or gm grams
[0214] micrograms
[0215] mg milligrams
[0216] kg kilograms
[0217] .mu.L and .mu.l microliters
[0218] mL and ml milliliters
[0219] mm millimeters
[0220] micrometer
[0221] M molar
[0222] mM millimolar
[0223] .mu.M micromolar
[0224] U units
[0225] sec seconds
[0226] min(s) minute/minutes
[0227] hr(s) hour/hours
[0228] DO dissolved oxygen
[0229] Ncm Newton centimeter
[0230] ETOH ethanol
[0231] eq. equivalents
[0232] N normal
[0233] uPWA variant .alpha.-amylase derived from Pyrococcus
woesei
[0234] PWA .alpha.-amylase from Pyrococcus woesei
[0235] MWCO molecular weight cut-off
[0236] SSRL Stanford Synchrotron Radiation Lightsource
[0237] PDB Protein Database
[0238] CAZy Carbohydrate-Active Enzymes database
[0239] Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride
[0240] HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
1.2. Definitions
[0241] The terms "amylase" or "amylolytic enzyme" refer to an
enzyme that is, among other things, capable of catalyzing the
degradation of starch. .alpha.-Amylases are hydrolases that cleave
the .alpha.-D-(1.fwdarw.4) 0-glycosidic linkages in starch.
Generally, .alpha.-amylases (EC 3.2.1.1;
.alpha.-D-(1.fwdarw.4)-glucan glucanohydrolase) are defined as
endo-acting enzymes cleaving .alpha.-D-(1.fwdarw.4) 0-glycosidic
linkages within the starch molecule in a random fashion yielding
polysaccharides containing three or more (1-4)-.alpha.-linked
D-glucose units. In contrast, the exo-acting amylolytic enzymes,
such as .beta.-amylases (EC 3.2.1.2; .alpha.-D-(1.fwdarw.4)-glucan
maltohydrolase) and some product-specific amylases like maltogenic
.alpha.-amylase (EC 3.2.1.133) cleave the polysaccharide molecule
from the non-reducing end of the substrate. .beta.-amylases,
.alpha.-glucosidases (EC 3.2.1.20; .alpha.-D-glucoside
glucohydrolase), glucoamylase (EC 3.2.1.3;
.alpha.-D-(1.fwdarw.4)-glucan glucohydrolase), and product-specific
amylases like the maltotetraosidases (EC 3.2.1.60) and the
maltohexaosidases (EC 3.2.1.98) can produce malto-oligosaccharides
of a specific length or enriched syrups of specific
maltooligosaccharides.
[0242] "Enzyme units" herein refer to the amount of product formed
per time under the specified conditions of the assay. For example,
a "glucoamylase activity unit" (GAU) is defined as the amount of
enzyme that produces 1 g of glucose per hour from soluble starch
substrate (4% DS) at 60.degree. C., pH 4.2. A "soluble starch unit"
(SSU) is the amount of enzyme that produces 1 mg of glucose per
minute from soluble starch substrate (4% DS) at pH 4.5, 50.degree.
C. DS refers to "dry solids."
[0243] The term "starch" refers to any material comprised of the
complex polysaccharide carbohydrates of plants, comprised of
amylose and amylopectin with the formula (C6H10O5)x, wherein X can
be any number. The term includes plant-based materials such as
grains, cereal, grasses, tubers and roots, and more specifically
materials obtained from wheat, barley, corn, rye, rice, sorghum,
brans, cassava, millet, milo, potato, sweet potato, and tapioca.
The term "starch" includes granular starch. The term "granular
starch" refers to raw, i.e., uncooked starch, e.g., starch that has
not been subject to gelatinization.
[0244] The terms, "wild-type," "parental," or "reference," with
respect to a polypeptide, refer to a naturally-occurring
polypeptide that does not include a man-made substitution,
insertion, or deletion at one or more amino acid positions.
Similarly, the terms "wild-type," "parental," or "reference," with
respect to a polynucleotide, refer to a naturally-occurring
polynucleotide that does not include a man-made nucleoside change.
However, note that a polynucleotide encoding a wild-type, parental,
or reference polypeptide is not limited to a naturally-occurring
polynucleotide, and encompasses any polynucleotide encoding the
wild-type, parental, or reference polypeptide.
[0245] Reference to the wild-type polypeptide is understood to
include the mature form of the polypeptide. A "mature" polypeptide
or variant, thereof, is one in which a signal sequence is absent,
for example, cleaved from an immature form of the polypeptide
during or following expression of the polypeptide.
[0246] The term "variant," with respect to a polypeptide, refers to
a polypeptide that differs from a specified wild-type, parental, or
reference polypeptide in that it includes one or more
naturally-occurring or man-made substitutions, insertions, or
deletions of an amino acid. Similarly, the term "variant," with
respect to a polynucleotide, refers to a polynucleotide that
differs in nucleotide sequence from a specified wild-type,
parental, or reference polynucleotide. The identity of the
wild-type, parental, or reference polypeptide or polynucleotide
will be apparent from context.
[0247] In the case of the present .alpha.-amylases, "activity"
refers to .alpha.-amylase activity, which can be measured as
described, herein.
[0248] The term "performance benefit" refers to an improvement in a
desirable property of a molecule. Exemplary performance benefits
include, but are not limited to, increased hydrolysis of a starch
substrate, increased grain, cereal or other starch substrate
liquifaction performance, increased cleaning performance, increased
thermal stability, increased detergent stability, increased storage
stability, increased solubility, an altered pH profile, decreased
calcium dependence, increased specific activity, modified substrate
specificity, modified substrate binding, modified pH-dependent
activity, modified pH-dependent stability, increased oxidative
stability, and increased expression. In some cases, the performance
benefit is realized at a relatively low temperature. In some cases,
the performance benefit is realized at relatively high
temperature.
[0249] The terms "protease" and "proteinase" refer to an enzyme
protein that has the ability to perform "proteolysis" or
"proteolytic cleavage" which refers to hydrolysis of peptide bonds
that link amino acids together in a peptide or polypeptide chain
forming the protein. This activity of a protease as a
protein-digesting enzyme is referred to as "proteolytic activity."
Many well-known procedures exist for measuring proteolytic activity
(See e.g., Kalisz, "Microbial Proteinases," In: Fiechter (ed.),
Advances in Biochemical Engineering/Biotechnology, (1988)). For
example, proteolytic activity may be ascertained by comparative
assays which analyze the respective protease's ability to hydrolyze
a commercial substrate. Exemplary substrates useful in the analysis
of protease or proteolytic activity, include, but are not limited
to, di-methyl casein (Sigma C-9801), bovine collagen (Sigma
C-9879), bovine elastin (Sigma E-1625), and bovine keratin (ICN
Biomedical 902111). Colorimetric assays utilizing these substrates
are well known in the art (See e.g., WO 99/34011 and U.S. Pat. No.
6,376,450, both of which are incorporated herein by reference). The
pNA assay (See e.g., Del Mar et al., Anal. Biochem. 99:316-320
[1979]) also finds use in determining the active enzyme
concentration for fractions collected during gradient elution. This
assay measures the rate at which p-nitroaniline is released as the
enzyme hydrolyzes a soluble synthetic peptide substrate, such as
succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide
(suc-AAPF-pNA), and cleavage occurs between the C-terminal amino
acid (phenylalanine) and the p-NA, causing the production of yellow
color from the hydrolysis reaction, which is measured at 410 nm on
a spectrophotometer and is proportional to the active enzyme
concentration. Measurement of the color change allows calculation
of the rate of the reaction. In addition, absorbance measurements
at 280 nanometers (nm) can be used to determine the total protein
concentration. The active enzyme/total protein ratio gives the
enzyme purity when a reference standard is used.
[0250] The terms "serine protease" refers to enzymes that cleave
peptide bonds in proteins, in which enzymes serine serves as the
nucleophilic amino acid at the enzyme active site. Serine proteases
fall into two broad categories based on their structure:
chymotrypsin-like (trypsin-like) or subtilisin-like. Most commonly
used in laundry and dishwashing detergents are serine protease,
particularly subtlisins.
[0251] The term "TIM barrel" refers to a three dimensional
polypeptide structure that include eight .alpha.-helices and eight
parallel .beta.-strands that alternate along the peptide
backbone.
[0252] The term "surface-exposed" with respect to an amino acid
residue in a polypeptide refers to a residue that is on the
exterior surface of a polypeptide when the polypeptide is intact
and properly folded, i.e., not denatured or fragmented. In the case
of an .alpha.-amylase, the structure is referred to as a TIM
barrel.
[0253] The term "non-canonical" with reference to an amino acid
residue in a polypeptide refers to a residue that is not normally
found at a given position based on amino acid sequence alignments
of similar molecules using Clustal W with default parameter. In
some cases, the particular residue is found at a given position in
only 1 in 10, 1 in 20, 1 in 30, 1 in 50, or even 1 in 100 similar
molecules.
[0254] "Combinatorial variants" are variants comprising two or more
mutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more,
substitutions, deletions, and/or insertions.
[0255] "Combinable mutations" are mutations at any amino acid
position that can be used to make combinatorial variants.
Combinable mutations improve at least one desired property of the
molecule (in this case, an amylase), while not significantly
decreasing either expression, activity, or stability.
[0256] Terms, such as "a remaining non-G residue in the
calcium-binding loop," "a non-G amino acid residue remaining in the
calcium-binding loop," and similar terms, refer to an amino acid
residue in the calcium-binding loop of a variant .alpha.-amylase,
which remains in the variant following a deletion of at least one
amino acid residue in the calcium-binding loop of a parent
.alpha.-amylases, and which is not a glycine residue. The non-G
residue may be a member of an "XG" pair, of which there are two in
most .alpha.-amylases, and may be the remaining non-G residue
following a pair-wise deletion of one of the two XG residue pairs
in the calcium binding loop of a parent .alpha.-amylase.
[0257] A "stabilizing interaction" between the residue at position
132 (using SEQ ID NO: 1 for numbering) and the remaining non-G
residue in the X.sub.1G/S.sub.1X.sub.2G.sub.2 motif (corresponding
to residues at positions 178-181 of SEQ ID NO: 1) refers to a
hydrogen bond or a salt bridge formed between the side chains of
the subject amino acid residues. The stabilization can result from
charge balancing the interacting residues, such that if one residue
is positively charged at a preselected pH, the other is negatively
charged, and the overall charge is zero.
[0258] The term "recombinant," when used in reference to a subject
cell, nucleic acid, protein or vector, indicates that the subject
has been modified from its native state. Thus, for example,
recombinant cells express genes that are not found within the
native (non-recombinant) form of the cell, or express native genes
at different levels or under different conditions than found in
nature. Recombinant nucleic acids differ from a native sequence by
one or more nucleotides and/or are operably linked to heterologous
sequences, e.g., a heterologous promoter in an expression vector.
Recombinant proteins may differ from a native sequence by one or
more amino acids and/or are fused with heterologous sequences. A
vector comprising a nucleic acid encoding an amylase is a
recombinant vector.
[0259] The terms "recovered," "isolated," and "separated," refer to
a compound, protein (polypeptides), cell, nucleic acid, amino acid,
or other specified material or component that is removed from at
least one other material or component with which it is naturally
associated as found in nature. An "isolated" polypeptides, thereof,
includes, but is not limited to, a culture broth containing
secreted polypeptide expressed in a heterologous host cell.
[0260] The term "purified" refers to material (e.g., an isolated
polypeptide or polynucleotide) that is in a relatively pure state,
e.g., at least about 90% pure, at least about 95% pure, at least
about 98% pure, or even at least about 99% pure.
[0261] The term "enriched" refers to material (e.g., an isolated
polypeptide or polynucleotide) that is in about 50% pure, at least
about 60% pure, at least about 70% pure, or even at least about 70%
pure.
[0262] The terms "thermostable" and "thermostability," with
reference to an enzyme, refer to the ability of the enzyme to
retain activity after exposure to an elevated temperature. The
thermostability of an enzyme, such as an amylase enzyme, is
measured by its half-life (t1/2) given in minutes, hours, or days,
during which half the enzyme activity is lost under defined
conditions. The half-life may be calculated by measuring residual
.alpha.-amylase activity following exposure to (i.e., challenge by)
an elevated temperature.
[0263] A "pH range," with reference to an enzyme, refers to the
range of pH values under which the enzyme exhibits catalytic
activity.
[0264] The terms "pH stable" and "pH stability," with reference to
an enzyme, relate to the ability of the enzyme to retain activity
over a wide range of pH values for a predetermined period of time
(e.g., 15 min., 30 min., 1 hour).
[0265] The term "amino acid sequence" is synonymous with the terms
"polypeptide," "protein," and "peptide," and are used
interchangeably. Where such amino acid sequences exhibit activity,
they may be referred to as an "enzyme." The conventional one-letter
or three-letter codes for amino acid residues are used, with amino
acid sequences being presented in the standard amino-to-carboxy
terminal orientation (i.e., N.fwdarw.C).
[0266] The term "nucleic acid" encompasses DNA, RNA,
heteroduplexes, and synthetic molecules capable of encoding a
polypeptide. Nucleic acids may be single stranded or double
stranded, and may be chemical modifications. The terms "nucleic
acid" and "polynucleotide" are used interchangeably. Because the
genetic code is degenerate, more than one codon may be used to
encode a particular amino acid, and the present compositions and
methods encompass nucleotide sequences that encode a particular
amino acid sequence. Unless otherwise indicated, nucleic acid
sequences are presented in 5'-to-3' orientation.
[0267] "Hybridization" refers to the process by which one strand of
nucleic acid forms a duplex with, i.e., base pairs with, a
complementary strand, as occurs during blot hybridization
techniques and PCR techniques. Stringent hybridization conditions
are exemplified by hybridization under the following conditions:
65.degree. C. and 0.1.times.SSC (where 1.times.SSC=0.15 M NaCl,
0.015 M Na3 citrate, pH 7.0). Hybridized, duplex nucleic acids are
characterized by a melting temperature (Tm), where one half of the
hybridized nucleic acids are unpaired with the complementary
strand. Mismatched nucleotides within the duplex lower the Tm. A
nucleic acid encoding a variant .alpha.-amylase may have a Tm
reduced by 1.degree. C.-3.degree. C. or more compared to a duplex
formed between the nucleotide of SEQ ID NO: 2 and its identical
complement.
[0268] A "synthetic" molecule is produced by in vitro chemical or
enzymatic synthesis rather than by an organism.
[0269] The terms "transformed," "stably transformed," and
"transgenic," used with reference to a cell means that the cell
contains a non-native (e.g., heterologous) nucleic acid sequence
integrated into its genome or carried as an episome that is
maintained through multiple generations.
[0270] The term "introduced" in the context of inserting a nucleic
acid sequence into a cell, means "transfection", "transformation"
or "transduction," as known in the art.
[0271] A "host strain" or "host cell" is an organism into which an
expression vector, phage, virus, or other DNA construct, including
a polynucleotide encoding a polypeptide of interest (e.g., an
amylase) has been introduced. Exemplary host strains are
microorganism cells (e.g., bacteria, filamentous fungi, and yeast)
capable of expressing the polypeptide of interest and/or fermenting
saccharides. The term "host cell" includes protoplasts created from
cells.
[0272] The term "heterologous" with reference to a polynucleotide
or protein refers to a polynucleotide or protein that does not
naturally occur in a host cell.
[0273] The term "endogenous" with reference to a polynucleotide or
protein refers to a polynucleotide or protein that occurs naturally
in the host cell.
[0274] The term "expression" refers to the process by which a
polypeptide is produced based on a nucleic acid sequence. The
process includes both transcription and translation.
[0275] A "selective marker" or "selectable marker" refers to a gene
capable of being expressed in a host to facilitate selection of
host cells carrying the gene. Examples of selectable markers
include but are not limited to antimicrobials (e.g., hygromycin,
bleomycin, or chloramphenicol) and/or genes that confer a metabolic
advantage, such as a nutritional advantage on the host cell.
[0276] A "vector" refers to a polynucleotide sequence designed to
introduce nucleic acids into one or more cell types. Vectors
include cloning vectors, expression vectors, shuttle vectors,
plasmids, phage particles, cassettes and the like.
[0277] An "expression vector" refers to a DNA construct comprising
a DNA sequence encoding a polypeptide of interest, which coding
sequence is operably linked to a suitable control sequence capable
of effecting expression of the DNA in a suitable host. Such control
sequences may include a promoter to effect transcription, an
optional operator sequence to control transcription, a sequence
encoding suitable ribosome binding sites on the mRNA, enhancers and
sequences which control termination of transcription and
translation.
[0278] The term "operably linked" means that specified components
are in a relationship (including but not limited to juxtaposition)
permitting them to function in an intended manner. For example, a
regulatory sequence is operably linked to a coding sequence such
that expression of the coding sequence is under control of the
regulatory sequences.
[0279] A "signal sequence" is a sequence of amino acids attached to
the N-terminal portion of a protein, which facilitates the
secretion of the protein outside the cell. The mature form of an
extracellular protein lacks the signal sequence, which is cleaved
off during the secretion process.
[0280] "Biologically active" refer to a sequence having a specified
biological activity, such an enzymatic activity.
[0281] The term "specific activity" refers to the number of moles
of substrate that can be converted to product by an enzyme or
enzyme preparation per unit time under specific conditions.
Specific activity is generally expressed as units (U)/mg of
protein.
[0282] As used herein, "water hardness" is a measure of the
minerals (e.g., calcium and magnesium) present in water.
[0283] A "swatch" is a piece of material such as a fabric that has
a stain applied thereto. The material can be, for example, fabrics
made of cotton, polyester or mixtures of natural and synthetic
fibers. The swatch can further be paper, such as filter paper or
nitrocellulose, or a piece of a hard material such as ceramic,
metal, or glass. For amylases, the stain is starch based, but can
include blood, milk, ink, grass, tea, wine, spinach, gravy,
chocolate, egg, cheese, clay, pigment, oil, or mixtures of these
compounds.
[0284] A "smaller swatch" is a section of the swatch that has been
cut with a single hole punch device, or has been cut with a custom
manufactured 96-hole punch device, where the pattern of the
multi-hole punch is matched to standard 96-well microtiter plates,
or the section has been otherwise removed from the swatch. The
swatch can be of textile, paper, metal, or other suitable material.
The smaller swatch can have the stain affixed either before or
after it is placed into the well of a 24-, 48- or 96-well
microtiter plate. The smaller swatch can also be made by applying a
stain to a small piece of material. For example, the smaller swatch
can be a stained piece of fabric 5/8'' or 0.25'' in diameter. The
custom manufactured punch is designed in such a manner that it
delivers 96 swatches simultaneously to all wells of a 96-well
plate. The device allows delivery of more than one swatch per well
by simply loading the same 96-well plate multiple times. Multi-hole
punch devices can be conceived of to deliver simultaneously
swatches to any format plate, including but not limited to 24-well,
48-well, and 96-well plates. In another conceivable method, the
soiled test platform can be a bead made of metal, plastic, glass,
ceramic, or another suitable material that is coated with the soil
substrate. The one or more coated beads are then placed into wells
of 96-, 48-, or 24-well plates or larger formats, containing
suitable buffer and enzyme.
[0285] "A cultured cell material comprising an amylase" or similar
language, refers to a cell lysate or supernatant (including media)
that includes an amylase as a component. The cell material may be
from a heterologous host that is grown in culture for the purpose
of producing the amylase.
[0286] "Percent sequence identity" means that a particular sequence
has at least a certain percentage of amino acid residues identical
to those in a specified reference sequence, when aligned using the
CLUSTAL W algorithm with default parameters. See Thompson et al.
(1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the
CLUSTAL W algorithm are: [0287] Gap opening penalty: 10.0 [0288]
Gap extension penalty: 0.05 [0289] Protein weight matrix: BLOSUM
series [0290] DNA weight matrix: IUB [0291] Delay divergent
sequences %: 40 [0292] Gap separation distance: 8 [0293] DNA
transitions weight: 0.50 [0294] List hydrophilic residues:
GPSNDQEKR [0295] Use negative matrix: OFF [0296] Toggle Residue
specific penalties: ON [0297] Toggle hydrophilic penalties: ON
[0298] Toggle end gap separation penalty OFF.
[0299] Deletions are counted as non-identical residues, compared to
a reference sequence. Deletions occurring at either termini are
included. For example, a variant with five amino acid deletions of
the C-terminus of the mature CspAmy2 polypeptide of SEQ ID NO: 1
would have a percent sequence identity of 99% (612/617 identical
residues.times.100, rounded to the nearest whole number) relative
to the mature polypeptide. Such a variant would be encompassed by a
variant having "at least 99% sequence identity" to a mature amylase
polypeptide.
[0300] "Fused" polypeptide sequences are connected, i.e., operably
linked, via a peptide bond between two subject polypeptide
sequences.
[0301] The term "filamentous fungi" refers to all filamentous forms
of the subdivision Eumycotina, particulary Pezizomycotina
species.
[0302] The term "degree of polymerization" (DP) refers to the
number (n) of anhydro-glucopyranose units in a given saccharide.
Examples of DP1 are the monosaccharides glucose and fructose.
Examples of DP2 are the disaccharides maltose and sucrose. The term
"DE," or "dextrose equivalent," is defined as the percentage of
reducing sugar, i.e., D-glucose, as a fraction of total
carbohydrate in a syrup.
[0303] The term "dry solids content" (ds) refers to the total
solids of a slurry in a dry weight percent basis. The term "slurry"
refers to an aqueous mixture containing insoluble solids.
[0304] The phrase "simultaneous saccharification and fermentation
(SSF)" refers to a process in the production of biochemicals in
which a microbial organism, such as an ethanologenic microorganism,
and at least one enzyme, such as an amylase, are present during the
same process step. SSF includes the contemporaneous hydrolysis of
starch substrates (granular, liquefied, or solubilized) to
saccharides, including glucose, and the fermentation of the
saccharides into alcohol or other biochemical or biomaterial in the
same reactor vessel.
[0305] An "ethanologenic microorganism" refers to a microorganism
with the ability to convert a sugar or oligosaccharide to
ethanol.
[0306] The term "fermented beverage" refers to any beverage
produced by a method comprising a fermentation process, such as a
microbial fermentation, e.g., a bacterial and/or fungal
fermentation. "Beer" is an example of such a fermented beverage,
and the term "beer" is meant to comprise any fermented wort
produced by fermentation/brewing of a starch-containing plant
material. Often, beer is produced exclusively from malt or adjunct,
or any combination of malt and adjunct. Examples of beers include:
full malted beer, beer brewed under the "Reinheitsgebot," ale,
India pale ale, lager, pilsner, bitter, Happoshu (second beer),
third beer, dry beer, near beer, light beer, low alcohol beer, low
calorie beer, porter, bock, dopplebock, stout, porter, malt liquor,
non-alcoholic beer, non-alcoholic malt liquor and the like, but
also alternative cereal and malt beverages such as fruit flavored
malt beverages, e.g., citrus flavored, such as lemon-, orange-,
lime-, or berry-flavored malt beverages, liquor flavored malt
beverages, e.g., vodka-, rum-, or tequila-flavored malt liquor, or
coffee flavored malt beverages, such as caffeine-flavored malt
liquor, and the like.
[0307] The term "malt" refers to any malted cereal grain, such as
malted barley or wheat.
[0308] The term "adjunct" refers to any starch and/or sugar
containing plant material that is not malt, such as barley or wheat
malt. Examples of adjuncts include common corn grits, refined corn
grits, brewer's milled yeast, rice, sorghum, refined corn starch,
barley, barley starch, dehusked barley, wheat, wheat starch,
torrified cereal, cereal flakes, rye, oats, potato, tapioca,
cassava and syrups, such as corn syrup, sugar cane syrup, inverted
sugar syrup, barley and/or wheat syrups, and the like.
[0309] The term "mash" refers to an aqueous slurry of any starch
and/or sugar containing plant material, such as grist, e.g.,
comprising crushed barley malt, crushed barley, and/or other
adjunct or a combination thereof, mixed with water later to be
separated into wort and spent grains.
[0310] The term "wort" refers to the unfermented liquor run-off
following extracting the grist during mashing.
[0311] "Iodine-positive starch" or "IPS" refers to (1) amylose that
is not hydrolyzed after liquefaction and saccharification, or (2) a
retrograded starch polymer. When saccharified starch or saccharide
liquor is tested with iodine, the high DPn amylose or the
retrograded starch polymer binds iodine and produces a
characteristic blue color. The saccharide liquor is thus termed
"iodine-positive saccharide," "blue saccharide," or "blue sac."
[0312] The terms "retrograded starch" or "starch retrogradation"
refer to changes that occur spontaneously in a starch paste or gel
on ageing.
[0313] The term "about" refers to .+-.15% to the referenced
value.
2. .alpha.-Amylase Variants
[0314] An aspect of the present compositions and methods is variant
amylase enzymes that include combinations of mutations that improve
their performance in industrial applications. The combinatorial
variants were initially discovered using an .alpha.-amylase from
Cytophaga sp. (herein, "CspAmy2 amylase"), which was previously
described by Jeang, C-L et al. ((2002) Applied and Environmental
Microbiology, 68:3651-54). The amino acid sequence of the mature
form of the CspAmy2 .alpha.-amylase polypeptide is shown below as
SEQ ID NO: 1:
TABLE-US-00001 AATNGTMMQY FEWYVPNDGQ QWNRLRTDAP YLSSVGITAV
WTPPAYKGTS QADVGYGPYD LYDLGEFNQK GTVRTKYGTK GELKSAVNTL HSNGIQVYGD
VVMNHKAGAD YTENVTAVEV NPSNRNQETS GEYNIQAWTG FNFPGRGTTY SNFKWQWFHF
DGTDWDQSRS LSRIFKFRGT GKAWDWEVSS ENGNYDYLMY ADIDYDHPDV VNEMKKWGVW
YANEVGLDGY RLDAVKHIKF SFLKDWVDNA RAATGKEMFT VGEYWQNDLG ALNNYLAKVN
YNQSLFDAPL HYNFYAASTG GGYYDMRNIL NNTLVASNPT KAVTLVENHD TQPGQSLEST
VQPWFKPLAY AFILTRSGGY PSVFYGDMYG TKGTTTREIP ALKSKIEPLL KARKDYAYGT
QRDYIDNPDV IGWTREGDST KAKSGLATVI TDGPGGSKRM YVGTSNAGEI WYDLTGNRTD
KITIGSDGYA TFPVNGGSVS VWVQQ
[0315] In SEQ ID NO: 1, R178 and G179 are underlined. A variant of
the Cytophaga sp. .alpha.-amylase having a deletion of both R178
and G179 (herein, "CspAmy2-v1") has also been described (Shiau,
R-J. et al. (2003) Applied and Environmental Microbiology,
69:2383-85). The amino acid sequence of the mature CspAmy2-v1
.alpha.-amylase polypeptide is shown below as SEQ ID NO: 2:
TABLE-US-00002 AATNGTMMQY FEWYVPNDGQ QWNRLRTDAP YLSSVGITAV
WTPPAYKGTS QADVGYGPYD LYDLGEFNQK GTVRTKYGTK GELKSAVNTL HSNGIQVYGD
VVMNHKAGAD YTENVTAVEV NPSNRNQETS GEYNIQAWTG FNFPGRGTTY SNFKWQWFHF
DGTDWDQSRS LSRIFKFTGK AWDWEVSSEN GNYDYLMYAD IDYDHPDVVN EMKKWGVWYA
NEVGLDGYRL DAVKHIKFSF LKDWVDNARA ATGKEMFTVG EYWQNDLGAL NNYLAKVNYN
QSLFDAPLHY NFYAASTGGG YYDMRNILNN TLVASNPTKA VTLVENHDTQ PGQSLESTVQ
PWFKPLAYAF ILTRSGGYPS VFYGDMYGTK GTTTREIPAL KSKIEPLLKA RKDYAYGTQR
DYIDNPDVIG WTREGDSTKA KSGLATVITD GPGGSKRMYV GTSNAGEIWY DLTGNRTDKI
TIGSDGYATF PVNGGSVSVW VQQ
[0316] Using SEQ ID NO: 2 as a starting point, a number of
combinatorial CspAmy2 variants were initially made and tested as
described in the Examples section. The best performing variants
generally included a stabilizing mutation at an amino acid position
corresponding to either E187 or S241, but not both positions, and
at least one additional performance-enhancing mutation at amino
acid position selected from the group consisting of N126, Y150,
F153, L171, T180, and, I203 (using SEQ ID NO: 1 for numbering).
[0317] It is known that many bacterial (and other) .alpha.-amylases
share the same fold, often share significant amino acid sequence
identity, and sometimes benefit from the same mutations. In the
present case, corresponding amino acid positions were identified by
amino acid sequence alignment in an .alpha.-amylase from
Paenibacillus curdlanolyticus (i.e., PcuAmy1; SEQ ID NO: 3) and a
C-terminal-truncated version of the Bacillus sp. TS-23
.alpha.-amylase (i.e., "BASE;" SEQ ID NO: 5; see, e.g.,
US20120045817 and WO2010/115028).
[0318] The amino acid sequence of the mature form of the PcuAmy1
.alpha.-amylase polypeptide is shown below as SEQ ID NO: 3:
TABLE-US-00003 ADNGTIMQYF EWYLPNDGAH WNRLNNDAQN LKNVGITAVW
IPPAYKGGSS ADVGYGVYDT YDLGEFNQKG TVRTKYGTKS ELISAVNNLH AKGIAVYGDV
VLNHRMNADA TELVDAVEVD PNNRNVETTS TYQIQAWTQY DFPGRGNTYS SFKWRWYHFD
GVDWDQSRGL NRIYKLRGDG KDWDWEVDSE YGNYDYLMGA DLDFNHPDVV NETKTWGKWF
VNTVNLDGVR LDAVKHIKFD FMRDWVNNVR STTGKNLFAV GEYWHYDVNK LNSYITKTNG
TMSLFDVPLH FRFYDASNGG GGYDMRNLLN NTLMSSNPMK AVTFVENHDT QPTQALQSTV
QSWFKPLAYA TILTREQGYP CVFYGDYYGT SDGKISSYKP IMDKLLNARK VYAYGTQRDY
FDHPDIVGWT REGDAAHAGS GLATLITDGP GGSKWMYVGT SKAGQVWTDK TGNRSGTVTI
DANGWGNFWV NGGSVSVWAK
[0319] In SEQ ID NO: 3, R177 and R178 are underlined. The amino
acid sequence of a variant form of PcuAmy1 .alpha.-amylase having a
deletion of both R177 and R178 (herein, "PcuAmy1-v1") is shown
below as SEQ ID NO: 4:
TABLE-US-00004 ADNGTIMQYF EWYLPNDGAH WNRLNNDAQN LKNVGITAVW
IPPAYKGGSS ADVGYGVYDT YDLGEFNQKG TVRTKYGTKS ELISAVNNLH AKGIAVYGDV
VLNHRMNADA TELVDAVEVD PNNRNVETTS TYQIQAWTQY DFPGRGNTYS SFKWRWYHFD
GVDWDQSRGL NRIYKLDGKD WDWEVDSEYG NYDYLMGADL DFNHPDVVNE TKTWGKWFVN
TVNLDGVRLD AVKHIKFDFM RDWVNNVRST TGKNLFAVGE YWHYDVNKLN SYITKTNGTM
SLFDVPLHFR FYDASNGGGG YDMRNLLNNT LMSSNPMKAV TFVENHDTQP TQALQSTVQS
WFKPLAYATI LTREQGYPCV FYGDYYGTSD GKISSYKPIM DKLLNARKVY AYGTQRDYFD
HPDIVGWTRE GDAAHAGSGL ATLITDGPGG SKWMYVGTSK AGQVWTDKTG NRSGTVTIDA
NGWGNFWVNG GSVSVWAK
[0320] The amino acid sequence of a C-terminal-truncated version of
the Bacillus sp. TS-23 .alpha.-amylase (herein, "BASE;" see, e.g.,
US20120045817 and WO2010/115028) is shown, below as SEQ ID NO:
5:
TABLE-US-00005 NTAPINETMM QYFEWDLPND GTLWTKVKNE AANLSSLGIT
ALWLPPAYKG TSQSDVGYGV YDLYDLGEFN QKGTIRTKYG TKTQYIQAIQ AAKAAGMQVY
ADVVFNHKAG ADGTEFVDAV EVDPSNRNQE TSGTYQIQAW TKFDFPGRGN TYSSFKWRWY
HFDGTDWDES RKLNRIYKFR STGKAWDWEV DTENGNYDYL MFADLDMDHP EVVTELKNWG
TWYVNTTNID GFRLDAVKHI KYSFFPDWLT YVRNQTGKNL FAVGEFWSYD VNKLHNYITK
TNGSMSLFDA PLHNNFYTAS KSSGYFDMRY LLNNTLMKDQ PSLAVTLVDN HDTQPGQSLQ
SWVEPWFKPL AYAFILTRQE GYPCVFYGDY YGIPKYNIPG LKSKIDPLLI ARRDYAYGTQ
RDYIDHQDII GWTREGIDTK PNSGLAALIT DGPGGSKWMY VGKKHAGKVF YDLTGNRSDT
VTINADGWGE FKVNGGSVSI WVAK
[0321] In SEQ ID NO: 5, R180 and S181 are underlined. The amino
acid sequence of a variant form of BASE .alpha.-amylase having a
deletion of both R180 and S181 (herein, "ACE") is shown, below as
SEQ ID NO: 6:
TABLE-US-00006 NTAPINETMM QYFEWDLPND GTLWTKVKNE AANLSSLGIT
ALWLPPAYKG TSQSDVGYGV YDLYDLGEFN QKGTIRTKYG TKTQYIQAIQ AAKAAGMQVY
ADVVFNHKAG ADGTEFVDAV EVDPSNRNQE TSGTYQIQAW TKFDFPGRGN TYSSFKWRWY
HFDGTDWDES RKLNRIYKFT GKAWDWEVDT ENGNYDYLMF ADLDMDHPEV VTELKNWGTW
YVNTTNIDGF RLDAVKHIKY SFFPDWLTYV RNQTGKNLFA VGEFWSYDVN KLHNYITKTN
GSMSLFDAPL HNNFYTASKS SGYFDMRYLL NNTLMKDQPS LAVTLVDNHD TQPGQSLQSW
VEPWFKPLAY AFILTRQEGY PCVFYGDYYG IPKYNIPGLK SKIDPLLIAR RDYAYGTQRD
YIDHQDIIGW TREGIDTKPN SGLAALITDG PGGSKWMYVG KKHAGKVFYD LTGNRSDTVT
INADGWGEFK VNGGSVSIWV AK
[0322] An amino acid sequence alignment of CspAmy2 (SEQ ID NO: 1),
PcuAmy1 (SEQ ID NO: 3), and BASE (SEQ ID NO: 5), using Clustal W
with default parameters, is shown in FIG. 1. Positions N126, Y150,
F153, L171, R178, G179, T180, E187, 1203, and S241 in CspAmy2
correspond to positions N125, Y149, F152, L170, R177, G178, D179,
E186, L202, and D240 in PcuAmy1, respectively, and positions N128,
Y152, F155, L173, R180, S181, T182, E189, L205, and S243,
respectively in BASE. Numbering for other positions through out the
molecules can be determined using the alignment and information,
herein.
[0323] Based on experimental data obtained using the aforementioned
three parent .alpha.-amylases, embodiments of the present variant
.alpha.-amylases include variants having a mutation at an amino
acid position corresponding to E187 or S241 in combination with at
least one mutation at an amino acid position corresponding to a
position selected from N126, Y150, F153, L171, T180, and I203
(using SEQ ID NO: 1 for numbering), wherein the mutations provide
at least one performance benefit to the resulting variant.
[0324] Referring to SEQ ID NO: 1 for numbering, exemplary mutations
at amino acid position E187 include E187V and E187P. Exemplary
mutations at amino acid position S241 include S241Q and S241A. In
some embodiments, mutations are made in only one of these
positions. Exemplary mutations at amino acid position N126 includes
N126Y. Exemplary mutations at amino acid position Y150 include
Y150F, Y150H, and Y150W. Exemplary mutations at amino acid position
F153 include F153H, F153W, and F153Y. Exemplary mutations at amino
acid position L171 include L171F, L171G, L171I, L171M, L171R,
L171V, L171W, L171Y, L171H, L171K, L171N, L171Q, and L171S.
Exemplary mutations at amino acid position T180 include T180D and
T180H. Exemplary mutations at amino acid position I203 includes
I203C, I203V, I203F, I203L, I203M, and I203Y.
[0325] In some embodiments, the variant .alpha.-amylases further
include a mutation in amino acid residues corresponding to E132,
Q167, A277, and/or T400, using SEQ ID NO: 1 for numbering.
Exemplary mutations at amino acid position E132 include E132A,
E132C, E132D, E132F, E132G, E132H, E132I, E132K, E132L, E132M,
E132N, E132P, E132Q, E132R, E132V, and E132W. Exemplary mutations
at amino acid position Q167 include Q167A, Q167D, Q167E, Q167G,
Q167H, Q167K, Q167M, Q167N, Q167P, Q167S, Q167T, and Q167V.
Exemplary mutations at amino acid position A277 include A277C,
A277D, A277E, A277F, A277G, A277I, A277K, A277L, A277M, A277N,
A277Q, A277R, A277S, A277T, A277V, A277W, and A277Y. Exemplary
mutations at amino acid position T400 include T400A, T400C, T400D,
T400F, T400G, T400I, T400K, T400L, T400M, T400N, T400Q, T400R,
T400W, and T400Y.
[0326] In some embodiments, the variant .alpha.-amylases further
include a mutation in an amino acid residue corresponding to G476,
using SEQ ID NO: 1 for numbering. Exemplary mutations at amino acid
position G476 include G476A, G476C, G476H, G476K, G476N, G476P,
G476Q, G476R, G476S, G476T, G476V, and G476Y.
[0327] In some embodiments, the variant .alpha.-amylases are those
that include, or further include, mutations in both amino acid
residues corresponding to G476 and G477, using SEQ ID NO: 1 for
numbering. Surprisingly, experimental evidence suggests that any
combination of residues at these positions, other than two adjacent
glycines as are present in many naturally occurring
.alpha.-amylases, increases improve starch hydrolysis activity,
particularly in cleaning applications.
[0328] In some embodiments, the variant .alpha.-amylases further
include mutations in amino acid residues corresponding to R458,
T459, and/or D460. Exemplary mutations are R458N, T459S, and D460T,
respectively.
[0329] In some embodiments, the variant .alpha.-amylases further
include a deletion in the X.sub.1G/S.sub.1X.sub.2G.sub.2 motif
adjacent to the calcium-binding loop corresponding to R178, G179,
T180, and G181, using SEQ ID NO: 1 for numbering. In some
embodiments, the variant .alpha.-amylases include adjacent,
pair-wise deletions of amino acid residues corresponding to R178
and G179, or T180 and G181. A deletion in amino acid residues
corresponding to R178 and G179 may be referred to as ".DELTA.RG,"
while a deletion in amino acid residues corresponding to T180 and
G181 ".DELTA.TG." This nomenclature will naturally change depending
on the amino acid residues originally present in the parent
molecule.
[0330] In some embodiments, the variant .alpha.-amylases include
mutations at positions corresponding to E132 and/or T180 (using SEQ
ID NO: 1 for numbering), in combination with an RG-deletion or a
TG-deletion (or equivalent deletion based on the sequence of the
parent .alpha.-amylases), such that a stabilizing interaction can
occur between the remaining non-G residue in the
X.sub.1G/S.sub.1X.sub.2G.sub.2 motif and the residue at position
132. In some embodiments, the residue at position 132 is negatively
charged (i.e., D or E) and the remaining non-G residue is
positively charged (i.e., H, R, or K). In some embodiments, the
residue at position 132 is positively charged (i.e., H, R, or K)
and the remaining non-G residue is negatively charged (i.e., D or
E).
[0331] Exemplary combinations of mutation (using SEQ ID NO: 1 for
numbering) are shown, below:
E187P+I203Y+G476K (i.e., CspAmy2-v5);
E187P+I203Y+G476K+R458N+T459S+D460T (i.e., CspAmy2-v6);
[0332] T180D+E187P+I203Y+G476K (i.e., CspAmy2 v171);
N126Y+T180D+E187P+I203Y+G476K (i.e., CspAmy2 v172);
N126Y+T180D+E187P+I203Y+Y303D+G476T+G477E, (i.e., CspAmy2 v179);
N126Y+T180D+E187P+I203Y+Y303D+N475E+G477Q, (i.e., CspAmy2 v180);
N126Y+T180D+E187P+I203Y+Y303R+N475E+G476T+G477R, (i.e., CspAmy2
v181);
T038N+N88H+N126Y+T129I+N134M+F153W+L171R+T180D+E187P+I203Y+G476K+G477E,
(i.e., CspAmy2 v186);
N126Y+E132H+T180D+E187P+I203Y+Y303D+G476T+G477E, (i.e., CspAmy2
v191);
N126Y+E187P+I203Y (i.e., CspAmy2-vC16A);
N126Y+I203Y+S241Q (i.e., CspAmy2-vC16B);
N126Y+T180H+E187P+I203Y (i.e., CspAmy2-vC16C);
N126Y+T180H+I203Y+S241Q (i.e., CspAmy2-vC16D);
N126Y+F153W+T180H+E187P+I203Y (i.e., CspAmy2-vC16E);
N126Y+F153W+T180H+I203Y+S241Q (i.e., CspAmy2-vC16F);
N126Y+Y150H+F153W+L171N+E187P+I203Y (i.e., CspAmy2-vC16G);
N126Y+Y150H+F153W+L171N+I203Y+S241Q (i.e., CspAmy2-vC16H);
N126Y+Y150H+F153W+L171N+T180H+E187P+I203Y (i.e.,
CspAmy2-vC16I);
N126Y+Y150H+F153W+L171N+T180H+I203Y+S241Q (i.e.,
CspAmy2-vC16J);
N126Y+F153W+T180D+I203Y+S241Q, (i.e., CspAmy2-v C18P);
N126Y+E132H+F153W+T180D+I203Y+S241Q+A277F (i.e., CspAmy2-C25F);
N126Y+E132H+F153W+Q167E+T180D+I203Y+S241Q+A277F (i.e.,
CspAmy2-C25B); and
N126Y+E132H+F153W+Q167E+T180D+I203Y+S241Q+A277F+T400K (i.e.,
CspAmy2-C25A).
[0333] All the above combinations of mutation are contemplated for
use in conjunction with the aforementioned deletions at positions
corresponding to R178, G179, T180, and/or G181. Such deletions may
be naturally occurring, as in the case of Bacillus licheniformis
.alpha.-amylase.
[0334] In addition to the aforementioned mutations, the PcuAmy1
variants may further include mutations at position T333, A335, and
Q337E (using SEQ ID NO: 3 numbering). These positions are in a
surface-exposed loop and mutations at these positions, particularly
at T333, impart protease resistance to PcuAmy1 but do not otherwise
affect performance. These mutations also appear to be fully
compatible with other mutations. A further mutation in PcuAmy1
variants is N205, which mutation changes the wild-type N residue to
D. D is the residue that typically occupies this position in
.alpha.-amylases.
[0335] Accordingly, the present .alpha.-amylases include the all
the exemplary combinations of mutations shown above in the context
of CspAmy2, as well as the following exemplary combinations (using
SEQ ID NO: 3 numbering) are shown, below:
N125Y+E186P+T333G+A335S+Q337E+G472K (i.e., PcuAmy1-v1A);
N125Y+F152W+E186P+T333G+A335S+Q337E+G472K (i.e., PcuAmy1-v6);
N125Y+F152W+E186P+T333G+A335S+Q337E+G472R+G473R (i.e., PcuAmy1-v8);
and
[0336] N125Y+F152W+E186P+N205D+T333G+A335S+Q337E+G472K (i.e.,
PcuAmy1-v16).
[0337] All the above combinations of mutations are contemplated for
use in conjunction with deletions at positions corresponding to
R177, G178, D179, and/or G180, and such deletions may be naturally
occurring, as in the case of Bacillus licheniformis
.alpha.-amylase.
[0338] The present variants can also be described in terms of BASE
numbering (i.e., SEQ ID NO: 5), for example,
N128Y+E189P+G475R (i.e., BASE-V28);
F155W+E189P+G475R (i.e., BASE-V29);
T134E+T182H+E189P+G475R (i.e., BASE-V30);
N128Y+T134E+T182H+E189P+G475R (i.e., BASE-V31);
N128Y+F155W+E189P+G475R (i.e., BASE-V32);
T134E+F155W+T182H+E189P+G475R (i.e., BASE-V33);
N128Y+T134E+F155W+T182H+E189P+G475R (i.e., BASE-V34);
N128Y+T134H+F155W+T182D+E189P+G475R (i.e., BASE-V35); and
N128Y+T134E+F155W+T182G+E189P+G457R (i.e., BASE-V36).
[0339] All the above combinations of mutations are contemplated for
use in conjunction with deletions at positions corresponding to
R180, S181, T182, and/or G183, and such deletions may be naturally
occurring, as in the case of Bacillus licheniformis
.alpha.-amylase.
[0340] Corresponding amino acid positions in other .alpha.-amylases
be identified by amino acid sequence alignment using CspAmy2 (SEQ
ID NO: 1), PcuAmy1 (SEQ ID NO: 3), or BASE (SEQ ID NO: 5), using
Clustal W with default parameters. .alpha.-amylases in which the
foregoing mutations are likely to produce a performance benefit
include those having a similar fold and/or having 60% or greater
amino acid sequence identity to any of the well-known Bacillus
amylases (e.g., from B. lichenifomis (i.e., BLA and LAT), B.
stearothermophilus (i.e., BSG), and B. amyloliquifaciens (i.e.,
P00692, BACAM, and BAA)), Carbohydrate-Active Enzymes database
(CAZy) Family 13 amylases, or any amylase that has heretofore been
referred to by the descriptive term, "Termamyl-like." Exemplary
.alpha.-amylases include but are not limited to those from Bacillus
sp. SG-1, Bacillus sp. 707, Bacillus sp. DSM12368 (i.e., A7-7),
Bacillus sp. DSM 12649 (i.e., AA560), Bacillus sp. SP722, Bacillus
megaterium (DSM90 14), and KSM AP1378.
[0341] The reader will appreciate that where an .alpha.-amylase
naturally has a mutation listed above (i.e., where the wild-type
.alpha.-amylase already comprised a residue identified as a
mutation), then that particular mutation does not apply to that
.alpha.-amylase. However, other described mutations may work in
combination with the naturally occurring residue at that
position.
[0342] In some embodiments, the present .alpha.-amylase variants
have the indicated combinations of mutations and a defined degree
of amino acid sequence homology/identity to SEQ ID NO: 1, SEQ ID
NO: 3, or SEQ ID NO: 5, for example, at least 60%, at least 65%, at
least 70%, at least 75%, at least 76%, at least 77%, at least 78%,
at least 79%, at least 80%, at least 81%, at least 82%, at least
83%, at least 84%, at least 85%, at least 86%, at least 87%, at
least 88%, at least 89%, at least 90%, at least 91%, at least 92%,
at least 93%, at least 94%, at least 95%, at least 96%, at least
97%, at least 98% or even at least 99% amino acid sequence
homology/identity.
[0343] In some embodiments, the present .alpha.-amylase variants
have the indicated combinations of mutations and are derived from a
parental amylase having a defined degree of amino acid sequence
homology/identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5,
for example, at least 60%, at least 65%, at least 70%, at least
75%, at least 76%, at least 77%, at least 78%, at least 79%, at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%
or even at least 99% amino acid sequence homology/identity.
[0344] In some embodiments, in addition to the mutations described
above, the present .alpha.-amylases further include one or more
mutations that provide a further performance benefit. Additional
mutations that were experimentally determined to provide at least
one performance advantage when combined with the aforementioned
combinatorial variants include mutations at positions corresponding
to 6, 7, 8, 11, 14, 15, 20, 21, 23, 26, 27, 28, 37, 38, 39, 40, 42,
45, 46, 48, 49, 50, 51, 52, 53, 54, 58, 61, 62, 68, 70, 71, 72, 73,
79, 80, 81, 82, 84, 85, 87, 88, 89, 92, 93, 94, 95, 96, 97, 98,
101, 108, 111, 112, 113, 114, 115, 116, 117, 118, 120, 122, 123,
124, 126, 127, 129, 130, 131, 132, 133, 134, 136, 137, 138, 140,
142, 143, 144, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,
158, 159, 165, 167, 168, 170, 171, 172, 175, 176, 177, 180, 181,
182, 187, 190, 191, 193, 199, 200, 201, 203, 206, 208, 210, 211,
212, 214, 215, 216, 219, 221, 223, 225, 226, 227, 235, 238, 239,
240, 241, 242, 243, 245, 246, 247, 248, 249, 250, 252, 253, 254,
256, 257, 258, 260, 261, 262, 266, 267, 268, 269, 270, 271, 273,
276, 277, 279, 280, 282, 284, 285, 286, 288, 296, 299, 300, 301,
302, 303, 304, 307, 308, 310, 311, 312, 313, 316, 317, 318, 320,
321, 325, 327, 335, 338, 342, 348, 349, 352, 356, 357, 360, 362,
363, 368, 369, 377, 381, 382, 383, 384, 385, 388, 390, 392, 394,
395, 396, 397, 398, 400, 401, 402, 403, 404, 405, 407, 408, 410,
414, 415, 416, 418, 419, 420, 421, 422, 423, 424, 426, 428, 429,
430, 431, 434, 435, 436, 439, 441, 442, 444, 445, 446, 447, 448,
449, 450, 451, 454, 455, 457, 460, 461, 462, 463, 464, 465, 466,
467, 469, 470, 471, 473, 474, 475, 476, 477, 479, 480, 481, 482,
483, and 484, using SEQ ID NO: 1 for numbering. Specific mutations
are T6A, T6D, T6E, T6G, T6K, T6M, T6N, T6Q, T6S, M7A, M7V, MBC,
MBF, M8I, M8L, M8Y, F11V, Y14A, Y14I, Y14Q, Y14T, Y14V, V15C, V15D,
V15I, V15N, V15T, Q20A, Q20C, Q20D, Q20H, Q20K, Q20M, Q20N, Q20R,
Q205, Q20Y, Q21F, Q21W, N23A, N23C, N23D, N23E, N23H, N23K, N23Q,
N23R, N23S, N23T, N23V, R26C, R26E, R26G, R26K, R26M, R26S, R26T,
T27A, T27C, T27D, T27E, T27F, T27H, T27I, T27K, T27L, T27M, T27N,
T27Q, T27R, T27S, T27V, T27Y, D28A, D28C, D28T, I37F, I37V, T38D,
T38N, A39L, V40A, V40C, V40D, V40G, V40H, V40I, V40K, V40M, V40P,
V40Q, V40R, V405, V40T, V40W, V40Y, T42A, T42C, T42I, T42M, T42V,
A45C, A45G, Y46F, G48A, T49I, S50A, S50C, S50E, S50G, S50K, S50M,
S50N, S50Q, S50R, S50T, S50Y, Q51C, Q51D, Q51E, Q51S, Q51V, A52C,
A52D, A52E, A52F, A52G, A52H, A52K, A52L, A52M, A52R, A52S, A52T,
A52V, A52Y, D53E, V54N, V54T, P58A, P58C, P58H, P58I, P58S, P58T,
P58V, L61M, L61V, Y62A, Y62C, Y62D, Y62F, Y62G, Y62H, Y62I, Y62K,
Y62L, Y62M, Y62N, Y62P, Y62Q, Y62R, Y62S, Y62V, N68C, N68D, N68E,
N68F, N68H, N68L, N68P, N68Q, N68R, N68S, N68V, N68W, N68Y, K70R,
G71A, G71C, G71D, G71E, G71K, G71R, G71S, T72G, T72S, V73S, T79F,
T791, T79L, T79M, T79N, T79S, T79Y, K80A, K80C, K80D, K80F, K80H,
K80I, K80M, K80N, K80Q, K80R, K80S, K80T, K80V, K80Y, G81A, G81D,
G81E, G81F, G81H, G81I, G81K, G81N, G81P, G81R, G81S, G81T, E82A,
E82D, E82M, E82Q, K84A, K84C, K84E, K84I, K84Q, K84R, K84S, K84T,
K84Y, S85A, S85C, S85D, S85E, S85G, S85H, S851, S85L, S85M, S85N,
S85Q, S85R, S85T, S85V, S85Y, V871, V87T, N88C, N88D, N88E, N88G,
N88H, N88I, N88K, N88L, N88M, N88Q, N88R, N88S, N88T, N88V, N88W,
N88Y, T89A, T89D, T89E, T89F, T89H, T891, T89K, T89L, T89M, T89N,
T89Q, T89R, T89S, T89Y, S92A, S92C, S92D, S92E, S92F, S92G, S92H,
S92L, S92M, S92N, S92Q, S92R, S92T, S92W, S92Y, N93A, N93C, N93E,
N93F, N93H, N93I, N93K, N93L, N93Q, N93S, N93T, N93Y, G94A, G94C,
G94N, I95M, Q96A, Q96E, Q96H, Q961, Q96K, Q96L, Q96M, Q96N, Q96R,
Q96V, Q96Y, V971, V97T, Y98F, Y981, Y98L, Y98V, V101C, V101T,
G108A, G108S, Y111D, Y111E, Y111L, Y111N, Y111S, Y111T, Y111V,
T112A, T112F, T112H, T112I, T112K, T112L, T112M, T112N, T112P,
T112R, T112V, T112W, E113D, E113N, E113Q, E113T, N114A, N114C,
N114D, N114E, N114F, N114G, N114H, N114I, N114L, N114P, N114Q,
N114R, N114S, N114T, N114V, N114W, N114Y, V115A, V115I, T116A,
T116C, T116D, T116G, T116H, T116I, T116K, T116N, T116P, T116Q,
T116R, T116S, A117C, A117I, A117S, A117V, V118A, V118C, V118E,
V118F, V118H, V118I, V118L, V118M, V118N, V118Q, V118R, V118S,
V118W, V118Y, V120A, V120C, V120I, V120M, V120T, P122A, P122D,
P122E, P122G, P122H, P122N, P122R, P122S, P122T, P122W, S123A,
S123C, S123G, S123K, S123L, N124A, N124D, N124F, N124G, N124L,
N124R, N124S, N124V, Y126A, Y126C, Y126D, Y126E, Y126G, Y126H,
Y126I, Y126K, Y126L, Y126M, Y126N, Y126Q, Y126R, Y126S, Y126T,
Y126V, Y126W, Q127A, Q127C, Q127D, Q127E, Q127F, Q127H, Q127I,
Q127K, Q127L, Q127M, Q127N, Q127R, Q127S, Q127T, Q127V, Q127W,
Q127Y, T129A, T129C, T129D, T129E, T129F, T129G, T129I, T129K,
T129L, T129M, T129N, T129Q, T129R, T129S, T129V, T129W, T129Y,
S130A, S130G, S130I, S130K, S130L, S130M, S130N, S130P, S130R,
S130T, S130V, S130W, G131A, G131C, G131D, G131F, G131H, G131I,
G131K, G131L, G131M, G131P, G131Q, G131V, G131W, G131Y, E132A,
E132C, E132D, E132F, E132G, E132H, E132I, E132K, E132L, E132M,
E132N, E132P, E132Q, E132R, E132V, E132W, Y133A, Y133D, Y133E,
Y133G, Y133K, Y133L, Y133M, Y133R, Y133S, Y133T, Y133W, N134A,
N134D, N134F, N134G, N134H, N134I, N134K, N134L, N134M, N134R,
N134S, N134V, N134W, N134Y, Q136A, Q136C, Q136D, Q136E, Q136F,
Q136H, Q136K, Q136L, Q136M, Q136N, Q136R, Q136S, Q136T, Q136V,
Q136W, Q136Y, A137S, A137T, A137V, W138F, W138Y, G140A, G140M,
N142F, N142K, N142L, N142M, N142P, N142V, N142W, N142Y, F143H,
F143Y, P144C, P144D, P144E, P144G, P144H, P1441, P144K, P144L,
P144M, P144N, P144Q, P144S, P144T, P144Y, G147A, G147C, G147H,
G147K, G147L, G147M, G147N, G147Q, G147R, T148A, T148D, T148E,
T148F, T1481, T148K, T148L, T148R, T148S, T148V, T148W, T149A,
T149C, T149D, T149E, T149F, T149H, T149K, T149L, T149M, T149N,
T149Q, T149V, T149W, T149Y, Y150H, S151A, S151E, S151F, S151H,
S1511, S151K, S151L, S151M, S151Q, S151R, S151V, N152A, N152C,
N152D, N152E, N152G, N152H, N152K, N152M, N152P, N152Q, N152R,
N152S, N152T, W153F, W153H, W153Q, W153R, W153T, W153Y, K154A,
K154C, K154D, K154E, K154G, K1541, K154L, K154M, K154N, K154R,
K154T, K154Y, W155P, Q156A, Q156D, Q156E, Q156F, Q156G, Q156H,
Q1561, Q156L, Q156M, Q156N, Q156R, Q156S, Q156Y, F158C, F158D,
F158K, F158L, F158M, F158N, F158P, F158Q, F158R, F158S, F158V,
H159M, H159Y, W165C, W165D, W165E, W165F, W165H, W1651, W165K,
W165L, W165M, W165Q, W165R, W165T, W165Y, Q167A, Q167D, Q167E,
Q167G, Q167H, Q167K, Q167M, Q167N, Q167P, Q167S, Q167T, Q167V,
S168A, S168D, S168F, S168H, S1681, S168K, S168M, S168N, S168Q,
S168R, S168T, S168V, S168W, S168Y, S170A, S170C, S170D, S170E,
S170F, S170L, S170M, S170N, S170Q, S170R, S170T, L171A, L171C,
L171F, L171G, L171H, L171I, L171N, L171Q, L171R, L171T, L171V,
L171W, L171Y, S172A, S172D, S172H, S172R, S172T, F175M, F175Y,
K176I, K176T, F177L, F177V, F177W, D180A, D180C, D180F, D180G,
D180H, D180I, D180L, D180M, D180N, D180Q, D180R, D180S, D180T,
D180V, D180W, D180Y, G181A, G181C, G181D, G181E, G181F, G181H,
G181K, G181L, G181M, G181N, G181Q, G181R, G181S, G181T, G181V,
G181Y, K182A, K182P, E187D, E187I, E187K, E187M, E187N, E187P,
E187R, E187S, E187T, E187V, E187Y, S190A, S190C, S190D, S190F,
S190L, S190N, S190P, S190Q, E191A, E191C, E191F, E191G, E191H,
E191I, E191K, E191N, E191Q, E191R, E191S, E191T, E191W, E191Y,
G193A, G193C, G193D, G193E, G193F, G193H, G193K, G193L, G193M,
G193N, G193Q, G193R, G193S, G193T, G193V, G193W, M199L, Y200F,
A201M, Y2031, Y203L, Y203V, D206A, D206E, D206G, D206H, D206M,
D206N, D206Q, D206R, D206S, D206T, P208A, P208E, P208F, P2081,
P208K, P208L, P208T, P208V, P208Y, V210A, V210E, V210H, V210K,
V210N, V210Q, V210R, V210S, V210T, V211A, V211E, V211H, V2111,
V211Q, V211R, N212E, N212F, N212G, N212L, N212M, N212R, N212V,
M2141, M214L, K215C, K215E, K215F, K215M, K215N, K215R, K215Y,
K216F, V2191, V219T, Y221F, Y2211, Y221L, N223C, N223E, N223I,
N223K, N223Q, N223R, N223S, N223T, N223V, N223W, N223Y, V225A,
V2251, V225L, V225M, G226D, G226M, G226Q, G226R, G226S, L227F,
L227I, L227W, L227Y, V235A, I238A, I238L, I238M, K239D, K239E,
K239P, K239Q, K239R, K239S, K239T, F240K, F240L, F240M, F240Q,
F240R, Q241A, Q241C, Q241D, Q241E, Q241G, Q241H, Q241K, Q241L,
Q241M, Q241N, Q241P, Q241R, Q241S, Q241T, Q241V, Q241W, Q241Y,
F242V, L243C, L243Y, D245A, D245C, D245E, D245G, D245L, D245M,
D245N, W246F, V247I, V247L, D248E, D248H, D248N, D248T, D248V,
N249A, N249E, N249G, N249H, N249Q, N249Y, A250M, A250S, A250V,
A252C, A252D, A252E, A252G, A252H, A252I, A252K, A252L, A252M,
A252N, A252Q, A252S, A252V, A252W, A252Y, A253E, A253I, A253K,
A253L, A253M, A253Q, A253S, A253T, A253V, A253Y, T254F, T254K,
T254S, K256A, K256M, K256N, K256S, E257Q, E257S, M258L, T260A,
T260C, T260S, T260V, V261I, V261W, G262A, Q266A, Q266D, Q266E,
Q266H, Q266I, Q266M, Q266N, Q266S, Q266T, Q266V, Q266Y, N267H,
N267I, N267Q, N267R, N267S, N267T, N267V, N267Y, D268G, D268N,
L269C, L269D, L269I, L269K, L269Q, L269S, L269T, L269Y, G270A,
G270D, G270E, G270F, G270H, G270I, G270L, G270M, G270Q, G270T,
G270V, G270Y, A271C, A271D, A271E, A271H, A271K, A271M, A271Q,
A271R, A271S, A271T, A271V, A271Y, N273C, N273G, N273H, N273I,
N273K, N273R, L276I, L276M, A277C, A277D, A277E, A277F, A277G,
A277I, A277K, A277L, A277M, A277N, A277Q, A277R, A277S, A277T,
A277V, A277W, A277Y, V279C, V279T, N280A, N282S, N282T, S284T,
S284Y, L285A, L285C, L285I, L285V, F286M, A288C, A296C, A296D,
A296E, A296F, A296G, A296H, A296I, A296L, A296M, A296N, A296Q,
A296R, A296S, A296V, A296W, A296Y, T299A, T299D, T299E, T299F,
T299G, T299K, T299L, T299M, T299R, T299S, T299V, T299W, G300A,
G300C, G300D, G300E, G300F, G300H, G300K, G300M, G300Q, G300R,
G300V, G300W, G301A, G301C, G301D, G301E, G301F, G301H, G301K,
G301L, G301M, G301Q, G301R, G301S, G301T, G301V, G301W, G301Y,
G302S, Y303A, Y303C, Y303D, Y303E, Y303F, Y303G, Y303H, Y3031,
Y303K, Y303L, Y303M, Y303N, Y303Q, Y303R, Y303S, Y303T, Y303V,
Y303W, Y304F, Y304K, Y304W, R307A, R307C, R307E, R307G, R307H,
R307K, R307M, R307N, R307Q, R307S, R307T, N308C, N308D, N308E,
N308G, N308L, N308M, N308T, N308V, L310A, L310C, L310D, L310E,
L310H, L310I, L310M, L310P, L310W, L310Y, N311C, N311E, N311G,
N311H, N311K, N311Q, N311R, N311S, N311V, N311W, N311Y, N312D,
N312F, N312G, N312H, N312K, N312Q, N312R, T313A, T313S, A316D,
A316E, A316G, A316H, A316K, A316Q, A316R, A316Y, S317C, S317D,
S317G, S317H, S317K, S317L, S317N, S317Q, S317R, S317T, S317W,
S317Y, N318A, N318C, N318F, N318G, N318I, N318K, N318L, N318M,
N318Q, N318R, N318S, N318T, N318V, N318W, T320A, T320C, T320D,
T320E, T320G, T320H, T3201, T320K, T320N, T320P, T320Q, T320R,
T320V, T320W, T320Y, K321C, K321F, K321H, K321N, K321S, K321Y,
L325A, L325C, L325F, L3251, L325M, L325Q, L325V, E327D, E327L,
Q335C, Q335E, E338A, E338D, E338F, E338G, E338H, E3381, E338K,
E338P, E338Q, E338R, E338T, E338V, E338Y, Q342A, Q342C, Q342G,
Q342L, Q342M, Q342R, Q342S, Q342T, Q342V, Q342W, L348A, L348C,
L348H, L3481, L348M, L348Q, L348S, L348T, A349G, A349R, F3521,
F352L, F352M, F352T, F352V, R356Q, S357A, S357C, S357D, S357E,
S357F, S357H, S3571, S357K, S357L, S357N, S357Q, S357T, S357V,
S357W, S357Y, Y360F, Y3601, Y360L, Y360M, Y360V, S362A, S362C,
S362E, S3621, S362T, S362V, V3631, V363L, M368F, M3681, M368L,
M368Y, Y369A, Y369E, Y3691, Y369L, Y369N, Y369V, R377A, R377C,
R377D, R377E, R377F, R377G, R377H, R3771, R377K, R377L, R377N,
R377Q, R377S, R377T, R377V, R377W, R377Y, A381C, A381E, A381G,
A381H, A381K, A381L, A381M, A381N, A381P, A381Q, A381V, A381W,
A381Y, L382F, L382H, L382Q, L382S, K383A, K383C, K383D, K383E,
K383H, K3831, K383L, K383M, K383N, K383Q, K383R, K383S, K383W,
K383Y, S384A, S384C, S384D, S384F, S384G, S384H, S3841, S384L,
S384N, S384R, S384V, S384Y, K385A, K385D, K385E, K385F, K385G,
K385H, K385L, K385M, K385Q, K385R, K385T, K385V, K385Y, P388A,
P388C, P388D, P388I, P388L, P388N, P388R, P388S, P388T, P388V,
L390M, L390V, A392C, A392S, K394A, K394C, K394E, K394F, K394G,
K394H, K3941, K394L, K394Q, K394R, K394S, K394T, K394V, K394W,
K394Y, D395A, D395E, D395N, D395S, D395T, Y396A, Y396D, Y396F,
Y396K, Y396M, Y396N, Y396Q, Y396T, Y396V, Y396W, A397D, A397E,
A397H, A397K, A397M, A397N, A397Q, A397V, Y398A, Y398C, Y398F,
Y398H, Y3981, Y398L, Y398W, T400A, T400C, T400D, T400F, T400G,
T400I, T400K, T400L, T400M, T400N, T400Q, T400R, T400W, T400Y,
Q401A, Q401C, Q401F, Q401H, Q401I, Q401L, Q401M, Q401N, R402C,
R402D, R402F, R402K, R402L, R402M, R402N, R402Q, R402S, R402W,
D403E, D403N, D403S, Y404E, Y404G, Y404K, Y404M, Y404N, Y404R,
Y404W, I405C, I405F, I405L, I405M, I405V, N407A, N407C, N407D,
N407E, N407G, N407K, N407M, N407Q, N407R, P408A, P408C, P408D,
P4081, P408K, P408L, P408M, P408N, P408Q, P408V, P408W, V410A,
V410C, V410D, V410E, V410F, V410H, V4101, V410L, V410M, V410N,
V410Q, V410S, V410T, T414A, T4141, T414S, T414V, R415M, E416C,
E416D, E416F, E416H, E4161, E416K, E416L, E416M, E416N, E416Q,
E416R, E416T, E416V, E416W, E416Y, D418A, D418E, D418G, D418H,
D418I, D418K, D418L, D418M, D418N, D418Q, D418S, D418T, D418V,
D418W, S419A, S419C, S419E, S419G, S419L, S419M, S419N, S419R,
S419V, S419W, S419Y, T420A, T420C, T420D, T420E, T420G, T420H,
T420I, T420K, T420M, T420P, T420S, T420V, T420W, T420Y, K421A,
K421D, K421E, K421H, K421I, K421L, K421M, K421N, K421P, K421Q,
K421R, K421T, K421V, K421W, K421Y, A422C, A422D, A422E, A422F,
A422G, A422I, A422L, A422N, A422P, A422Q, A422R, A422S, A422Y,
K423A, K423D, K423E, K423F, K423H, K423I, K423L, K423M, K423N,
K423Q, K423R, K423S, K423T, K423V, K423W, K423Y, S424A, S424C,
S424G, S424K, S424N, S424Q, S424R, S424T, L426S, L426T, L426V,
T428G, T428V, V429A, V429C, V429I, V429L, I430C, I430G, I430L,
I430M, I430Q, I430V, T431A, T431C, T431S, P434A, P434C, P434D,
P434E, P434F, P434H, P434I, P434K, P434L, P434M, P434N, P434Q,
P434R, P434S, P434V, P434Y, G435A, G435C, G435D, G435E, G435F,
G435H, G435I, G435K, G435M, G435N, G435P, G435Q, G435R, G435S,
G435T, G435W, G436F, G436I, G436M, G436N, G436Q, G436S, G436V,
R439A, R439D, R439G, R439H, R439K, R439M, R439N, R439P, R439Q,
R439S, R439V, R439W, R439Y, Y441A, Y441C, Y441D, Y441F, Y441G,
Y441H, Y441K, Y441L, Y441M, Y441N, Y441P, Y441R, Y441S, Y441T,
Y441W, V442A, V442C, V442I, V442T, T444C, T444D, T444E, T444F,
T444G, T444H, T444I, T444K, T444L, T444M, T444N, T444P, T444R,
T444S, T444W, S445A, S445C, S445E, S445G, S445H, S445K, S445L,
S445M, S445N, S445T, S445V, N446A, N446C, N446H, N446K, A447C,
A447D, A447F, A447H, A447L, A447M, A447N, A447Q, A447R, A447S,
A447Y, G448A, G448C, G448D, G448E, G448H, G448K, G448L, G448M,
G448N, G448Q, G448R, G448S, G448T, G448W, E449D, E449H, E449K,
E449T, I450A, I450C, I450D, I450E, I450G, I450K, I450L, I450M,
I450N, I450Q, I450S, I450T, I450W, I450Y, W451Y, L454A, L454I,
L454K, L454M, L454W, T455A, T455I, T455L, T455S, N457H, N457K,
N457R, N457T, N457V, N457Y, D460A, D460E, D460G, D460M, D460N,
D460Q, D460S, D460V, K461C, K461H, K461L, K461M, K461N, K461Q,
K461T, K461Y, I462A, I462L, I462M, I462Q, I462T, I462V, T463D,
T463E, T463H, T463P, T463Q, T463R, T463V, T463Y, I464P, I464T,
G465A, G465C, G465D, G465E, G465K, G465L, G465M, G465N, G465Q,
G465W, G465Y, S466A, S466C, S466D, S466G, S466H, S466K, S466L,
S466M, S466N, S466T, S466W, S466Y, D467E, D467G, D467L, Y469A,
Y469D, Y469E, Y469I, Y469M, Y469N, Y469R, Y469S, Y469T, Y469V,
Y469W, A470S, A470V, T471A, T471C, T471E, T471G, T471H, T471I,
T471L, T471M, T471N, T471S, T471V, T471W, P473C, P473D, P473E,
P473G, P473I, P473K, P473L, P473R, P473T, P473W, V474A, V474C,
V474L, V474S, N475A, N475C, N475E, N475F, N475H, N475K, N475L,
N475M, N475P, N475Q, N475R, N475S, N475T, N475V, G476A, G476D,
G476E, G476F, G476H, G4761, G476L, G476M, G476N, G476P, G476Q,
G476R, G476S, G476T, G476V, G476W, G476Y, G477A, G477D, G477F,
G477H, G4771, G477K, G477L, G477M, G477Q, G477S, G477T, G477V,
G477W, G477Y, V479C, V479D, V479E, V479F, V479H, V4791, V479N,
V479P, V479Y, S480A, S480C, S480H, V481A, V481C, V481N, W482Y,
V483A, V483G, V4831, V483K, V483L, V483M, V483R, V483Y, Q484A,
Q484C, Q484F, Q484G, Q484H, Q484K, Q484L, Q484M, Q484P, Q484R,
Q484T, and Q484Y.
[0345] Furthermore, the present amylases may include any number of
conservative amino acid substitutions. Exemplary conservative amino
acid substitutions are listed in Table 1 Table 1. Conservative
amino acid substitutions
TABLE-US-00007 For Amino Acid Code Replace with any of Alanine A
D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys,
homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine
N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp,
D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met,
D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp,
D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln
Glycine G Ala, D-Ala, Pro, D-Pro, b-Ala, Acp Isoleucine I D-Ile,
Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val,
Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg,
D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met,
S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe,
Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or
5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro,
L-I-thioazolidine-4-carboxylic acid, D-or
L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,
allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T
D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val,
D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V
D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met
[0346] The reader will appreciate that some of the above mentioned
conservative mutations can be produced by genetic manipulation,
while others are produced by introducing synthetic amino acids into
a polypeptide by genetic or other means.
[0347] The present amylase may be "precursor," "immature," or
"full-length," in which case they include a signal sequence, or
"mature," in which case they lack a signal sequence. Mature forms
of the polypeptides are generally the most useful. Unless otherwise
noted, the amino acid residue numbering used herein refers to the
mature forms of the respective amylase polypeptides. The present
amylase polypeptides may also be truncated to remove the N or
C-termini, so long as the resulting polypeptides retain amylase
activity.
[0348] The present amylase may be a "chimeric" or "hybrid"
polypeptide, in that it includes at least a portion of a first
amylase polypeptide, and at least a portion of a second amylase
polypeptide (such chimeric amylases have recently been
"rediscovered" as domain-swap amylases). The present amylases may
further include heterologous signal sequence, an epitope to allow
tracking or purification, or the like. Exemplary heterologous
signal sequences are from B. licheniformis amylase (LAT), B.
subtilis (AmyE or AprE), and Streptomyces CelA.
2.5. Nucleotides Encoding Variant Amylase Polypeptides
[0349] In another aspect, nucleic acids encoding a variant amylase
polypeptide are provided. The nucleic acid may encode a particular
amylase polypeptide, or an amylase having a specified degree of
amino acid sequence identity to the particular amylase.
[0350] In one example, the nucleic acid encodes an amylase having
at least 60%, at least 65%, at least 70%, at least 75%, at least
76%, at least 77%, at least 78%, at least 79%, at least 80%, at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98% or even at
least 99% homology/identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ
ID NO: 5 (excluding the portion of the nucleic acid that encodes
the signal sequence). It will be appreciated that due to the
degeneracy of the genetic code, a plurality of nucleic acids may
encode the same polypeptide.
[0351] In another example, the nucleic acid hybridizes under
stringent or very stringent conditions to a nucleic acid encoding
(or complementary to a nucleic acid encoding) an amylase having at
least 60%, at least 65%, at least 70%, at least 75%, at least 76%,
at least 77%, at least 78%, at least 79%, at least 80%, at least
81%, at least 82%, at least 83%, at least 84%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98% or even at least 99%
homology/identity to SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5
(excluding the portion of the nucleic acid that encodes the signal
sequence).
[0352] In some embodiments, the nucleic acid hybridizes under
stringent or very stringent conditions to the nucleic acid of SEQ
ID NO: 7, SEQ ID NO: 33, or SEQ ID NO: 38, or to a nucleic acid
complementary to these nucleic acids.
[0353] Nucleic acids may encode a "full-length" ("fl" or "FL")
amylase, which includes a signal sequence, only the mature form of
an amylase, which lacks the signal sequence, or a truncated form of
an amylase, which lacks the N or C-terminus of the mature form.
[0354] A nucleic acid that encodes a .alpha.-amylase can be
operably linked to various promoters and regulators in a vector
suitable for expressing the .alpha.-amylase in host cells.
Exemplary promoters are from B. licheniformis amylase (LAT), B.
subtilis (AmyE or AprE), and Streptomyces CelA. Such a nucleic acid
can also be linked to other coding sequences, e.g., to encode a
chimeric polypeptide.
3. Production of Variant Amylases
[0355] The present variant amylases can be produced in host cells,
for example, by secretion or intracellular expression. A cultured
cell material (e.g., a whole-cell broth) comprising a variant
amylase can be obtained following secretion of the variant amylase
into the cell medium. Optionally, the variant amylase can be
isolated from the host cells, or even isolated from the cell broth,
depending on the desired purity of the final variant amylase. A
gene encoding a variant amylase can be cloned and expressed
according to methods well known in the art. Suitable host cells
include bacterial, fungal (including yeast and filamentous fungi),
and plant cells (including algae). Particularly useful host cells
include Aspergillus niger, Aspergillus oryzae or Trichoderma
reesei. Other host cells include bacterial cells, e.g., Bacillus
subtilis or B. licheniformis, as well as Streptomyces.
[0356] The host cell further may express a nucleic acid encoding a
homologous or heterologous glucoamylase, i.e., a glucoamylase that
is not the same species as the host cell, or one or more other
enzymes. The glucoamylase may be a variant glucoamylase, such as
one of the glucoamylase variants disclosed in U.S. Pat. No.
8,058,033 (Danisco US Inc.), for example. Additionally, the host
may express one or more accessory enzymes, proteins, peptides.
These may benefit liquefaction, saccharification, fermentation,
SSF, etc processes. Furthermore, the host cell may produce
biochemicals in addition to enzymes used to digest the various
feedstock(s). Such host cells may be useful for fermentation or
simultaneous saccharification and fermentation processes to reduce
or eliminate the need to add enzymes.
3.1. Vectors
[0357] A DNA construct comprising a nucleic acid encoding variant
amylases can be constructed to be expressed in a host cell.
Representative nucleic acids that encode variant amylases include
SEQ ID NO: 4. Because of the well-known degeneracy in the genetic
code, variant polynucleotides that encode an identical amino acid
sequence can be designed and made with routine skill. It is also
well-known in the art to optimize codon use for a particular host
cell. Nucleic acids encoding variant amylases can be incorporated
into a vector. Vectors can be transferred to a host cell using
well-known transformation techniques, such as those disclosed
below.
[0358] The vector may be any vector that can be transformed into
and replicated within a host cell. For example, a vector comprising
a nucleic acid encoding a variant amylase can be transformed and
replicated in a bacterial host cell as a means of propagating and
amplifying the vector. The vector also may be transformed into an
expression host, so that the encoding nucleic acids can be
expressed as a functional amylase. Host cells that serve as
expression hosts can include filamentous fungi, for example. The
Fungal Genetics Stock Center (FGSC) Catalogue of Strains lists
suitable vectors for expression in fungal host cells. See FGSC,
Catalogue of Strains, University of Missouri, at www.fgsc.net (last
modified Jan. 17, 2007). A representative vector is pJG153, a
promoterless Cre expression vector that can be replicated in a
bacterial host. See Harrison et al. (June 2011) Applied Environ.
Microbiol. 77: 3916-22. pJG153 can be modified with routine skill
to comprise and express a nucleic acid encoding an amylase
variant.
[0359] A nucleic acid encoding a variant amylase can be operably
linked to a suitable promoter, which allows transcription in the
host cell. The promoter may be any DNA sequence that shows
transcriptional activity in the host cell of choice and may be
derived from genes encoding proteins either homologous or
heterologous to the host cell. Exemplary promoters for directing
the transcription of the DNA sequence encoding a variant amylase,
especially in a bacterial host, are the promoter of the lac operon
of E. coli, the Streptomyces coelicolor agarase gene dagA or celA
promoters, the promoters of the Bacillus licheniformis
.alpha.-amylase gene (amyL), the promoters of the Bacillus
stearothermophilus maltogenic amylase gene (amyM), the promoters of
the Bacillus amyloliquefaciens .alpha.-amylase (amyQ), the
promoters of the Bacillus subtilis xylA and xylB genes etc. For
transcription in a fungal host, examples of useful promoters are
those derived from the gene encoding Aspergillus oryzae TAKA
amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger
neutral .alpha.-amylase, A. niger acid stable .alpha.-amylase, A.
niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline
protease, A. oryzae triose phosphate isomerase, or A. nidulans
acetamidase. When a gene encoding an amylase is expressed in a
bacterial species such as E. coli, a suitable promoter can be
selected, for example, from a bacteriophage promoter including a T7
promoter and a phage lambda promoter. Examples of suitable
promoters for the expression in a yeast species include but are not
limited to the Gal 1 and Gal 10 promoters of Saccharomyces
cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters. cbh1 is
an endogenous, inducible promoter from T. reesei. See Liu et al.
(2008) "Improved heterologous gene expression in Trichoderma reesei
by cellobiohydrolase I gene (cbh1) promoter optimization," Acta
Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.
[0360] The coding sequence can be operably linked to a signal
sequence. The DNA encoding the signal sequence may be the DNA
sequence naturally associated with the amylase gene to be expressed
or from a different Genus or species. A signal sequence and a
promoter sequence comprising a DNA construct or vector can be
introduced into a fungal host cell and can be derived from the same
source. For example, the signal sequence is the cbh1 signal
sequence that is operably linked to a cbh1 promoter.
[0361] An expression vector may also comprise a suitable
transcription terminator and, in eukaryotes, polyadenylation
sequences operably linked to the DNA sequence encoding a variant
amylase. Termination and polyadenylation sequences may suitably be
derived from the same sources as the promoter.
[0362] The vector may further comprise a DNA sequence enabling the
vector to replicate in the host cell. Examples of such sequences
are the origins of replication of plasmids pUC19, pACYC177, pUB110,
pE194, pAMB1, and pIJ702.
[0363] The vector may also comprise a selectable marker, e.g., a
gene the product of which complements a defect in the isolated host
cell, such as the dal genes from B. subtilis or B. licheniformis,
or a gene that confers antibiotic resistance such as, e.g.,
ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
Furthermore, the vector may comprise Aspergillus selection markers
such as amdS, argB, niaD and xxsC, a marker giving rise to
hygromycin resistance, or the selection may be accomplished by
co-transformation, such as known in the art. See e.g.,
International PCT Application WO 91/17243.
[0364] Intracellular expression may be advantageous in some
respects, e.g., when using certain bacteria or fungi as host cells
to produce large amounts of amylase for subsequent enrichment or
purification. Extracellular secretion of amylase into the culture
medium can also be used to make a cultured cell material comprising
the isolated amylase.
[0365] The expression vector typically includes the components of a
cloning vector, such as, for example, an element that permits
autonomous replication of the vector in the selected host organism
and one or more phenotypically detectable markers for selection
purposes. The expression vector normally comprises control
nucleotide sequences such as a promoter, operator, ribosome binding
site, translation initiation signal and optionally, a repressor
gene or one or more activator genes. Additionally, the expression
vector may comprise a sequence coding for an amino acid sequence
capable of targeting the amylase to a host cell organelle such as a
peroxisome, or to a particular host cell compartment. Such a
targeting sequence includes but is not limited to the sequence,
SKL. For expression under the direction of control sequences, the
nucleic acid sequence of the amylase is operably linked to the
control sequences in proper manner with respect to expression.
[0366] The procedures used to ligate the DNA construct encoding an
amylase, the promoter, terminator and other elements, respectively,
and to insert them into suitable vectors containing the information
necessary for replication, are well known to persons skilled in the
art (see, e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORY
MANUAL, 2.sup.nd ed., Cold Spring Harbor, 1989, and 3.sup.rd ed.,
2001).
3.2. Transformation and Culture of Host Cells
[0367] An isolated cell, either comprising a DNA construct or an
expression vector, is advantageously used as a host cell in the
recombinant production of an amylase. The cell may be transformed
with the DNA construct encoding the enzyme, conveniently by
integrating the DNA construct (in one or more copies) in the host
chromosome. This integration is generally considered to be an
advantage, as the DNA sequence is more likely to be stably
maintained in the cell. Integration of the DNA constructs into the
host chromosome may be performed according to conventional methods,
e.g., by homologous or heterologous recombination. Alternatively,
the cell may be transformed with an expression vector as described
above in connection with the different types of host cells.
[0368] Examples of suitable bacterial host organisms are Gram
positive bacterial species such as Bacillaceae including Bacillus
subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis,
Geobacillus (formerly Bacillus) stearothermophilus, Bacillus
alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans,
Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis;
Streptomyces species such as Streptomyces murinus; lactic acid
bacterial species including Lactococcus sp. such as Lactococcus
lactis; Lactobacillus sp. including Lactobacillus reuteri;
Leuconostoc sp.; Pediococcus sp.; and Streptococcus sp.
Alternatively, strains of a Gram negative bacterial species
belonging to Enterobacteriaceae including E. coli, or to
Pseudomonadaceae can be selected as the host organism.
[0369] A suitable yeast host organism can be selected from the
biotechnologically relevant yeasts species such as but not limited
to yeast species such as Pichia sp., Hansenula sp., or
Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species
of Saccharomyces, including Saccharomyces cerevisiae or a species
belonging to Schizosaccharomyces such as, for example, S. pombe. A
strain of the methylotrophic yeast species, Pichia pastoris, can be
used as the host organism. Alternatively, the host organism can be
a Hansenula species. Suitable host organisms among filamentous
fungi include species of Aspergillus, e.g., Aspergillus niger,
Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or
Aspergillus nidulans. Alternatively, strains of a Fusarium sp.,
e.g., Fusarium oxysporum or of a Rhizomucor sp. such as Rhizomucor
miehei can be used as the host organism. Other suitable strains
include Thermomyces and Mucor sp. In addition, Trichoderma sp. can
be used as a host. A suitable procedure for transformation of
Aspergillus host cells includes, for example, that described in
EP238023. An amylase expressed by a fungal host cell can be
glycosylated, i.e., will comprise a glycosyl moiety. The
glycosylation pattern can be the same or different as present in
the wild-type amylase. The type and/or degree of glycosylation may
impart changes in enzymatic and/or biochemical properties.
[0370] It is advantageous to delete genes from expression hosts,
where the gene deficiency can be cured by the transformed
expression vector. Known methods may be used to obtain a fungal
host cell having one or more inactivated genes. Gene inactivation
may be accomplished by complete or partial deletion, by insertional
inactivation or by any other means that renders a gene
nonfunctional for its intended purpose, such that the gene is
prevented from expression of a functional protein. Any gene from a
Trichoderma sp. or other filamentous fungal host that has been
cloned can be deleted, for example, cbh1, cbh2, egl1, and egl2
genes. Gene deletion may be accomplished by inserting a form of the
desired gene to be inactivated into a plasmid by methods known in
the art.
[0371] Introduction of a DNA construct or vector into a host cell
includes techniques such as transformation; electroporation;
nuclear microinjection; transduction; transfection, e.g.,
lipofection mediated and DEAE-Dextrin mediated transfection;
incubation with calcium phosphate DNA precipitate; high velocity
bombardment with DNA-coated microprojectiles; and protoplast
fusion. General transformation techniques are known in the art.
See, e.g., Sambrook et al. (2001), supra. The expression of
heterologous protein in Trichoderma is described, for example, in
U.S. Pat. No. 6,022,725. Reference is also made to Cao et al.
(2000) Science 9:991-1001 for transformation of Aspergillus
strains. Genetically stable transformants can be constructed with
vector systems whereby the nucleic acid encoding an amylase is
stably integrated into a host cell chromosome. Transformants are
then selected and purified by known techniques.
[0372] The preparation of Trichoderma sp. for transformation, for
example, may involve the preparation of protoplasts from fungal
mycelia. See Campbell et al. (1989) Curr. Genet. 16: 53-56. The
mycelia can be obtained from germinated vegetative spores. The
mycelia are treated with an enzyme that digests the cell wall,
resulting in protoplasts. The protoplasts are protected by the
presence of an osmotic stabilizer in the suspending medium. These
stabilizers include sorbitol, mannitol, potassium chloride,
magnesium sulfate, and the like. Usually the concentration of these
stabilizers varies between 0.8 M and 1.2 M, e.g., a 1.2 M solution
of sorbitol can be used in the suspension medium.
[0373] Uptake of DNA into the host Trichoderma sp. strain depends
upon the calcium ion concentration. Generally, between about 10-50
mM CaCl2 is used in an uptake solution. Additional suitable
compounds include a buffering system, such as TE buffer (10 mM
Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 and polyethylene
glycol. The polyethylene glycol is believed to fuse the cell
membranes, thus permitting the contents of the medium to be
delivered into the cytoplasm of the Trichoderma sp. strain. This
fusion frequently leaves multiple copies of the plasmid DNA
integrated into the host chromosome.
[0374] Usually transformation of Trichoderma sp. uses protoplasts
or cells that have been subjected to a permeability treatment,
typically at a density of 10.sup.5 to 10.sup.7/mL, particularly
2.times.10.sup.6/mL. A volume of 100 .mu.L of these protoplasts or
cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM
CaCl.sub.2)) may be mixed with the desired DNA. Generally, a high
concentration of PEG is added to the uptake solution. From 0.1 to 1
volume of 25% PEG 4000 can be added to the protoplast suspension;
however, it is useful to add about 0.25 volumes to the protoplast
suspension. Additives, such as dimethyl sulfoxide, heparin,
spermidine, potassium chloride and the like, may also be added to
the uptake solution to facilitate transformation. Similar
procedures are available for other fungal host cells. See, e.g.,
U.S. Pat. No. 6,022,725.
3.3. Expression
[0375] A method of producing an amylase may comprise cultivating a
host cell as described above under conditions conducive to the
production of the enzyme and recovering the enzyme from the cells
and/or culture medium.
[0376] The medium used to cultivate the cells may be any
conventional medium suitable for growing the host cell in question
and obtaining expression of an amylase. Suitable media and media
components are available from commercial suppliers or may be
prepared according to published recipes (e.g., as described in
catalogues of the American Type Culture Collection).
[0377] An enzyme secreted from the host cells can be used in a
whole broth preparation. In the present methods, the preparation of
a spent whole fermentation broth of a recombinant microorganism can
be achieved using any cultivation method known in the art resulting
in the expression of an .alpha.-amylase. Fermentation may,
therefore, be understood as comprising shake flask cultivation,
small- or large-scale fermentation (including continuous, batch,
fed-batch, or solid state fermentations) in laboratory or
industrial fermenters performed in a suitable medium and under
conditions allowing the amylase to be expressed or isolated. The
term "spent whole fermentation broth" is defined herein as
unfractionated contents of fermentation material that includes
culture medium, extracellular proteins (e.g., enzymes), and
cellular biomass. It is understood that the term "spent whole
fermentation broth" also encompasses cellular biomass that has been
lysed or permeabilized using methods well known in the art.
[0378] An enzyme secreted from the host cells may conveniently be
recovered from the culture medium by well-known procedures,
including separating the cells from the medium by centrifugation or
filtration, and precipitating proteinaceous components of the
medium by means of a salt such as ammonium sulfate, followed by the
use of chromatographic procedures such as ion exchange
chromatography, affinity chromatography, or the like.
[0379] The polynucleotide encoding an amylase in a vector can be
operably linked to a control sequence that is capable of providing
for the expression of the coding sequence by the host cell, i.e.
the vector is an expression vector. The control sequences may be
modified, for example by the addition of further transcriptional
regulatory elements to make the level of transcription directed by
the control sequences more responsive to transcriptional
modulators. The control sequences may in particular comprise
promoters.
[0380] Host cells may be cultured under suitable conditions that
allow expression of an amylase. Expression of the enzymes may be
constitutive such that they are continually produced, or inducible,
requiring a stimulus to initiate expression. In the case of
inducible expression, protein production can be initiated when
required by, for example, addition of an inducer substance to the
culture medium, for example dexamethasone or IPTG or Sophorose.
Polypeptides can also be produced recombinantly in an in vitro
cell-free system, such as the TNT.TM. (Promega) rabbit reticulocyte
system.
[0381] An expression host also can be cultured in the appropriate
medium for the host, under aerobic conditions. Shaking or a
combination of agitation and aeration can be provided, with
production occurring at the appropriate temperature for that host,
e.g., from about 25.degree. C. to about 75.degree. C. (e.g.,
30.degree. C. to 45.degree. C.), depending on the needs of the host
and production of the desired variant amylase. Culturing can occur
from about 12 to about 100 hours or greater (and any hour value
there between, e.g., from 24 to 72 hours). Typically, the culture
broth is at a pH of about 4.0 to about 8.0, again depending on the
culture conditions needed for the host relative to production of an
amylase.
3.4. Identification of Amylase Activity
[0382] To evaluate the expression of an amylase in a host cell,
assays can measure the expressed protein, corresponding mRNA, or
.alpha.-amylase activity. For example, suitable assays include
Northern blotting, reverse transcriptase polymerase chain reaction,
and in situ hybridization, using an appropriately labeled
hybridizing probe. Suitable assays also include measuring amylase
activity in a sample, for example, by assays directly measuring
reducing sugars such as glucose in the culture media. For example,
glucose concentration may be determined using glucose reagent kit
No. 15-UV (Sigma Chemical Co.) or an instrument, such as Technicon
Autoanalyzer. .alpha.-amylase activity also may be measured by any
known method, such as the PAHBAH or ABTS assays, described
below.
3.5. Methods for Enriching and Purifying Variants Amylases
[0383] Fermentation, separation, and concentration techniques are
well known in the art and conventional methods can be used in order
to prepare a concentrated a variant .alpha.-amylase
polypeptide-containing solution.
[0384] After fermentation, a fermentation broth is obtained, the
microbial cells and various suspended solids, including residual
raw fermentation materials, are removed by conventional separation
techniques in order to obtain an amylase solution. Filtration,
centrifugation, microfiltration, rotary vacuum drum filtration,
ultrafiltration, centrifugation followed by ultra-filtration,
extraction, or chromatography, or the like, are generally used.
[0385] It is desirable to concentrate a variant .alpha.-amylase
polypeptide-containing solution in order to optimize recovery. Use
of unconcentrated solutions requires increased incubation time in
order to collect the enriched or purified enzyme precipitate.
[0386] The enzyme containing solution is concentrated using
conventional concentration techniques until the desired enzyme
level is obtained. Concentration of the enzyme containing solution
may be achieved by any of the techniques discussed herein.
Exemplary methods of enrichment and purification include but are
not limited to rotary vacuum filtration and/or ultrafiltration.
[0387] The enzyme solution is concentrated into a concentrated
enzyme solution until the enzyme activity of the concentrated
variant .alpha.-amylase polypeptide-containing solution is at a
desired level.
[0388] Concentration may be performed using, e.g., a precipitation
agent, such as a metal halide precipitation agent. Metal halide
precipitation agents include but are not limited to alkali metal
chlorides, alkali metal bromides and blends of two or more of these
metal halides. Exemplary metal halides include sodium chloride,
potassium chloride, sodium bromide, potassium bromide and blends of
two or more of these metal halides. The metal halide precipitation
agent, sodium chloride, can also be used as a preservative.
[0389] The metal halide precipitation agent is used in an amount
effective to precipitate an amylase. The selection of at least an
effective amount and an optimum amount of metal halide effective to
cause precipitation of the enzyme, as well as the conditions of the
precipitation for maximum recovery including incubation time, pH,
temperature and concentration of enzyme, will be readily apparent
to one of ordinary skill in the art, after routine testing.
[0390] Generally, at least about 5% w/v (weight/volume) to about
25% w/v of metal halide is added to the concentrated enzyme
solution, and usually at least 8% w/v. Generally, no more than
about 25% w/v of metal halide is added to the concentrated enzyme
solution and usually no more than about 20% w/v. The optimal
concentration of the metal halide precipitation agent will depend,
among others, on the nature of the specific variant .alpha.-amylase
polypeptide and on its concentration in the concentrated enzyme
solution.
[0391] Another alternative way to precipitate the enzyme is to use
organic compounds. Exemplary organic compound precipitating agents
include: 4-hydroxybenzoic acid, alkali metal salts of
4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and
blends of two or more of these organic compounds. The addition of
the organic compound precipitation agents can take place prior to,
simultaneously with or subsequent to the addition of the metal
halide precipitation agent, and the addition of both precipitation
agents, organic compound and metal halide, may be carried out
sequentially or simultaneously.
[0392] Generally, the organic precipitation agents are selected
from the group consisting of alkali metal salts of 4-hydroxybenzoic
acid, such as sodium or potassium salts, and linear or branched
alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group
contains from 1 to 12 carbon atoms, and blends of two or more of
these organic compounds. The organic compound precipitation agents
can be, for example, linear or branched alkyl esters of
4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to
10 carbon atoms, and blends of two or more of these organic
compounds. Exemplary organic compounds are linear alkyl esters of
4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 6
carbon atoms, and blends of two or more of these organic compounds.
Methyl esters of 4-hydroxybenzoic acid, propyl esters of
4-hydroxybenzoic acid, butyl ester of 4-hydroxybenzoic acid, ethyl
ester of 4-hydroxybenzoic acid and blends of two or more of these
organic compounds can also be used. Additional organic compounds
also include but are not limited to 4-hydroxybenzoic acid methyl
ester (named methyl PARABEN), 4-hydroxybenzoic acid propyl ester
(named propyl PARABEN), which also are both amylase preservative
agents. For further descriptions, see, e.g., U.S. Pat. No.
5,281,526.
[0393] Addition of the organic compound precipitation agent
provides the advantage of high flexibility of the precipitation
conditions with respect to pH, temperature, variant amylase
concentration, precipitation agent concentration, and time of
incubation.
[0394] The organic compound precipitation agent is used in an
amount effective to improve precipitation of the enzyme by means of
the metal halide precipitation agent. The selection of at least an
effective amount and an optimum amount of organic compound
precipitation agent, as well as the conditions of the precipitation
for maximum recovery including incubation time, pH, temperature and
concentration of enzyme, will be readily apparent to one of
ordinary skill in the art, in light of the present disclosure,
after routine testing.
[0395] Generally, at least about 0.01% w/v of organic compound
precipitation agent is added to the concentrated enzyme solution
and usually at least about 0.02% w/v. Generally, no more than about
0.3% w/v of organic compound precipitation agent is added to the
concentrated enzyme solution and usually no more than about 0.2%
w/v.
[0396] The concentrated polypeptide solution, containing the metal
halide precipitation agent, and the organic compound precipitation
agent, can be adjusted to a pH, which will, of necessity, depend on
the enzyme to be enriched or purified. Generally, the pH is
adjusted at a level near the isoelectric point of the amylase. The
pH can be adjusted at a pH in a range from about 2.5 pH units below
the isoelectric point (pI) up to about 2.5 pH units above the
isoelectric point.
[0397] The incubation time necessary to obtain an enriched or
purified enzyme precipitate depends on the nature of the specific
enzyme, the concentration of enzyme, and the specific precipitation
agent(s) and its (their) concentration. Generally, the time
effective to precipitate the enzyme is between about 1 to about 30
hours; usually it does not exceed about 25 hours. In the presence
of the organic compound precipitation agent, the time of incubation
can still be reduced to less about 10 hours and in most cases even
about 6 hours.
[0398] Generally, the temperature during incubation is between
about 4.degree. C. and about 50.degree. C. Usually, the method is
carried out at a temperature between about 10.degree. C. and about
45.degree. C. (e.g., between about 20.degree. C. and about
40.degree. C.). The optimal temperature for inducing precipitation
varies according to the solution conditions and the enzyme or
precipitation agent(s) used.
[0399] The overall recovery of enriched or purified enzyme
precipitate, and the efficiency with which the process is
conducted, is improved by agitating the solution comprising the
enzyme, the added metal halide and the added organic compound. The
agitation step is done both during addition of the metal halide and
the organic compound, and during the subsequent incubation period.
Suitable agitation methods include mechanical stirring or shaking,
vigorous aeration, or any similar technique.
[0400] After the incubation period, the enriched or purified enzyme
is then separated from the dissociated pigment and other impurities
and collected by conventional separation techniques, such as
filtration, centrifugation, microfiltration, rotary vacuum
filtration, ultrafiltration, press filtration, cross membrane
microfiltration, cross flow membrane microfiltration, or the like.
Further enrichment or purification of the enzyme precipitate can be
obtained by washing the precipitate with water. For example, the
enriched or purified enzyme precipitate is washed with water
containing the metal halide precipitation agent, or with water
containing the metal halide and the organic compound precipitation
agents.
[0401] During fermentation, a variant .alpha.-amylase polypeptide
accumulates in the culture broth. For the isolation, enrichment, or
purification of the desired variant .alpha.-amylase, the culture
broth is centrifuged or filtered to eliminate cells, and the
resulting cell-free liquid is used for enzyme enrichment or
purification. In one embodiment, the cell-free broth is subjected
to salting out using ammonium sulfate at about 70% saturation; the
70% saturation-precipitation fraction is then dissolved in a buffer
and applied to a column such as a Sephadex G-100 column, and eluted
to recover the enzyme-active fraction. For further enrichment or
purification, a conventional procedure such as ion exchange
chromatography may be used.
[0402] Enriched or purified enzymes are useful for laundry and
cleaning applications. For example, they can be used in laundry
detergents and spot removers. They can be made into a final product
that is either liquid (solution, slurry) or solid (granular,
powder).
[0403] A more specific example of enrichment or purification, is
described in Sumitani et al. (2000) "New type of starch-binding
domain: the direct repeat motif in the C-terminal region of
Bacillus sp. 195 .alpha.-amylase contributes to starch binding and
raw starch degrading," Biochem. J. 350: 477-484, and is briefly
summarized here. The enzyme obtained from 4 liters of a
Streptomyces lividans TK24 culture supernatant was treated with
(NH.sub.4).sub.2SO.sub.4 at 80% saturation. The precipitate was
recovered by centrifugation at 10,000.times.g (20 min. and
4.degree. C.) and re-dissolved in 20 mM Tris/HCl buffer (pH 7.0)
containing 5 mM CaCl.sub.2). The solubilized precipitate was then
dialyzed against the same buffer. The dialyzed sample was then
applied to a Sephacryl S-200 column, which had previously been
equilibrated with 20 mM Tris/HCl buffer, (pH 7.0), 5 mM CaCl2, and
eluted at a linear flow rate of 7 mL/hr with the same buffer.
Fractions from the column were collected and assessed for activity
as judged by enzyme assay and SDS-PAGE. The protein was further
purified as follows. A Toyopearl HW55 column (Tosoh Bioscience,
Montgomeryville, Pa.; Cat. No. 19812) was equilibrated with 20 mM
Tris/HCl buffer (pH 7.0) containing 5 mM CaCl2 and 1.5 M (NH4)2SO4.
The enzyme was eluted with a linear gradient of 1.5 to 0 M
(NH.sub.4).sub.2SO.sub.4 in 20 mM Tris/HCL buffer, pH 7.0
containing 5 mM CaCl.sub.2). The active fractions were collected,
and the enzyme precipitated with (NH.sub.4).sub.2SO.sub.4 at 80%
saturation. The precipitate was recovered, re-dissolved, and
dialyzed as described above. The dialyzed sample was then applied
to a Mono Q HR5/5 column (Amersham Pharmacia; Cat. No. 17-5167-01)
previously equilibrated with 20 mM Tris/HCl buffer (pH 7.0)
containing 5 mM CaCl.sub.2), at a flow rate of 60 mL/hour. The
active fractions are collected and added to a 1.5 M
(NH.sub.4).sub.2SO.sub.4 solution. The active enzyme fractions were
re-chromatographed on a Toyopearl HW55 column, as before, to yield
a homogeneous enzyme as determined by SDS-PAGE. See, e.g., Sumitani
et al. (2000) Biochem. J. 350: 477-484, for general discussion of
the method and variations thereon.
[0404] For production scale recovery, variant .alpha.-amylase
polypeptides can be enriched or partially purified as generally
described above by removing cells via flocculation with polymers.
Alternatively, the enzyme can be enriched or purified by
microfiltration followed by concentration by ultrafiltration using
available membranes and equipment. However, for some applications,
the enzyme does not need to be enriched or purified, and whole
broth culture can be lysed and used without further treatment. The
enzyme can then be processed, for example, into granules.
4. Compositions and Uses of Variant Amylases
[0405] Variants amylases are useful for a variety of industrial
applications. For example, variant amylases are useful in a starch
conversion process, particularly in a saccharification process of a
starch that has undergone liquefaction. The desired end-product may
be any product that may be produced by the enzymatic conversion of
the starch substrate. For example, the desired product may be a
syrup rich in glucose and maltose, which can be used in other
processes, such as the preparation of HFCS, or which can be
converted into a number of other useful products, such as ascorbic
acid intermediates (e.g., gluconate; 2-keto-L-gulonic acid;
5-keto-gluconate; and 2,5-diketogluconate); 1,3-propanediol;
aromatic amino acids (e.g., tyrosine, phenylalanine and
tryptophan); organic acids (e.g., lactate, pyruvate, succinate,
isocitrate, and oxaloacetate); amino acids (e.g., serine and
glycine); antibiotics; antimicrobials; enzymes; vitamins; and
hormones.
[0406] The starch conversion process may be a precursor to, or
simultaneous with, a fermentation process designed to produce
alcohol for fuel or drinking (i.e., potable alcohol). One skilled
in the art is aware of various fermentation conditions that may be
used in the production of these end-products. Variant amylases are
also useful in compositions and methods of food preparation. These
various uses of variant amylases are described in more detail
below.
4.1. Preparation of Starch Substrates
[0407] Those of general skill in the art are well aware of
available methods that may be used to prepare starch substrates for
use in the processes disclosed herein. For example, a useful starch
substrate may be obtained from tubers, roots, stems, legumes,
cereals or whole grain. More specifically, the granular starch may
be obtained from corn, cobs, wheat, barley, rye, triticale, milo,
sago, millet, cassava, tapioca, sorghum, rice, peas, bean, banana,
or potatoes. Corn contains about 60-68% starch; barley contains
about 55-65% starch; millet contains about 75-80% starch; wheat
contains about 60-65% starch; and polished rice contains 70-72%
starch. Specifically contemplated starch substrates are corn starch
and wheat starch. The starch from a grain may be ground or whole
and includes corn solids, such as kernels, bran and/or cobs. The
starch may also be highly refined raw starch or feedstock from
starch refinery processes. Various starches also are commercially
available. For example, corn starch is available from Cerestar,
Sigma, and Katayama Chemical Industry Co. (Japan); wheat starch is
available from Sigma; sweet potato starch is available from Wako
Pure Chemical Industry Co. (Japan); and potato starch is available
from Nakaari Chemical Pharmaceutical Co. (Japan).
[0408] The starch substrate can be a crude starch from milled whole
grain, which contains non-starch fractions, e.g., germ residues and
fibers. Milling may comprise either wet milling or dry milling or
grinding. In wet milling, whole grain is soaked in water or dilute
acid to separate the grain into its component parts, e.g., starch,
protein, germ, oil, kernel fibers. Wet milling efficiently
separates the germ and meal (i.e., starch granules and protein) and
is especially suitable for production of syrups. In dry milling or
grinding, whole kernels are ground into a fine powder and often
processed without fractionating the grain into its component parts.
In some cases, oils from the kernels are recovered. Dry ground
grain thus will comprise significant amounts of non-starch
carbohydrate compounds, in addition to starch. Dry grinding of the
starch substrate can be used for production of ethanol and other
biochemicals. The starch to be processed may be a highly refined
starch quality, for example, at least 90%, at least 95%, at least
97%, or at least 99.5% pure.
4.2. Gelatinization and Liquefaction of Starch
[0409] As used herein, the term "liquefaction" or "liquefy" means a
process by which starch is converted to less viscous and shorter
chain dextrins. Generally, this process involves gelatinization of
starch simultaneously with or followed by the addition of an
.alpha.-amylase, although additional liquefaction-inducing enzymes
optionally may be added. In some embodiments, the starch substrate
prepared as described above is slurried with water. The starch
slurry may contain starch as a weight percent of dry solids of
about 10-55%, about 20-45%, about 30-45%, about 30-40%, or about
30-35%. .alpha.-amylase may be added to the slurry, with a metering
pump, for example. The .alpha.-amylase typically used for this
application is a thermally stable, bacterial .alpha.-amylase, such
as a Geobacillus stearothermophilus .alpha.-amylase. The
.alpha.-amylase is usually supplied, for example, at about 1500
units per kg dry matter of starch. To optimize .alpha.-amylase
stability and activity, the pH of the slurry typically is adjusted
to about pH 5.5-6.5 and about 1 mM of calcium (about 40 ppm free
calcium ions) can also be added. Bacterial .alpha.-amylase
remaining in the slurry following liquefaction may be deactivated
via a number of methods, including lowering the pH in a subsequent
reaction step or by removing calcium from the slurry in cases where
the enzyme is dependent upon calcium.
[0410] The slurry of starch plus the .alpha.-amylase may be pumped
continuously through a jet cooker, which is steam heated to
105.degree. C. Gelatinization occurs rapidly under these
conditions, and the enzymatic activity, combined with the
significant shear forces, begins the hydrolysis of the starch
substrate. The residence time in the jet cooker is brief. The
partly gelatinized starch may be passed into a series of holding
tubes maintained at 105-110.degree. C. and held for 5-8 min. to
complete the gelatinization process ("primary liquefaction").
Hydrolysis to the required DE is completed in holding tanks at
85-95.degree. C. or higher temperatures for about 1 to 2 hours
("secondary liquefaction"). These tanks may contain baffles to
discourage back mixing. As used herein, the term "minutes of
secondary liquefaction" refers to the time that has elapsed from
the start of secondary liquefaction to the time that the Dextrose
Equivalent (DE) is measured. The slurry is then allowed to cool to
room temperature. This cooling step can be 30 minutes to 180
minutes, e.g., 90 minutes to 120 minutes. The liquefied starch
typically is in the form of a slurry having a dry solids content
(w/w) of about 10-50%; about 10-45%; about 15-40%; about 20-40%;
about 25-40%; or about 25-35%.
[0411] Liquefaction with variant amylases advantageously can be
conducted at low pH, eliminating the requirement to adjust the pH
to about pH 5.5-6.5. Variants amylases can be used for liquefaction
at a pH range of 2 to 7, e.g., pH 3.0-7.5, pH 4.0-6.0, or pH
4.5-5.8. Variant amylases can maintain liquefying activity at a
temperature range of about 85.degree. C.-95.degree. C., e.g.,
85.degree. C., 90.degree. C., or 95.degree. C. For example,
liquefaction can be conducted with 800 .mu.g an amylase in a
solution of 25% DS corn starch for 10 min at pH 5.8 and 85.degree.
C., or pH 4.5 and 95.degree. C., for example. Liquefying activity
can be assayed using any of a number of known viscosity assays in
the art.
[0412] In particular embodiments using the present amylase
variants, starch liquifaction is performed at a temperature range
of 90-115.degree. C., for the purpose of producing high-purity
glucose syrups, HFCS, maltodextrins, etc.
4.3. Saccharification
[0413] The liquefied starch can be saccharified into a syrup rich
in lower DP (e.g., DP1+DP2) saccharides, using variant amylases,
optionally in the presence of another enzyme(s). The exact
composition of the products of saccharification depends on the
combination of enzymes used, as well as the type of granular starch
processed. Advantageously, the syrup obtainable using the provided
variant amylases may contain a weight percent of DP2 of the total
oligosaccharides in the saccharified starch exceeding 30%, e.g.,
45%-65% or 55%-65%. The weight percent of (DP1+DP2) in the
saccharified starch may exceed about 70%, e.g., 75%-85% or 80%-85%.
The present amylases also produce a relatively high yield of
glucose, e.g., DP1>20%, in the syrup product.
[0414] Whereas liquefaction is generally run as a continuous
process, saccharification is often conducted as a batch process.
Saccharification typically is most effective at temperatures of
about 60-65.degree. C. and a pH of about 4.0-4.5, e.g., pH 4.3,
necessitating cooling and adjusting the pH of the liquefied starch.
Saccharification may be performed, for example, at a temperature
between about 40.degree. C., about 50.degree. C., or about
55.degree. C. to about 60.degree. C. or about 65.degree. C.
Saccharification is normally conducted in stirred tanks, which may
take several hours to fill or empty. Enzymes typically are added
either at a fixed ratio to dried solids as the tanks are filled or
added as a single dose at the commencement of the filling stage. A
saccharification reaction to make a syrup typically is run over
about 24-72 hours, for example, 24-48 hours. When a maximum or
desired DE has been attained, the reaction is stopped by heating to
85.degree. C. for 5 min., for example. Further incubation will
result in a lower DE, eventually to about 90 DE, as accumulated
glucose re-polymerizes to isomaltose and/or other reversion
products via an enzymatic reversion reaction and/or with the
approach of thermodynamic equilibrium. When using an amylase,
saccharification optimally is conducted at a temperature range of
about 30.degree. C. to about 75.degree. C., e.g., 45.degree.
C.-75.degree. C. or 47.degree. C.-74.degree. C. The saccharifying
may be conducted over a pH range of about pH 3 to about pH 7, e.g.,
pH 3.0-pH 7.5, pH 3.5-pH 5.5, pH 3.5, pH 3.8, or pH 4.5.
[0415] An .alpha.-amylase may be added to the slurry in the form of
a composition. An .alpha.-amylase can be added to a slurry of a
granular starch substrate in an amount of about 0.6-10 ppm ds,
e.g., 2 ppm ds. An .alpha.-amylase can be added as a whole broth,
clarified, enriched, partially purified, or purified enzyme. The
specific activity of the amylase may be about 300 U/mg of enzyme,
for example, measured with the PAHBAH assay. The .alpha.-amylase
also can be added as a whole broth product.
[0416] An .alpha.-amylase may be added to the slurry as an isolated
enzyme solution. For example, an .alpha.-amylase can be added in
the form of a cultured cell material produced by host cells
expressing an amylase. An .alpha.-amylase may also be secreted by a
host cell into the reaction medium during the fermentation or SSF
process, such that the enzyme is provided continuously into the
reaction. The host cell producing and secreting amylase may also
express an additional enzyme, such as a glucoamylase. For example,
U.S. Pat. No. 5,422,267 discloses the use of a glucoamylase in
yeast for production of alcoholic beverages. For example, a host
cell, e.g., Trichoderma reesei or Aspergillus niger, may be
engineered to co-express an .alpha.-amylase and a glucoamylase,
e.g., HgGA, TrGA, or a TrGA variant, during saccharification. The
host cell can be genetically modified so as not to express its
endogenous glucoamylase and/or other enzymes, proteins or other
materials. The host cell can be engineered to express a broad
spectrum of various saccharolytic enzymes. For example, the
recombinant yeast host cell can comprise nucleic acids encoding a
glucoamylase, an alpha-glucosidase, an enzyme that utilizes pentose
sugar, an .alpha.-amylase, a pullulanase, an isoamylase, and/or an
isopullulanase. See, e.g., WO 2011/153516 A2.
4.4. Isomerization
[0417] The soluble starch hydrolysate produced by treatment with
amylase can be converted into high fructose starch-based syrup
(HFSS), such as high fructose corn syrup (HFCS). This conversion
can be achieved using a glucose isomerase, particularly a glucose
isomerase immobilized on a solid support. The pH is increased to
about 6.0 to about 8.0, e.g., pH 7.5 (depending on the isomerase),
and Ca.sup.2+ is removed by ion exchange. Suitable isomerases
include SWEETZYME.RTM., IT (Novozymes A/S); G-ZYME.RTM. IMGI, and
G-ZYME.RTM. G993, KETOMAX.RTM., G-ZYME.RTM. G993, G-ZYME.RTM. G993
liquid, and GENSWEET.RTM. IGI. Following isomerization, the mixture
typically contains about 40-45% fructose, e.g., 42% fructose.
4.5. Fermentation
[0418] The soluble starch hydrolysate, particularly a glucose rich
syrup, can be fermented by contacting the starch hydrolysate with a
fermenting organism typically at a temperature around 32.degree.
C., such as from 30.degree. C. to 35.degree. C. for
alcohol-producing yeast. The temperature and pH of the fermentation
will depend upon the fermenting organism. EOF products include
metabolites, such as citric acid, lactic acid, succinic acid,
monosodium glutamate, gluconic acid, sodium gluconate, calcium
gluconate, potassium gluconate, itaconic acid and other carboxylic
acids, glucono delta-lactone, sodium erythorbate, lysine and other
amino acids, omega 3 fatty acid, butanol, isoprene, 1,3-propanediol
and other biomaterials.
[0419] Ethanologenic microorganisms include yeast, such as
Saccharomyces cerevisiae and bacteria, e.g., Zymomonas moblis,
expressing alcohol dehydrogenase and pyruvate decarboxylase. The
ethanologenic microorganism can express xylose reductase and
xylitol dehydrogenase, which convert xylose to xylulose. Improved
strains of ethanologenic microorganisms, which can withstand higher
temperatures, for example, are known in the art and can be used.
See Liu et al. (2011) Sheng Wu Gong Cheng Xue Bao 27(7): 1049-56.
Commercial sources of yeast include ETHANOL RED.RTM. (LeSaffre);
Thermosacc.RTM. (Lallemand); RED STAR.RTM. (Red Star); FERMIOL.RTM.
(DSM Specialties); and SUPERSTART.RTM. (Alltech). Microorganisms
that produce other metabolites, such as citric acid and lactic
acid, by fermentation are also known in the art. See, e.g.,
Papagianni (2007) "Advances in citric acid fermentation by
Aspergillus niger: biochemical aspects, membrane transport and
modeling," Biotechnol. Adv. 25(3): 244-63; John et al. (2009)
"Direct lactic acid fermentation: focus on simultaneous
saccharification and lactic acid production," Biotechnol. Adv.
27(2): 145-52.
[0420] The saccharification and fermentation processes may be
carried out as an SSF process. Fermentation may comprise subsequent
enrichment, purification, and recovery of ethanol, for example.
During the fermentation, the ethanol content of the broth or "beer"
may reach about 8-18% v/v, e.g., 14-15% v/v. The broth may be
distilled to produce enriched, e.g., 96% pure, solutions of
ethanol. Further, CO2 generated by fermentation may be collected
with a CO2 scrubber, compressed, and marketed for other uses, e.g.,
carbonating beverage or dry ice production. Solid waste from the
fermentation process may be used as protein-rich products, e.g.,
livestock feed.
[0421] As mentioned above, an SSF process can be conducted with
fungal cells that express and secrete amylase continuously
throughout SSF. The fungal cells expressing amylase also can be the
fermenting microorganism, e.g., an ethanologenic microorganism.
Ethanol production thus can be carried out using a fungal cell that
expresses sufficient amylase so that less or no enzyme has to be
added exogenously. The fungal host cell can be from an
appropriately engineered fungal strain. Fungal host cells that
express and secrete other enzymes, in addition to amylase, also can
be used. Such cells may express glucoamylase and/or a pullulanase,
phytase, alpha-glucosidase, isoamylase, beta-amylase cellulase,
xylanase, other hemicellulases, protease, beta-glucosidase,
pectinase, esterase, redox enzymes, transferase, or other
enzyme.
[0422] A variation on this process is a "fed-batch fermentation"
system, where the substrate is added in increments as the
fermentation progresses. Fed-batch systems are useful when
catabolite repression may inhibit the metabolism of the cells and
where it is desirable to have limited amounts of substrate in the
medium. The actual substrate concentration in fed-batch systems is
estimated by the changes of measurable factors such as pH,
dissolved oxygen and the partial pressure of waste gases, such as
CO2. Batch and fed-batch fermentations are common and well known in
the art.
[0423] Continuous fermentation is an open system where a defined
fermentation medium is added continuously to a bioreactor, and an
equal amount of conditioned medium is removed simultaneously for
processing. Continuous fermentation generally maintains the
cultures at a constant high density where cells are primarily in
log phase growth. Continuous fermentation permits modulation of
cell growth and/or product concentration. For example, a limiting
nutrient such as the carbon source or nitrogen source is maintained
at a fixed rate and all other parameters are allowed to moderate.
Because growth is maintained at a steady state, cell loss due to
medium being drawn off should be balanced against the cell growth
rate in the fermentation. Methods of optimizing continuous
fermentation processes and maximizing the rate of product formation
are well known in the art of industrial microbiology.
4.6. Compositions Comprising Variants Amylases
[0424] Variant amylases may be combined with a glucoamylase (EC
3.2.1.3), e.g., a Trichoderma glucoamylase or variant thereof. An
exemplary glucoamylase is Trichoderma reesei glucoamylase (TrGA)
and variants thereof that possess superior specific activity and
thermal stability. See U.S. Published Applications Nos.
2006/0094080, 2007/0004018, and 2007/0015266 (Danisco US Inc.).
Suitable variants of TrGA include those with glucoamylase activity
and at least 80%, at least 90%, or at least 95% sequence identity
to wild-type TrGA. Variant amylases advantageously increase the
yield of glucose produced in a saccharification process catalyzed
by TrGA.
[0425] Alternatively, the glucoamylase may be another glucoamylase
derived from plants (including algae), fungi, or bacteria. For
example, the glucoamylases may be Aspergillus niger G1 or G2
glucoamylase or its variants (e.g., Boel et al. (1984) EMBO J. 3:
1097-1102; WO 92/00381; WO 00/04136 (Novo Nordisk A/S)); and A.
awamori glucoamylase (e.g., WO 84/02921 (Cetus Corp.)). Other
contemplated Aspergillus glucoamylase include variants with
enhanced thermal stability, e.g., G137A and G139A (Chen et al.
(1996) Prot. Eng. 9: 499-505); D257E and D293E/Q (Chen et al.
(1995) Prot. Eng. 8: 575-582); N182 (Chen et al. (1994) Biochem. J.
301: 275-281); A246C (Fierobe et al. (1996) Biochemistry, 35:
8698-8704); and variants with Pro residues in positions A435 and
5436 (Li et al. (1997) Protein Eng. 10: 1199-1204). Other
contemplated glucoamylases include Talaromyces glucoamylases, in
particular derived from T. emersonii (e.g., WO 99/28448 (Novo
Nordisk A/S), T. leycettanus (e.g., U.S. Pat. No. RE 32,153 (CPC
International, Inc.)), T. duponti, or T. thermophilus (e.g., U.S.
Pat. No. 4,587,215). Contemplated bacterial glucoamylases include
glucoamylases from the genus Clostridium, in particular C.
thermoamylolyticum (e.g., EP 135,138 (CPC International, Inc.) and
C. thermohydrosulfuricum (e.g., WO 86/01831 (Michigan Biotechnology
Institute)). Suitable glucoamylases include the glucoamylases
derived from Aspergillus oryzae, such as a glucoamylase shown in
SEQ ID NO:2 in WO 00/04136 (Novo Nordisk A/S). Also suitable are
commercial glucoamylases, such as AMG 200L; AMG 300 L; SAN.TM.
SUPER and AMG.TM. E (Novozymes); OPTIDEX.RTM. 300 and OPTIDEX L-400
(Danisco US Inc.); AMIGASE.TM. and AMIGASE.TM. PLUS (DSM);
G-ZYME.RTM. G900 (Enzyme Bio-Systems); and G-ZYME.RTM. G990 ZR (A.
niger glucoamylase with a low protease content). Still other
suitable glucoamylases include Aspergillus fumigatus glucoamylase,
Talaromyces glucoamylase, Thielavia glucoamylase, Trametes
glucoamylase, Thermomyces glucoamylase, Athelia glucoamylase, or
Humicola glucoamylase (e.g., HgGA). Glucoamylases typically are
added in an amount of about 0.1-2 glucoamylase units (GAU)/g ds,
e.g., about 0.16 GAU/g ds, 0.23 GAU/g ds, or 0.33 GAU/g ds.
[0426] Other suitable enzymes that can be used with amylase include
a phytase, protease, pullulanase, .beta.-amylase, isoamylase, a
different .alpha.-amylase, alpha-glucosidase, cellulase, xylanase,
other hemicellulases, beta-glucosidase, transferase, pectinase,
lipase, cutinase, esterase, redox enzymes, or a combination
thereof. For example, a debranching enzyme, such as an isoamylase
(EC 3.2.1.68), may be added in effective amounts well known to the
person skilled in the art. A pullulanase (EC 3.2.1.41), e.g.,
PROMOZYME.RTM., is also suitable. Pullulanase typically is added at
100 U/kg ds. Further suitable enzymes include proteases, such as
fungal and bacterial proteases. Fungal proteases include those
obtained from Aspergillus, such as A. niger, A. awamori, A. oryzae;
Mucor (e.g., M. miehei); Rhizopus; and Trichoderma.
[0427] .beta.-Amylases (EC 3.2.1.2) are exo-acting maltogenic
amylases, which catalyze the hydrolysis of 1,4-.alpha.-glucosidic
linkages into amylopectin and related glucose polymers, thereby
releasing maltose. .beta.-Amylases have been isolated from various
plants and microorganisms. See Fogarty et al. (1979) in Progress in
Industrial Microbiology, Vol. 15, pp. 112-115. These (3-Amylases
have optimum temperatures in the range from 40.degree. C. to
65.degree. C. and optimum pH in the range from about 4.5 to about
7.0. Contemplated .beta.-amylases include, but are not limited to,
.beta.-amylases from barley SPEZYME.RTM. BBA 1500, SPEZYME.RTM.
DBA, OPTIMALT.TM. ME, OPTIMALT.TM. BBA (Danisco US Inc.); and
NOVOZYM.TM. WBA (Novozymes A/S).
[0428] Compositions comprising the present amylases may be aqueous
or non-aqueous formulations, granules, powders, gels, slurries,
pastes, etc., which may further comprise any one or more of the
additional enzymes listed, herein, along with buffers, salts,
preservatives, water, co-solvents, surfactants, and the like. Such
compositions may work in combination with endogenous enzymes or
other ingredients already present in a slurry, water bath, washing
machine, food or drink product, etc, for example, endogenous plant
(including algal) enzymes, residual enzymes from a prior processing
step, and the like.
5. Compositions and Methods for Baking and Food Preparation
[0429] The present invention also relates to a "food composition,"
including but not limited to a food product, animal feed and/or
food/feed additives, comprising an amylase, and methods for
preparing such a food composition comprising mixing variant amylase
with one or more food ingredients, or uses thereof.
[0430] Furthermore, the present invention relates to the use of an
amylase in the preparation of a food composition, wherein the food
composition is baked subsequent to the addition of the polypeptide
of the invention. As used herein the term "baking composition"
means any composition and/or additive prepared in the process of
providing a baked food product, including but not limited to bakers
flour, a dough, a baking additive and/or a baked product. The food
composition or additive may be liquid or solid.
[0431] As used herein, the term "flour" means milled or ground
cereal grain. The term "flour" also may mean Sago or tuber products
that have been ground or mashed. In some embodiments, flour may
also contain components in addition to the milled or mashed cereal
or plant matter. An example of an additional component, although
not intended to be limiting, is a leavening agent. Cereal grains
include wheat, oat, rye, and barley. Tuber products include tapioca
flour, cassava flour, and custard powder. The term "flour" also
includes ground corn flour, maize-meal, rice flour, whole-meal
flour, self-rising flour, tapioca flour, cassava flour, ground
rice, enriched flower, and custard powder.
[0432] For the commercial and home use of flour for baking and food
production, it is important to maintain an appropriate level of
.alpha.-amylase activity in the flour. A level of activity that is
too high may result in a product that is sticky and/or doughy and
therefore unmarketable. Flour with insufficient .alpha.-amylase
activity may not contain enough sugar for proper yeast function,
resulting in dry, crumbly bread, or baked products. Accordingly, an
amylase, by itself or in combination with another
.alpha.-amylase(s), may be added to the flour to augment the level
of endogenous .alpha.-amylase activity in flour.
[0433] An amylase can further be added alone or in a combination
with other amylases to prevent or retard staling, i.e., crumb
firming of baked products. The amount of anti-staling amylase will
typically be in the range of 0.01-10 mg of enzyme protein per kg of
flour, e.g., 0.5 mg/kg ds. Additional anti-staling amylases that
can be used in combination with an amylase include an endo-amylase,
e.g., a bacterial endo-amylase from Bacillus. The additional
amylase can be another maltogenic .alpha.-amylase (EC 3.2.1.133),
e.g., from Bacillus. NOVAMYL.RTM. is an exemplary maltogenic
.alpha.-amylase from B. stearothermophilus strain NCIB 11837 and is
described in Christophersen et al. (1997) Starch 50: 39-45. Other
examples of anti-staling endo-amylases include bacterial
.alpha.-amylases derived from Bacillus, such as B. licheniformis or
B. amyloliquefaciens. The anti-staling amylase may be an
exo-amylase, such as .beta.-amylase, e.g., from plant sources, such
as soy bean, or from microbial sources, such as Bacillus.
[0434] The baking composition comprising an amylase further can
comprise a phospholipase or enzyme with phospholipase activity. An
enzyme with phospholipase activity has an activity that can be
measured in Lipase Units (LU). The phospholipase may have A1 or A2
activity to remove fatty acid from the phospholipids, forming a
lysophospholipid. It may or may not have lipase activity, i.e.,
activity on triglyceride substrates. The phospholipase typically
has a temperature optimum in the range of 30-90.degree. C., e.g.,
30-70.degree. C. The added phospholipases can be of animal origin,
for example, from pancreas, e.g., bovine or porcine pancreas, snake
venom or bee venom. Alternatively, the phospholipase may be of
microbial origin, e.g., from filamentous fungi, yeast or bacteria,
for example.
[0435] The phospholipase is added in an amount that improves the
softness of the bread during the initial period after baking,
particularly the first 24 hours. The amount of phospholipase will
typically be in the range of 0.01-10 mg of enzyme protein per kg of
flour, e.g., 0.1-5 mg/kg. That is, phospholipase activity generally
will be in the range of 20-1000 LU/kg of flour, where a Lipase Unit
is defined as the amount of enzyme required to release 1 .mu.mol
butyric acid per minute at 30.degree. C., pH 7.0, with gum arabic
as emulsifier and tributyrin as substrate.
[0436] Compositions of dough generally comprise wheat meal or wheat
flour and/or other types of meal, flour or starch such as corn
flour, cornstarch, rye meal, rye flour, oat flour, oatmeal, soy
flour, sorghum meal, sorghum flour, potato meal, potato flour or
potato starch. The dough may be fresh, frozen or par-baked. The
dough can be a leavened dough or a dough to be subjected to
leavening. The dough may be leavened in various ways, such as by
adding chemical leavening agents, e.g., sodium bicarbonate or by
adding a leaven, i.e., fermenting dough. Dough also may be leavened
by adding a suitable yeast culture, such as a culture of
Saccharomyces cerevisiae (baker's yeast), e.g., a commercially
available strain of S. cerevisiae.
[0437] The dough may also comprise other conventional dough
ingredients, e.g., proteins, such as milk powder, gluten, and soy;
eggs (e.g., whole eggs, egg yolks or egg whites); an oxidant, such
as ascorbic acid, potassium bromate, potassium iodate,
azodicarbonamide (ADA) or ammonium persulfate; an amino acid such
as L-cysteine; a sugar; or a salt, such as sodium chloride, calcium
acetate, sodium sulfate or calcium sulfate. The dough further may
comprise fat, e.g., triglyceride, such as granulated fat or
shortening. The dough further may comprise an emulsifier such as
mono- or diglycerides, diacetyl tartaric acid esters of mono- or
diglycerides, sugar esters of fatty acids, polyglycerol esters of
fatty acids, lactic acid esters of monoglycerides, acetic acid
esters of monoglycerides, polyoxyethylene stearates, or
lysolecithin. In particular, the dough can be made without addition
of emulsifiers.
[0438] The dough product may be any processed dough product,
including fried, deep fried, roasted, baked, steamed and boiled
doughs, such as steamed bread and rice cakes. In one embodiment,
the food product is a bakery product. Typical bakery (baked)
products include bread--such as loaves, rolls, buns, bagels, pizza
bases etc. pastry, pretzels, tortillas, cakes, cookies, biscuits,
crackers etc.
[0439] Optionally, an additional enzyme may be used together with
the anti-staling amylase and the phospholipase. The additional
enzyme may be a second amylase, such as an amyloglucosidase, a
.beta.-amylase, a cyclodextrin glucanotransferase, or the
additional enzyme may be a peptidase, in particular an
exopeptidase, a transglutaminase, a lipase, a cellulase, a
xylanase, a protease, a protein disulfide isomerase, e.g., a
protein disulfide isomerase as disclosed in WO 95/00636, for
example, a glycosyltransferase, a branching enzyme
(1,4-.alpha.-glucan branching enzyme), a
4-.alpha.-glucanotransferase (dextrin glycosyltransferase) or an
oxidoreductase, e.g., a peroxidase, a laccase, a glucose oxidase,
an amadoriase, a metalloproteinase, a pyranose oxidase, a
lipooxygenase, an L-amino acid oxidase or a carbohydrate oxidase.
The additional enzyme(s) may be of any origin, including mammalian
and plant, and particularly of microbial (bacterial, yeast or
fungal) origin and may be obtained by techniques conventionally
used in the art.
[0440] The xylanase is typically of microbial origin, e.g., derived
from a bacterium or fungus, such as a strain of Aspergillus.
Xylanases include PENTOPAN.RTM. and NOVOZYM 384.RTM., for example,
which are commercially available xylanase preparations produced
from Trichoderma reesei. The amyloglucosidase may be an A. niger
amyloglucosidase (such as AMG.RTM.). Other useful amylase products
include GRINDAMYL.RTM. A 1000 or A 5000 (Grindsted Products,
Denmark) and AMYLASE H.TM. or AMYLASE P.TM. (DSM). The glucose
oxidase may be a fungal glucose oxidase, in particular an
Aspergillus niger glucose oxidase (such as GLUZYME.RTM.). An
exemplary protease is NEUTRASE.RTM..
[0441] The process may be used for any kind of baked product
prepared from dough, either of a soft or a crisp character, either
of a white, light or dark type. Examples are bread, particularly
white, whole-meal or rye bread, typically in the form of loaves or
rolls, such as, but not limited to, French baguette-type bread,
pita bread, tortillas, cakes, pancakes, biscuits, cookies, pie
crusts, crisp bread, steamed bread, pizza and the like.
[0442] An amylase may be used in a pre-mix, comprising flour
together with an anti-staling amylase, a phospholipase, and/or a
phospholipid. The pre-mix may contain other dough-improving and/or
bread-improving additives, e.g., any of the additives, including
enzymes, mentioned above. An amylase can be a component of an
enzyme preparation comprising an anti-staling amylase and a
phospholipase, for use as a baking additive.
[0443] The enzyme preparation is optionally in the form of a
granulate or agglomerated powder. The preparation can have a narrow
particle size distribution with more than 95% (by weight) of the
particles in the range from 25 to 500 .mu.m. Granulates and
agglomerated powders may be prepared by conventional methods, e.g.,
by spraying an amylase onto a carrier in a fluid-bed granulator.
The carrier may consist of particulate cores having a suitable
particle size. The carrier may be soluble or insoluble, e.g., a
salt (such as NaCl or sodium sulfate), a sugar (such as sucrose or
lactose), a sugar alcohol (such as sorbitol), starch, rice, corn
grits, or soy.
[0444] Enveloped particles, i.e., .alpha.-amylase particles, can
comprise an amylase. To prepare enveloped .alpha.-amylase
particles, the enzyme is contacted with a food grade lipid in
sufficient quantity to suspend all of the .alpha.-amylase
particles. Food grade lipids, as used herein, may be any naturally
organic compound that is insoluble in water but is soluble in
non-polar organic solvents such as hydrocarbon or diethyl ether.
Suitable food grade lipids include, but are not limited to,
triglycerides either in the form of fats or oils that are either
saturated or unsaturated. Examples of fatty acids and combinations
thereof which make up the saturated triglycerides include, but are
not limited to, butyric (derived from milk fat), palmitic (derived
from animal and plant fat), and/or stearic (derived from animal and
plant fat). Examples of fatty acids and combinations thereof which
make up the unsaturated triglycerides include, but are not limited
to, palmitoleic (derived from animal and plant fat), oleic (derived
from animal and plant fat), linoleic (derived from plant oils),
and/or linolenic (derived from linseed oil). Other suitable food
grade lipids include, but are not limited to, monoglycerides and
diglycerides derived from the triglycerides discussed above,
phospholipids and glycolipids.
[0445] The food grade lipid, particularly in the liquid form, is
contacted with a powdered form of the .alpha.-amylase particles in
such a fashion that the lipid material covers at least a portion of
the surface of at least a majority, e.g., 100% of the
.alpha.-amylase particles. Thus, each .alpha.-amylase particle is
individually enveloped in a lipid. For example, all or
substantially all of the .alpha.-amylase particles are provided
with a thin, continuous, enveloping film of lipid. This can be
accomplished by first pouring a quantity of lipid into a container,
and then slurrying the .alpha.-amylase particles so that the lipid
thoroughly wets the surface of each .alpha.-amylase particle. After
a short period of stirring, the enveloped .alpha.-amylase
particles, carrying a substantial amount of the lipids on their
surfaces, are recovered. The thickness of the coating so applied to
the particles of .alpha.-amylase can be controlled by selection of
the type of lipid used and by repeating the operation in order to
build up a thicker film, when desired.
[0446] The storing, handling and incorporation of the loaded
delivery vehicle can be accomplished by means of a packaged mix.
The packaged mix can comprise the enveloped .alpha.-amylase.
However, the packaged mix may further contain additional
ingredients as required by the manufacturer or baker. After the
enveloped .alpha.-amylase has been incorporated into the dough, the
baker continues through the normal production process for that
product.
[0447] The advantages of enveloping the .alpha.-amylase particles
are two-fold. First, the food grade lipid protects the enzyme from
thermal denaturation during the baking process for those enzymes
that are heat labile. Consequently, while the .alpha.-amylase is
stabilized and protected during the proving and baking stages, it
is released from the protective coating in the final baked good
product, where it hydrolyzes the glucosidic linkages in
polyglucans. The loaded delivery vehicle also provides a sustained
release of the active enzyme into the baked good. That is,
following the baking process, active .alpha.-amylase is continually
released from the protective coating at a rate that counteracts,
and therefore reduces the rate of, staling mechanisms.
[0448] In general, the amount of lipid applied to the
.alpha.-amylase particles can vary from a few percent of the total
weight of the .alpha.-amylase to many times that weight, depending
upon the nature of the lipid, the manner in which it is applied to
the .alpha.-amylase particles, the composition of the dough mixture
to be treated, and the severity of the dough-mixing operation
involved.
[0449] The loaded delivery vehicle, i.e., the lipid-enveloped
enzyme, is added to the ingredients used to prepare a baked good in
an effective amount to extend the shelf-life of the baked good. The
baker computes the amount of enveloped .alpha.-amylase, prepared as
discussed above, that will be required to achieve the desired
anti-staling effect. The amount of the enveloped .alpha.-amylase
required is calculated based on the concentration of enzyme
enveloped and on the proportion of .alpha.-amylase to flour
specified. A wide range of concentrations has been found to be
effective, although, as has been discussed, observable improvements
in anti-staling do not correspond linearly with the .alpha.-amylase
concentration, but above certain minimal levels, large increases in
.alpha.-amylase concentration produce little additional
improvement. The .alpha.-amylase concentration actually used in a
particular bakery production could be much higher than the minimum
necessary to provide the baker with some insurance against
inadvertent under-measurement errors by the baker. The lower limit
of enzyme concentration is determined by the minimum anti-staling
effect the baker wishes to achieve.
[0450] A method of preparing a baked good may comprise: a)
preparing lipid-coated .alpha.-amylase particles, where
substantially all of the .alpha.-amylase particles are coated; b)
mixing a dough containing flour; c) adding the lipid-coated
.alpha.-amylase to the dough before the mixing is complete and
terminating the mixing before the lipid coating is removed from the
.alpha.-amylase; d) proofing the dough; and e) baking the dough to
provide the baked good, where the .alpha.-amylase is inactive
during the mixing, proofing and baking stages and is active in the
baked good.
[0451] The enveloped .alpha.-amylase can be added to the dough
during the mix cycle, e.g., near the end of the mix cycle. The
enveloped .alpha.-amylase is added at a point in the mixing stage
that allows sufficient distribution of the enveloped
.alpha.-amylase throughout the dough; however, the mixing stage is
terminated before the protective coating becomes stripped from the
.alpha.-amylase particle(s). Depending on the type and volume of
dough, and mixer action and speed, anywhere from one to six minutes
or more might be required to mix the enveloped .alpha.-amylase into
the dough, but two to four minutes is average. Thus, several
variables may determine the precise procedure. First, the quantity
of enveloped .alpha.-amylase should have a total volume sufficient
to allow the enveloped .alpha.-amylase to be spread throughout the
dough mix. If the preparation of enveloped .alpha.-amylase is
highly concentrated, additional oil may need to be added to the
pre-mix before the enveloped .alpha.-amylase is added to the dough.
Recipes and production processes may require specific
modifications; however, good results can generally be achieved when
25% of the oil specified in a bread dough formula is held out of
the dough and is used as a carrier for a concentrated enveloped
.alpha.-amylase when added near the end of the mix cycle. In bread
or other baked goods, particularly those having a low fat content,
e.g., French-style breads, an enveloped .alpha.-amylase mixture of
approximately 1% of the dry flour weight is sufficient to admix the
enveloped .alpha.-amylase properly with the dough. The range of
suitable percentages is wide and depends on the formula, finished
product, and production methodology requirements of the individual
baker. Second, the enveloped .alpha.-amylase suspension should be
added to the mix with sufficient time for complete mixture into the
dough, but not for such a time that excessive mechanical action
strips the protective lipid coating from the enveloped
.alpha.-amylase particles.
[0452] In a further aspect of the invention, the food composition
is an oil, meat, lard, composition comprising an amylase. In this
context the term "[oil/meat/lard] composition" means any
composition, based on, made from and/or containing oil, meat or
lard, respectively. Another aspect the invention relates to a
method of preparing an oil or meat or lard composition and/or
additive comprising an amylase, comprising mixing the polypeptide
of the invention with a oil/meat/lard composition and/or additive
ingredients.
[0453] In a further aspect of the invention, the food composition
is an animal feed composition, animal feed additive and/or pet food
comprising an amylase and variants thereof. The present invention
further relates to a method for preparing such an animal feed
composition, animal feed additive composition and/or pet food
comprising mixing an amylase and variants thereof with one or more
animal feed ingredients and/or animal feed additive ingredients
and/or pet food ingredients. Furthermore, the present invention
relates to the use of an amylase in the preparation of an animal
feed composition and/or animal feed additive composition and/or pet
food.
[0454] The term "animal" includes all non-ruminant and ruminant
animals. In a particular embodiment, the animal is a non-ruminant
animal, such as a horse and a mono-gastric animal. Examples of
mono-gastric animals include, but are not limited to, pigs and
swine, such as piglets, growing pigs, sows; poultry such as
turkeys, ducks, chicken, broiler chicks, layers; fish such as
salmon, trout, tilapia, catfish and carps; and crustaceans such as
shrimps and prawns. In a further embodiment the animal is a
ruminant animal including, but not limited to, cattle, young
calves, goats, sheep, giraffes, bison, moose, elk, yaks, water
buffalo, deer, camels, alpacas, llamas, antelope, pronghorn and
nilgai.
[0455] In the present context, it is intended that the term "pet
food" is understood to mean a food for a household animal such as,
but not limited to dogs, cats, gerbils, hamsters, chinchillas,
fancy rats, guinea pigs; avian pets, such as canaries, parakeets,
and parrots; reptile pets, such as turtles, lizards and snakes; and
aquatic pets, such as tropical fish and frogs.
[0456] The terms "animal feed composition," "feedstuff" and
"fodder" are used interchangeably and may comprise one or more feed
materials selected from the group comprising a) cereals, such as
small grains (e.g., wheat, barley, rye, oats and combinations
thereof) and/or large grains such as maize or sorghum; b) by
products from cereals, such as corn gluten meal, Distillers Dried
Grain Solubles (DDGS) (particularly corn based Distillers Dried
Grain Solubles (cDDGS), wheat bran, wheat middlings, wheat shorts,
rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c)
protein obtained from sources such as soya, sunflower, peanut,
lupin, peas, fava beans, cotton, canola, fish meal, dried plasma
protein, meat and bone meal, potato protein, whey, copra, sesame;
d) oils and fats obtained from vegetable and animal sources; e)
minerals and vitamins.
6. Textile Desizing Compositions and Use
[0457] Also contemplated are compositions and methods of treating
fabrics (e.g., to desize a textile) using an amylase.
Fabric-treating methods are well known in the art (see, e.g., U.S.
Pat. No. 6,077,316). For example, the feel and appearance of a
fabric can be improved by a method comprising contacting the fabric
with an amylase in a solution. The fabric can be treated with the
solution under pressure.
[0458] An amylase can be applied during or after the weaving of a
textile, or during the desizing stage, or 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 to increase their tensile
strength and to prevent breaking. An amylase can be applied during
or after the weaving to remove these sizing starch or starch
derivatives. After weaving, an amylase can be used to remove the
size coating before further processing the fabric to ensure a
homogeneous and wash-proof result.
[0459] An amylase can be used alone or with other desizing chemical
reagents and/or desizing enzymes to desize fabrics, including
cotton-containing fabrics, as detergent additives, e.g., in aqueous
compositions. An amylase also can be used in compositions and
methods for producing a stonewashed look on indigo-dyed denim
fabric and garments. For the manufacture of clothes, the fabric can
be cut and sewn into clothes or garments, which are afterwards
finished. 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 to provide softness to the fabric and make the
cotton more accessible to the subsequent enzymatic finishing steps.
An amylase can be used in methods of finishing denim garments
(e.g., a "bio-stoning process"), enzymatic desizing and providing
softness to fabrics, and/or finishing process.
7. Cleaning Compositions
[0460] An aspect of the present compositions and methods is a
cleaning composition that includes an amylase as a component. An
amylase polypeptide can be used as a component in detergent
compositions for, e.g., hand washing, laundry washing, dishwashing,
and other hard-surface cleaning. Such compositions include heavy
duty liquid (HDL), heavy duty dry (HDD), and hand (manual) laundry
detergent compositions, including unit dose format laundry
detergent compositions, and automatic dishwashing (ADW) and hand
(manual) dishwashing compositions, including unit dose format
dishwashing compositions.
7.1. Overview
[0461] Preferably, an amylase is incorporated into detergents at or
near a concentration conventionally used for amylase in detergents.
For example, an amylase polypeptide may be added in amount
corresponding to 0.00001-1 mg (calculated as pure enzyme protein)
of amylase per liter of wash/dishwash liquor. Exemplary
formulations are provided herein, as exemplified by the
following:
[0462] An amylase polypeptide may be a component of a detergent
composition, as the only enzyme or with other enzymes including
other amylolytic enzymes. As such, it may be included in the
detergent composition in the form of a non-dusting granulate, a
stabilized liquid, or a protected enzyme. Non-dusting granulates
may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and
4,661,452 and may optionally be coated by methods known in the art.
Examples of waxy coating materials are poly(ethylene oxide)
products (polyethyleneglycol, PEG) with mean molar weights of 1,000
to 20,000; ethoxylated nonylphenols having from 16 to 50 ethylene
oxide units; ethoxylated fatty alcohols in which the alcohol
contains from 12 to 20 carbon atoms and in which there are 15 to 80
ethylene oxide units; fatty alcohols; fatty acids; and mono- and
di- and triglycerides of fatty acids. Examples of film-forming
coating materials suitable for application by fluid bed techniques
are given in, for example, GB 1483591. Liquid enzyme preparations
may, for instance, be stabilized by adding a polyol such as
propylene glycol, a sugar or sugar alcohol, lactic acid or boric
acid according to established methods. Other enzyme stabilizers are
known in the art. Protected enzymes may be prepared according to
the method disclosed in for example EP 238 216. Polyols have long
been recognized as stabilizers of proteins, as well as improving
protein solubility.
[0463] The detergent composition may be in any useful form, e.g.,
as powders, granules, pastes, bars, or liquid. A liquid detergent
may be aqueous, typically containing up to about 70% of water and
0% to about 30% of organic solvent. It may also be in the form of a
compact gel type containing only about 30% water.
[0464] The detergent composition comprises one or more surfactants,
each of which may be anionic, nonionic, cationic, or zwitterionic.
The detergent will usually contain 0% to about 50% of anionic
surfactant, such as linear alkylbenzenesulfonate (LAS);
.alpha.-olefinsulfonate (AOS); alkyl sulfate (fatty alcohol
sulfate) (AS); alcohol ethoxysulfate (AEOS or AES); secondary
alkanesulfonates (SAS); .alpha.-sulfo fatty acid methyl esters;
alkyl- or alkenylsuccinic acid; or soap. The composition may also
contain 0% to about 40% of nonionic surfactant such as alcohol
ethoxylate (AEO or AE), carboxylated alcohol ethoxylates,
nonylphenol ethoxylate, alkylpolyglycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide,
fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide
(as described for example in WO 92/06154).
[0465] The detergent composition may additionally comprise one or
more other enzymes, such as proteases, another amylolytic enzyme,
cutinase, lipase, cellulase, pectate lyase, perhydrolase, xylanase,
peroxidase, and/or laccase in any combination.
[0466] The detergent may contain about 1% to about 65% of a
detergent builder or complexing agent such as zeolite, diphosphate,
triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA),
ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid (DTMPA), alkyl- or
alkenylsuccinic acid, soluble silicates or layered silicates (e.g.,
SKS-6 from Hoechst). The detergent may also be unbuilt, i.e.
essentially free of detergent builder. The enzymes can be used in
any composition compatible with the stability of the enzyme.
Enzymes generally can be protected against deleterious components
by known forms of encapsulation, for example, by granulation or
sequestration in hydro gels. Enzymes, and specifically amylases,
either with or without starch binding domains, can be used in a
variety of compositions including laundry and dishwashing
applications, surface cleaners, as well as in compositions for
ethanol production from starch or biomass.
[0467] The detergent may comprise one or more polymers. Examples
include carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),
polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA),
polycarboxylates such as polyacrylates, maleic/acrylic acid
copolymers and lauryl methacrylate/acrylic acid copolymers.
[0468] The detergent may contain a bleaching system, which may
comprise a H2O2 source such as perborate or percarbonate, which may
be combined with a peracid-forming bleach activator such as
tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate
(NOBS).
[0469] Alternatively, the bleaching system may comprise peroxyacids
(e.g., the amide, imide, or sulfone type peroxyacids). The
bleaching system can also be an enzymatic bleaching system, for
example, perhydrolase, such as that described in International PCT
Application WO 2005/056783.
[0470] The enzymes of the detergent composition may be stabilized
using conventional stabilizing agents, e.g., a polyol such as
propylene glycol or glycerol; a sugar or sugar alcohol; lactic
acid; boric acid or a boric acid derivative such as, e.g., an
aromatic borate ester; and the composition may be formulated as
described in, e.g., WO 92/19709 and WO 92/19708.
[0471] The detergent may also contain other conventional detergent
ingredients such as e.g., fabric conditioners including clays, foam
boosters, suds suppressors, anti-corrosion agents, soil-suspending
agents, anti-soil redeposition agents, dyes, bactericides, tarnish
inhibitors, optical brighteners, or perfumes.
[0472] The pH (measured in aqueous solution at use concentration)
is usually neutral or alkaline, e.g., pH about 7.0 to about
11.0.
[0473] Particular forms of detergent compositions for inclusion of
the present .alpha.-amylase are described, below. Many of these
composition can be provided in unit dose format for ease of use.
Unit dose formulations and packaging are described in, for example,
US20090209445A1, US20100081598A1, U.S. Pat. No. 7,001,878B2,
EP1504994B1, WO2001085888A2, WO2003089562A1, WO2009098659A1,
WO2009098660A1, WO2009112992A1, WO2009124160A1, WO2009152031A1,
WO2010059483A1, WO2010088112A1, WO2010090915A1, WO2010135238A1,
WO2011094687A1, WO2011094690A1, WO2011127102A1, WO2011163428A1,
WO2008000567A1, WO2006045391A1, WO2006007911A1, WO2012027404A1,
EP1740690B1, WO2012059336A1, U.S. Pat. No. 6,730,646B1,
WO2008087426A1, WO2010116139A1, and WO2012104613A1.
7.2. Heavy Duty Liquid (HDL) Laundry Detergent Composition
[0474] Exemplary HDL laundry detergent compositions includes a
detersive surfactant (10%-40% wt/wt), including an anionic
detersive surfactant (selected from a group of linear or branched
or random chain, substituted or unsubstituted alkyl sulphates,
alkyl sulphonates, alkyl alkoxylated sulphate, alkyl phosphates,
alkyl phosphonates, alkyl carboxylates, and/or mixtures thereof),
and optionally non-ionic surfactant (selected from a group of
linear or branched or random chain, substituted or unsubstituted
alkyl alkoxylated alcohol, for example a C8-C18 alkyl ethoxylated
alcohol and/or C6-C12 alkyl phenol alkoxylates), wherein the weight
ratio of anionic detersive surfactant (with a hydrophilic index
(HIc) of from 6.0 to 9) to non-ionic detersive surfactant is
greater than 1:1. Suitable detersive surfactants also include
cationic detersive surfactants (selected from a group of alkyl
pyridinium compounds, alkyl quarternary ammonium compounds, alkyl
quarternary phosphonium compounds, alkyl ternary sulphonium
compounds, and/or mixtures thereof); zwitterionic and/or amphoteric
detersive surfactants (selected from a group of alkanolamine
sulpho-betaines); ampholytic surfactants; semi-polar non-ionic
surfactants and mixtures thereof.
[0475] The composition may optionally include, a surfactancy
boosting polymer consisting of amphiphilic alkoxylated grease
cleaning polymers (selected from a group of alkoxylated polymers
having branched hydrophilic and hydrophobic properties, such as
alkoxylated polyalkylenimines in the range of 0.05 wt %-10 wt %)
and/or random graft polymers (typically comprising of hydrophilic
backbone comprising monomers selected from the group consisting of:
unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes,
ketones, esters, sugar units, alkoxy units, maleic anhydride,
saturated polyalcohols such as glycerol, and mixtures thereof and
hydrophobic side chain(s) selected from the group consisting of:
C4-C25 alkyl group, polypropylene, polybutylene, vinyl ester of a
saturated C1-C6 mono-carboxylic acid, C1-C6 alkyl ester of acrylic
or methacrylic acid, and mixtures thereof.
[0476] The composition may include additional polymers such as soil
release polymers (include anionically end-capped polyesters, for
example SRP1, polymers comprising at least one monomer unit
selected from saccharide, dicarboxylic acid, polyol and
combinations thereof, in random or block configuration, ethylene
terephthalate-based polymers and co-polymers thereof in random or
block configuration, for example Repel-o-tex SF, SF-2 and SRP6,
Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325,
Marloquest SL), anti-redeposition polymers (0.1 wt % to 10 wt %,
include carboxylate polymers, such as polymers comprising at least
one monomer selected from acrylic acid, maleic acid (or maleic
anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic
acid, citraconic acid, methylenemalonic acid, and any mixture
thereof, vinylpyrrolidone homopolymer, and/or polyethylene glycol,
molecular weight in the range of from 500 to 100,000 Da);
cellulosic polymer (including those selected from alkyl cellulose,
alkyl alkoxyalkyl cellulose, carboxyalkyl cellulose, alkyl
carboxyalkyl cellulose examples of which include carboxymethyl
cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl
carboxymethyl cellulose, and mixtures thereof) and polymeric
carboxylate (such as maleate/acrylate random copolymer or
polyacrylate homopolymer).
[0477] The composition may further include saturated or unsaturated
fatty acid, preferably saturated or unsaturated C12-C24 fatty acid
(0 wt % to 10 wt %); deposition aids (examples for which include
polysaccharides, preferably cellulosic polymers, poly diallyl
dimethyl ammonium halides (DADMAC), and co-polymers of DAD MAC with
vinyl pyrrolidone, acrylamides, imidazoles, imidazolinium halides,
and mixtures thereof, in random or block configuration, cationic
guar gum, cationic cellulose such as cationic hydoxyethyl
cellulose, cationic starch, cationic polyacylamides, and mixtures
thereof.
[0478] The composition may further include dye transfer inhibiting
agents, examples of which include manganese phthalocyanine,
peroxidases, polyvinylpyrrolidone polymers, polyamine N-oxide
polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole,
polyvinyloxazolidones and polyvinylimidazoles and/or mixtures
thereof; chelating agents, examples of which include
ethylene-diamine-tetraacetic acid (EDTA), diethylene triamine penta
methylene phosphonic acid (DTPMP), hydroxy-ethane diphosphonic acid
(HEDP), ethylenediamine N,N'-disuccinic acid (EDDS), methyl glycine
diacetic acid (MGDA), diethylene triamine penta acetic acid (DTPA),
propylene diamine tetracetic acid (PDT A),
2-hydroxypyridine-N-oxide (HPNO), or methyl glycine diacetic acid
(MGDA), glutamic acid N,N-diacetic acid (N,N-dicarboxymethyl
glutamic acid tetrasodium salt (GLDA), nitrilotriacetic acid (NTA),
4,5-dihydroxy-m-benzenedisulfonic acid, citric acid and any salts
thereof, N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),
triethylenetetraaminehexaacetic acid (TTHA),
N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine
(DHEG), ethylenediaminetetrapropionic acid (EDTP), and derivatives
thereof.
[0479] The composition preferably included enzymes (generally about
0.01 wt % active enzyme to 0.03 wt % active enzyme) selected from
proteases, amylases, lipases, cellulases, choline oxidases,
peroxidases/oxidases, pectate lyases, mannanases, cutinases,
laccases, phospholipases, lysophospholipases, acyltransferases,
perhydrolases, arylesterases, and any mixture thereof. The
composition may include an enzyme stabilizer (examples of which
include polyols such as propylene glycol or glycerol, sugar or
sugar alcohol, lactic acid, reversible protease inhibitor, boric
acid, or a boric acid derivative, e.g., an aromatic borate ester,
or a phenyl boronic acid derivative such as 4-formylphenyl boronic
acid).
[0480] The composition optionally include silicone or fatty-acid
based suds suppressors; hueing dyes, calcium and magnesium cations,
visual signaling ingredients, anti-foam (0.001 wt % to about 4.0 wt
%), and/or structurant/thickener (0.01 wt % to 5 wt %, selected
from the group consisting of diglycerides and triglycerides,
ethylene glycol distearate, microcrystalline cellulose, cellulose
based materials, microfiber cellulose, biopolymers, xanthan gum,
gellan gum, and mixtures thereof).
[0481] The composition can be any liquid form, for example a liquid
or gel form, or any combination thereof. The composition may be in
any unit dose form, for example a pouch.
7.3. Heavy Duty Dry/Solid (HDD) Laundry Detergent Composition
[0482] Exemplary HDD laundry detergent compositions includes a
detersive surfactant, including anionic detersive surfactants
(e.g., linear or branched or random chain, substituted or
unsubstituted alkyl sulphates, alkyl sulphonates, alkyl alkoxylated
sulphate, alkyl phosphates, alkyl phosphonates, alkyl carboxylates
and/or mixtures thereof), non-ionic detersive surfactant (e.g.,
linear or branched or random chain, substituted or unsubstituted
C8-C18 alkyl ethoxylates, and/or C6-C12 alkyl phenol alkoxylates),
cationic detersive surfactants (e.g., alkyl pyridinium compounds,
alkyl quaternary ammonium compounds, alkyl quaternary phosphonium
compounds, alkyl ternary sulphonium compounds, and mixtures
thereof), zwitterionic and/or amphoteric detersive surfactants
(e.g., alkanolamine sulpho-betaines), ampholytic surfactants,
semi-polar non-ionic surfactants, and mixtures thereof; builders
including phosphate free builders (for example zeolite builders
examples which include zeolite A, zeolite X, zeolite P and zeolite
MAP in the range of 0 wt % to less than 10 wt %), phosphate
builders (for example sodium tri-polyphosphate in the range of 0 wt
% to less than 10 wt %), citric acid, citrate salts and
nitrilotriacetic acid, silicate salt (e.g., sodium or potassium
silicate or sodium meta-silicate in the range of 0 wt % to less
than 10 wt %, or layered silicate (SKS-6)); carbonate salt (e.g.,
sodium carbonate and/or sodium bicarbonate in the range of 0 wt %
to less than 80 wt %); and bleaching agents including photobleaches
(e.g., sulfonated zinc phthalocyanines, sulfonated aluminum
phthalocyanines, xanthenes dyes, and mixtures thereof) hydrophobic
or hydrophilic bleach activators (e.g., dodecanoyl oxybenzene
sulfonate, decanoyl oxybenzene sulfonate, decanoyl oxybenzoic acid
or salts thereof, 3,5,5-trimethy hexanoyl oxybenzene sulfonate,
tetraacetyl ethylene diamine-TAED, nonanoyloxybenzene
sulfonate-NOBS, nitrile quats, and mixtures thereof), sources of
hydrogen peroxide (e.g., inorganic perhydrate salts examples of
which include mono or tetra hydrate sodium salt of perborate,
percarbonate, persulfate, perphosphate, or persilicate), preformed
hydrophilic and/or hydrophobic peracids (e.g., percarboxylic acids
and salts, percarbonic acids and salts, perimidic acids and salts,
peroxymonosulfuric acids and salts, and mixtures thereof), and/or
bleach catalysts (e.g., imine bleach boosters (examples of which
include iminium cations and polyions), iminium zwitterions,
modified amines, modified amine oxides, N-sulphonyl imines,
N-phosphonyl imines, N-acyl imines, thiadiazole dioxides,
perfluoroimines, cyclic sugar ketones, and mixtures thereof, and
metal-containing bleach catalysts (e.g., copper, iron, titanium,
ruthenium, tungsten, molybdenum, or manganese cations along with an
auxiliary metal cations such as zinc or aluminum and a sequestrate
such as ethylenediaminetetraacetic acid,
ethylenediaminetetra(methylenephosphonic acid), and water-soluble
salts thereof).
[0483] The composition preferably includes enzymes, e.g.,
proteases, amylases, lipases, cellulases, choline oxidases,
peroxidases/oxidases, pectate lyases, mannanases, cutinases,
laccases, phospholipases, lysophospholipases, acyltransferase,
perhydrolase, arylesterase, and any mixture thereof.
[0484] The composition may optionally include additional detergent
ingredients including perfume microcapsules, starch encapsulated
perfume accord, hueing agents, additional polymers, including
fabric integrity and cationic polymers, dye-lock ingredients,
fabric-softening agents, brighteners (for example C.I. Fluorescent
brighteners), flocculating agents, chelating agents, alkoxylated
polyamines, fabric deposition aids, and/or cyclodextrin.
7.4. Automatic Dishwashing (ADW) Detergent Composition
[0485] Exemplary ADW detergent composition includes non-ionic
surfactants, including ethoxylated non-ionic surfactants, alcohol
alkoxylated surfactants, epoxy-capped poly(oxyalkylated) alcohols,
or amine oxide surfactants present in amounts from 0 to 10% by
weight; builders in the range of 5-60% including phosphate builders
(e.g., mono-phosphates, di-phosphates, tri-polyphosphates, other
oligomeric-poylphosphates, sodium tripolyphosphate-STPP) and
phosphate-free builders (e.g., amino acid-based compounds including
methyl-glycine-diacetic acid (MGDA) and salts and derivatives
thereof, glutamic-N,N-diacetic acid (GLDA) and salts and
derivatives thereof, iminodisuccinic acid (IDS) and salts and
derivatives thereof, carboxy methyl inulin and salts and
derivatives thereof, nitrilotriacetic acid (NTA), diethylene
triamine penta acetic acid (DTPA), B-alaninediacetic acid (B-ADA)
and their salts, homopolymers and copolymers of poly-carboxylic
acids and their partially or completely neutralized salts,
monomeric polycarboxylic acids and hydroxycarboxylic acids and
their salts in the range of 0.5% to 50% by weight;
sulfonated/carboxylated polymers in the range of about 0.1% to
about 50% by weight to provide dimensional stability; drying aids
in the range of about 0.1% to about 10% by weight (e.g.,
polyesters, especially anionic polyesters, optionally together with
further monomers with 3 to 6 functionalities--typically acid,
alcohol or ester functionalities which are conducive to
polycondensation, polycarbonate-, polyurethane- and/or
polyurea-polyorganosiloxane compounds or precursor compounds,
thereof, particularly of the reactive cyclic carbonate and urea
type); silicates in the range from about 1% to about 20% by weight
(including sodium or potassium silicates for example sodium
disilicate, sodium meta-silicate and crystalline phyllosilicates);
inorganic bleach (e.g., perhydrate salts such as perborate,
percarbonate, perphosphate, persulfate and persilicate salts) and
organic bleach (e.g., organic peroxyacids, including diacyl and
tetraacylperoxides, especially diperoxydodecanedioc acid,
diperoxytetradecanedioc acid, and diperoxyhexadecanedioc acid);
bleach activators (i.e., organic peracid precursors in the range
from about 0.1% to about 10% by weight); bleach catalysts (e.g.,
manganese triazacyclononane and related complexes, Co, Cu, Mn, and
Fe bispyridylamine and related complexes, and pentamine acetate
cobalt(III) and related complexes); metal care agents in the range
from about 0.1% to 5% by weight (e.g., benzatriazoles, metal salts
and complexes, and/or silicates); enzymes in the range from about
0.01 to 5.0 mg of active enzyme per gram of automatic dishwashing
detergent composition (e.g., proteases, amylases, lipases,
cellulases, choline oxidases, peroxidases/oxidases, pectate lyases,
mannanases, cutinases, laccases, phospholipases,
lysophospholipases, acyltransferase, perhydrolase, arylesterase,
and mixtures thereof); and enzyme stabilizer components (e.g.,
oligosaccharides, polysaccharides, and inorganic divalent metal
salts).
7.5. Additional Detergent Compositions
[0486] Additional exemplary detergent formulations to which the
present amylase can be added are described, below, in the numbered
paragraphs.
[0487] 1) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising linear
alkylbenzenesulfonate (calculated as acid) about 7% to about 12%;
alcohol ethoxysulfate (e.g., C12-18 alcohol, 1-2 ethylene oxide
(EO)) or alkyl sulfate (e.g., C16-18) about 1% to about 4%; alcohol
ethoxylate (e.g., C14-15 alcohol, 7 EO) about 5% to about 9%;
sodium carbonate (e.g., Na2CO3) about 14% to about 20%; soluble
silicate (e.g., Na2O, 2SiO2) about 2 to about 6%; zeolite (e.g.,
NaAlSiO4) about 15% to about 22%; sodium sulfate (e.g., Na2SO4) 0%
to about 6%; sodium citrate/citric acid (e.g., C6H5Na3O7/C6H8O7)
about 0% to about 15%; sodium perborate (e.g., NaBO3H2O) about 11%
to about 18%; TAED about 2% to about 6%; carboxymethylcellulose
(CMC) and 0% to about 2%; polymers (e.g., maleic/acrylic acid,
copolymer, PVP, PEG) 0-3%; enzymes (calculated as pure enzyme)
0.0001-0.1% protein; and minor ingredients (e.g., suds suppressors,
perfumes, optical brightener, photobleach) 0-5%.
[0488] 2) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising linear
alkylbenzenesulfonate (calculated as acid) about 6% to about 11%;
alcohol ethoxysulfate (e.g., C12-18 alcohol, 1-2 EO) or alkyl
sulfate (e.g., C16-18) about 1% to about 3%; alcohol ethoxylate
(e.g., C14-15 alcohol, 7 EO) about 5% to about 9%; sodium carbonate
(e.g., Na2CO3) about 15% to about 21%; soluble silicate (e.g.,
Na2O, 2SiO2) about 1% to about 4%; zeolite (e.g., NaAlSiO4) about
24% to about 34%; sodium sulfate (e.g., Na2SO4) about 4% to about
10%; sodium citrate/citric acid (e.g., C6H5Na3O7/C6H8O7) 0% to
about 15%; carboxymethylcellulose (CMC) 0% to about 2%; polymers
(e.g., maleic/acrylic acid copolymer, PVP, PEG) 1-6%; enzymes
(calculated as pure enzyme protein) 0.0001-0.1%; minor ingredients
(e.g., suds suppressors, perfume) 0-5%.
[0489] 3) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising linear
alkylbenzenesulfonate (calculated as acid) about 5% to about 9%;
alcohol ethoxylate (e.g., C12-15 alcohol, 7 EO) about 7% to about
14%; Soap as fatty acid (e.g., C16-22 fatty acid) about 1 to about
3%; sodium carbonate (as Na2CO3) about 10% to about 17%; soluble
silicate (e.g., Na2O, 2SiO2) about 3% to about 9%; zeolite (as
NaAlSiO4) about 23% to about 33%; sodium sulfate (e.g., Na2SO4) 0%
to about 4%; sodium perborate (e.g., NaBO3H2O) about 8% to about
16%; TAED about 2% to about 8%; phosphonate (e.g., EDTMPA) 0% to
about 1%; carboxymethylcellulose (CMC) 0% to about 2%; polymers
(e.g., maleic/acrylic acid copolymer, PVP, PEG) 0-3%; enzymes
(calculated as pure enzyme protein) 0.0001-0.1%; minor ingredients
(e.g., suds suppressors, perfume, optical brightener) 0-5%.
[0490] 4) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising linear
alkylbenzenesulfonate (calculated as acid) about 8% to about 12%;
alcohol ethoxylate (e.g., C12-15 alcohol, 7 EO) about 10% to about
25%; sodium carbonate (as Na2CO3) about 14% to about 22%; soluble
silicate (e.g., Na2O, 2SiO2) about 1% to about 5%; zeolite (e.g.,
NaAlSiO4) about 25% to about 35%; sodium sulfate (e.g., Na2SO4) 0%
to about 10%; carboxymethylcellulose (CMC) 0% to about 2%; polymers
(e.g., maleic/acrylic acid copolymer, PVP, PEG) 1-3%; enzymes
(calculated as pure enzyme protein) 0.0001-0.1%; and minor
ingredients (e.g., suds suppressors, perfume) 0-5%.
[0491] 5) An aqueous liquid detergent composition comprising linear
alkylbenzenesulfonate (calculated as acid) about 15% to about 21%;
alcohol ethoxylate (e.g., C12-15 alcohol, 7 EO or C12-15 alcohol, 5
EO) about 12% to about 18%; soap as fatty acid (e.g., oleic acid)
about 3% to about 13%; alkenylsuccinic acid (C12-14) 0% to about
13%; aminoethanol about 8% to about 18%; citric acid about 2% to
about 8%; phosphonate 0% to about 3%; polymers (e.g., PVP, PEG) 0%
to about 3%; borate (e.g., B407) 0% to about 2%; ethanol 0% to
about 3%; propylene glycol about 8% to about 14%; enzymes
(calculated as pure enzyme protein) 0.0001-0.1%; and minor
ingredients (e.g., dispersants, suds suppressors, perfume, optical
brightener) 0-5%.
[0492] 6) An aqueous structured liquid detergent composition
comprising linear alkylbenzenesulfonate (calculated as acid) about
15% to about 21%; alcohol ethoxylate (e.g., C12-15 alcohol, 7 EO,
or C12-15 alcohol, 5 EO) 3-9%; soap as fatty acid (e.g., oleic
acid) about 3% to about 10%; zeolite (as NaAlSiO4) about 14% to
about 22%; potassium citrate about 9% to about 18%; borate (e.g.,
B407) 0% to about 2%; carboxymethylcellulose (CMC) 0% to about 2%;
polymers (e.g., PEG, PVP) 0% to about 3%; anchoring polymers such
as, e.g., lauryl methacrylate/acrylic acid copolymer; molar ratio
25:1, MW 3800) 0% to about 3%; glycerol 0% to about 5%; enzymes
(calculated as pure enzyme protein) 0.0001-0.1%; and minor
ingredients (e.g., dispersants, suds suppressors, perfume, optical
brighteners) 0-5%.
[0493] 7) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising fatty alcohol sulfate
about 5% to about 10%; ethoxylated fatty acid monoethanolamide
about 3% to about 9%; soap as fatty acid 0-3%; sodium carbonate
(e.g., Na2CO3) about 5% to about 10%; Soluble silicate (e.g., Na2O,
2SiO2) about 1% to about 4%; zeolite (e.g., NaAlSiO4) about 20% to
about 40%; Sodium sulfate (e.g., Na2SO4) about 2% to about 8%;
sodium perborate (e.g., NaBO3H2O) about 12% to about 18%; TAED
about 2% to about 7%; polymers (e.g., maleic/acrylic acid
copolymer, PEG) about 1% to about 5%; enzymes (calculated as pure
enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., optical
brightener, suds suppressors, perfume) 0-5%.
[0494] 8) A detergent composition formulated as a granulate
comprising linear alkylbenzenesulfonate (calculated as acid) about
8% to about 14%; ethoxylated fatty acid monoethanolamide about 5%
to about 11%; soap as fatty acid 0% to about 3%; sodium carbonate
(e.g., Na2CO3) about 4% to about 10%; soluble silicate (Na2O,
2SiO2) about 1% to about 4%; zeolite (e.g., NaAlSiO4) about 30% to
about 50%; sodium sulfate (e.g., Na2SO4) about 3% to about 11%;
sodium citrate (e.g., C6H5Na3O7) about 5% to about 12%; polymers
(e.g., PVP, maleic/acrylic acid copolymer, PEG) about 1% to about
5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and
minor ingredients (e.g., suds suppressors, perfume) 0-5%.
[0495] 9) A detergent composition formulated as a granulate
comprising linear alkylbenzenesulfonate (calculated as acid) about
6% to about 12%; nonionic surfactant about 1% to about 4%; soap as
fatty acid about 2% to about 6%; sodium carbonate (e.g., Na2CO3)
about 14% to about 22%; zeolite (e.g., NaAlSiO4) about 18% to about
32%; sodium sulfate (e.g., Na2SO4) about 5% to about 20%; sodium
citrate (e.g., C6H5Na3O7) about 3% to about 8%; sodium perborate
(e.g., NaBO3H2O) about 4% to about 9%; bleach activator (e.g., NOBS
or TAED) about 1% to about 5%; carboxymethylcellulose (CMC) 0% to
about 2%; polymers (e.g., polycarboxylate or PEG) about 1% to about
5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and
minor ingredients (e.g., optical brightener, perfume) 0-5%.
[0496] 10) An aqueous liquid detergent composition comprising
linear alkylbenzenesulfonate (calculated as acid) about 15% to
about 23%; alcohol ethoxysulfate (e.g., C12-15 alcohol, 2-3 EO)
about 8% to about 15%; alcohol ethoxylate (e.g., C12-15 alcohol, 7
EO, or C12-15 alcohol, 5 EO) about 3% to about 9%; soap as fatty
acid (e.g., lauric acid) 0% to about 3%; aminoethanol about 1% to
about 5%; sodium citrate about 5% to about 10%; hydrotrope (e.g.,
sodium toluensulfonate) about 2% to about 6%; borate (e.g., B407)
0% to about 2%; carboxymethylcellulose 0% to about 1%; ethanol
about 1% to about 3%; propylene glycol about 2% to about 5%;
enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor
ingredients (e.g., polymers, dispersants, perfume, optical
brighteners) 0-5%.
[0497] 11) An aqueous liquid detergent composition comprising
linear alkylbenzenesulfonate (calculated as acid) about 20% to
about 32%; alcohol ethoxylate (e.g., C12-15 alcohol, 7 EO, or
C12-15 alcohol, 5 EO) 6-12%; aminoethanol about 2% to about 6%;
citric acid about 8% to about 14%; borate (e.g., B407) about 1% to
about 3%; polymer (e.g., maleic/acrylic acid copolymer, anchoring
polymer such as, e.g., lauryl methacrylate/acrylic acid copolymer)
0% to about 3%; glycerol about 3% to about 8%; enzymes (calculated
as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g.,
hydrotropes, dispersants, perfume, optical brighteners) 0-5%.
[0498] 12) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising anionic surfactant
(linear alkylbenzenesulfonate, alkyl sulfate,
.alpha.-olefinsulfonate, .alpha.-sulfo fatty acid methyl esters,
alkanesulfonates, soap) about 25% to about 40%; nonionic surfactant
(e.g., alcohol ethoxylate) about 1% to about 10%; sodium carbonate
(e.g., Na2CO3) about 8% to about 25%; soluble silicates (e.g.,
Na2O, 2SiO2) about 5% to about 15%; sodium sulfate (e.g., Na2SO4)
0% to about 5%; zeolite (NaAlSiO4) about 15% to about 28%; sodium
perborate (e.g., NaBO3.4H2O) 0% to about 20%; bleach activator
(TAED or NOBS) about 0% to about 5%; enzymes (calculated as pure
enzyme protein) 0.0001-0.1%; minor ingredients (e.g., perfume,
optical brighteners) 0-3%.
[0499] 13) Detergent compositions as described in compositions
1)-12) supra, wherein all or part of the linear
alkylbenzenesulfonate is replaced by (C12-C18) alkyl sulfate.
[0500] 14) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising (C12-C18) alkyl
sulfate about 9% to about 15%; alcohol ethoxylate about 3% to about
6%; polyhydroxy alkyl fatty acid amide about 1% to about 5%;
zeolite (e.g., NaAlSiO4) about 10% to about 20%; layered disilicate
(e.g., SK56 from Hoechst) about 10% to about 20%; sodium carbonate
(e.g., Na2CO3) about 3% to about 12%; soluble silicate (e.g., Na2O,
2SiO2) 0% to about 6%; sodium citrate about 4% to about 8%; sodium
percarbonate about 13% to about 22%; TAED about 3% to about 8%;
polymers (e.g., polycarboxylates and PVP) 0% to about 5%; enzymes
(calculated as pure enzyme protein) 0.0001-0.1%; and minor
ingredients (e.g., optical brightener, photobleach, perfume, suds
suppressors) 0-5%.
[0501] 15) A detergent composition formulated as a granulate having
a bulk density of at least 600 g/L comprising (C12-C18) alkyl
sulfate about 4% to about 8%; alcohol ethoxylate about 11% to about
15%; soap about 1% to about 4%; zeolite MAP or zeolite A about 35%
to about 45%; sodium carbonate (as Na2CO3) about 2% to about 8%;
soluble silicate (e.g., Na2O, 2SiO2) 0% to about 4%; sodium
percarbonate about 13% to about 22%; TAED 1-8%;
carboxymethylcellulose (CMC) 0% to about 3%; polymers (e.g.,
polycarboxylates and PVP) 0% to about 3%; enzymes (calculated as
pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g.,
optical brightener, phosphonate, perfume) 0-3%.
[0502] 16) Detergent formulations as described in 1)-15) supra,
which contain a stabilized or encapsulated peracid, either as an
additional component or as a substitute for already specified
bleach systems.
[0503] 17) Detergent compositions as described supra in 1), 3), 7),
9), and 12), wherein perborate is replaced by percarbonate.
[0504] 18) Detergent compositions as described supra in 1), 3), 7),
9), 12), 14), and 15), which additionally contain a manganese
catalyst. The manganese catalyst for example is one of the
compounds described in "Efficient manganese catalysts for
low-temperature bleaching," Nature 369: 637-639 (1994).
[0505] 19) Detergent composition formulated as a non-aqueous
detergent liquid comprising a liquid nonionic surfactant such as,
e.g., linear alkoxylated primary alcohol, a builder system (e.g.,
phosphate), an enzyme(s), and alkali. The detergent may also
comprise anionic surfactant and/or a bleach system.
[0506] As above, the present amylase polypeptide may be
incorporated at a concentration conventionally employed in
detergents. It is at present contemplated that, in the detergent
composition, the enzyme may be added in an amount corresponding to
0.00001-1.0 mg (calculated as pure enzyme protein) of amylase
polypeptide per liter of wash liquor.
[0507] The detergent composition may also contain other
conventional detergent ingredients, e.g., deflocculant material,
filler material, foam depressors, anti-corrosion agents,
soil-suspending agents, sequestering agents, anti-soil redeposition
agents, dehydrating agents, dyes, bactericides, fluorescers,
thickeners, and perfumes.
[0508] The detergent composition may be formulated as a hand
(manual) or machine (automatic) laundry detergent composition,
including a laundry additive composition suitable for pre-treatment
of stained fabrics and a rinse added fabric softener composition,
or be formulated as a detergent composition for use in general
household hard surface cleaning operations, or be formulated for
manual or automatic dishwashing operations.
[0509] Any of the cleaning compositions described, herein, may
include any number of additional enzymes. In general the enzyme(s)
should be compatible with the selected detergent, (e.g., with
respect to pH-optimum, compatibility with other enzymatic and
non-enzymatic ingredients, and the like), and the enzyme(s) should
be present in effective amounts. The following enzymes are provided
as examples.
[0510] Proteases: Suitable proteases include those of animal,
vegetable or microbial origin. Chemically modified or protein
engineered mutants are included, as well as naturally processed
proteins. The protease may be a serine protease or a
metalloprotease, an alkaline microbial protease, a trypsin-like
protease, or a chymotrypsin-like protease. Examples of alkaline
proteases are subtilisins, especially those derived from Bacillus,
e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309,
subtilisin 147, and subtilisin 168 (see, e.g., WO 89/06279).
Additional examples include those mutant proteases described in
U.S. Pat. Nos. RE 34,606, 5,955,340, 5,700,676, 6,312,936, and
6,482,628, all of which are incorporated herein by reference.
Examples of trypsin-like proteases are trypsin (e.g., of porcine or
bovine origin), and Fusarium proteases (see, e.g., WO 89/06270 and
WO 94/25583). Examples of useful proteases also include but are not
limited to the variants described in WO 92/19729, WO 98/20115, WO
98/20116, and WO 98/34946. Commercially available protease enzymes
include but are not limited to: Alcalase.RTM., Savinase.RTM.,
Primase.TM., Duralase.TM., Esperase.RTM., BLAZE.TM. POLARZYME.RTM.,
OVOZYME.RTM., KANNASE.RTM., LIQUANASE.RTM., NEUTRASE.RTM.,
RELASE.RTM., and ESPERASE.RTM. (Novo Nordisk A/S and Novozymes
A/S), Maxatase.RTM., Maxacal.TM. Maxapem.TM., Properase.RTM.,
Purafect.RTM., Purafect OxP.TM., Purafect Prime.TM., FNA.TM.,
FN2.TM. FN3.TM., OPTICLEAN.RTM., OPTIMASE.RTM., PURAMAX.TM.,
EXCELLASE.TM., and PURAFAST.TM. (Danisco US Inc./DuPont Industrial
Biosciences, Palo Alto, Calif., USA), BLAP.TM. and BLAP.TM.
variants (Henkel Kommanditgesellschaft auf Aktien, Duesseldorf,
Germany), and KAP (B. alkalophilus subtilisin; Kao Corp., Tokyo,
Japan). Another exemplary proteases NprE from Bacillus
amyloliquifaciens and ASP from Cellulomonas sp. strain 69B4
(Danisco US Inc./DuPont Industrial Biosciences, Palo Alto, Calif.,
USA). Various proteases are described in WO95/23221, WO 92/21760,
WO 09/149200, WO 09/149144, WO 09/149145, WO 11/072099, WO
10/056640, WO 10/056653, WO 11/140364, WO 12/151534, U.S. Pat.
Publ. No. 2008/0090747, and U.S. Pat. Nos. 5,801,039, 5,340,735,
5,500,364, 5,855,625, US RE 34,606, 5,955,340, 5,700,676,
6,312,936, and 6,482,628, and various other patents. In some
further embodiments, metalloproteases find use in the present
invention, including but not limited to the neutral metalloprotease
described in WO 07/044993. Suitable proteases include naturally
occurring proteases or engineered variants specifically selected or
engineered to work at relatively low temperatures.
[0511] Lipases: Suitable lipases include those of bacterial or
fungal origin. Chemically modified, proteolytically modified, or
protein engineered mutants are included. Examples of useful lipases
include but are not limited to lipases from Humicola (synonym
Thermomyces), e.g., from H. lanuginosa (T. lanuginosus) (see e.g.,
EP 258068 and EP 305216), from H. insolens (see e.g., WO 96/13580);
a Pseudomonas lipase (e.g., from P. alcaligenes or P.
pseudoalcaligenes; see, e.g., EP 218 272), P. cepacia (see e.g., EP
331 376), P. stutzeri (see e.g., GB 1,372,034), P. fluorescens,
Pseudomonas sp. strain SD 705 (see e.g., WO 95/06720 and WO
96/27002), P. wisconsinensis (see e.g., WO 96/12012); a Bacillus
lipase (e.g., from B. subtilis; see e.g., Dartois et al. Biochemica
et Biophysica Acta, 1131: 253-360 (1993)), B. stearothermophilus
(see e.g., JP 64/744992), or B. pumilus (see e.g., WO 91/16422).
Additional lipase variants contemplated for use in the formulations
include those described for example in: WO 92/05249, WO 94/01541,
WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO
95/22615, WO 97/04079, WO 97/07202, EP 407225, and EP 260105. Some
commercially available lipase enzymes include Lipolase.RTM. and
Lipolase Ultra.TM. (Novo Nordisk A/S and Novozymes A/S).
[0512] Polyesterases: Suitable polyesterases can be included in the
composition, such as those described in, for example, WO 01/34899,
WO 01/14629, and U.S. Pat. No. 6,933,140.
[0513] Amylases: The present compositions can be combined with
other amylases, including other .alpha.-amylases. Such a
combination is particularly desirable when different
.alpha.-amylases demonstrate different performance characteristics
and the combination of a plurality of different .alpha.-amylases
results in a composition that provides the benefits of the
different .alpha.-amylases. Other amylases include commercially
available amylases, such as but not limited to STAINZYME.RTM.,
NATALASE.RTM., DURAMYL.RTM., TERMAMYL.RTM., FUNGAMYL.RTM. and
BAN.TM. (Novo Nordisk A/S and Novozymes A/S); RAPIDASE.RTM.,
POWERASE.RTM., PURASTAR.RTM., and PREFERENZ.TM. (from DuPont
Industrial Biosciences).
[0514] Cellulases: Cellulases can be added to the compositions.
Suitable cellulases include those of bacterial or fungal origin.
Chemically modified or protein engineered mutants are included.
Suitable cellulases include cellulases from the genera Bacillus,
Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the
fungal cellulases produced from Humicola insolens, Myceliophthora
thermophila and Fusarium oxysporum disclosed for example in U.S.
Pat. Nos. 4,435,307; 5,648,263; 5,691,178; 5,776,757; and WO
89/09259. Exemplary cellulases contemplated for use are those
having color care benefit for the textile. Examples of such
cellulases are cellulases described in for example EP 0495257, EP
0531372, WO 96/11262, WO 96/29397, and WO 98/08940. Other examples
are cellulase variants, such as those described in WO 94/07998; WO
98/12307; WO 95/24471; PCT/DK98/00299; EP 531315; U.S. Pat. Nos.
5,457,046; 5,686,593; and 5,763,254. Commercially available
cellulases include CELLUZYME.RTM. and CAREZYME.RTM. (Novo Nordisk
A/S and Novozymes A/S); CLAZINASE.RTM. and PURADAX HA.RTM. (DuPont
Industrial Biosciences); and KAC-500(B).TM. (Kao Corporation).
[0515] Peroxidases/Oxidases: Suitable peroxidases/oxidases
contemplated for use in the compositions include those of plant,
bacterial or fungal origin. Chemically modified or protein
engineered mutants are included. Examples of useful peroxidases
include peroxidases from Coprinus, e.g., from C. cinereus, and
variants thereof as those described in WO 93/24618, WO 95/10602,
and WO 98/15257. Commercially available peroxidases include for
example GUARDZYME.TM. (Novo Nordisk A/S and Novozymes A/S).
[0516] The detergent composition can also comprise
2,6-.beta.-D-fructan hydrolase, which is effective for
removal/cleaning of biofilm present on household and/or industrial
textile/laundry.
[0517] The detergent enzyme(s) may be included in a detergent
composition by adding separate additives containing one or more
enzymes, or by adding a combined additive comprising all of these
enzymes. A detergent additive, i.e. a separate additive or a
combined additive, can be formulated e.g., as a granulate, a
liquid, a slurry, and the like. Exemplary detergent additive
formulations include but are not limited to granulates, in
particular non-dusting granulates, liquids, in particular
stabilized liquids or slurries.
[0518] Non-dusting granulates may be produced, e.g., as disclosed
in U.S. Pat. Nos. 4,106,991 and 4,661,452 and may optionally be
coated by methods known in the art. Examples of waxy coating
materials are poly(ethylene oxide) products (e.g.,
polyethyleneglycol, PEG) with mean molar weights of 1,000 to
20,000; ethoxylated nonylphenols having from 16 to 50 ethylene
oxide units; ethoxylated fatty alcohols in which the alcohol
contains from 12 to 20 carbon atoms and in which there are 15 to 80
ethylene oxide units; fatty alcohols; fatty acids; and mono- and
di- and triglycerides of fatty acids. Examples of film-forming
coating materials suitable for application by fluid bed techniques
are given in, for example, GB 1483591. Liquid enzyme preparations
may, for instance, be stabilized by adding a polyol such as
propylene glycol, a sugar or sugar alcohol, lactic acid or boric
acid according to established methods. Protected enzymes may be
prepared according to the method disclosed in EP 238,216.
[0519] The detergent composition may be in any convenient form,
e.g., a bar, a tablet, a powder, a granule, a paste, or a liquid. A
liquid detergent may be aqueous, typically containing up to about
70% water, and 0% to about 30% organic solvent. Compact detergent
gels containing about 30% or less water are also contemplated. The
detergent composition can optionally comprise one or more
surfactants, which may be non-ionic, including semi-polar and/or
anionic and/or cationic and/or zwitterionic. The surfactants can be
present in a wide range, from about 0.1% to about 60% by
weight.
[0520] When included therein the detergent will typically contain
from about 1% to about 40% of an anionic surfactant, such as linear
alkylbenzenesulfonate, .alpha.-olefinsulfonate, alkyl sulfate
(fatty alcohol sulfate), alcohol ethoxysulfate, secondary
alkanesulfonate, .alpha.-sulfo fatty acid methyl ester, alkyl- or
alkenylsuccinic acid, or soap.
[0521] When included therein, the detergent will usually contain
from about 0.2% to about 40% of a non-ionic surfactant such as
alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside,
alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide,
fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or
N-acyl-N-alkyl derivatives of glucosamine ("glucamides").
[0522] The detergent may contain 0% to about 65% of a detergent
builder or complexing agent such as zeolite, diphosphate,
triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic
acid, ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid,
soluble silicates or layered silicates (e.g., SKS-6 from
Hoechst).
[0523] The detergent may comprise one or more polymers. Exemplary
polymers include carboxymethylcellulose (CMC),
poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) (PEG),
poly(vinyl alcohol) (PVA), poly(vinylpyridine-N-oxide),
poly(vinylimidazole), polycarboxylates e.g., polyacrylates,
maleic/acrylic acid copolymers), and lauryl methacrylate/acrylic
acid copolymers.
[0524] The enzyme(s) of the detergent composition may be stabilized
using conventional stabilizing agents, e.g., as polyol (e.g.,
propylene glycol or glycerol), a sugar or sugar alcohol, lactic
acid, boric acid, or a boric acid derivative (e.g., an aromatic
borate ester), or a phenyl boronic acid derivative (e.g.,
4-formylphenyl boronic acid). The composition may be formulated as
described in WO 92/19709 and WO 92/19708.
[0525] It is contemplated that in the detergent compositions, in
particular the enzyme variants, may be added in an amount
corresponding to about 0.01 to about 100 mg of enzyme protein per
liter of wash liquor (e.g., about 0.05 to about 5.0 mg of enzyme
protein per liter of wash liquor or 0.1 to about 1.0 mg of enzyme
protein per liter of wash liquor).
[0526] Numerous exemplary detergent formulations to which the
present amylases can be added (or is in some cases are identified
as a component of) are described in WO2013063460. These include
commercially available unit dose detergent formulations/packages
such as PUREX.RTM. UltraPacks (Henkel), FINISH.RTM. Quantum
(Reckitt Benckiser), CLOROX.TM. 2 Packs (Clorox), OxiClean Max
Force Power Paks (Church & Dwight), TIDE.RTM. Stain Release,
CASCADE.RTM. ActionPacs, and TIDE.RTM. Pods.TM. (Procter &
Gamble), PS.
7.6. Methods of Assessing Amylase Activity in Detergent
Compositions
[0527] Numerous .alpha.-amylase cleaning assays are known in the
art, including swatch and micro-swatch assays. The appended
Examples describe only a few such assays.
[0528] In order to further illustrate the compositions and methods,
and advantages thereof, the following specific examples are given
with the understanding that they are illustrative rather than
limiting.
8. Brewing Compositions
[0529] The present variant amylase may be a component of a brewing
composition used in a process of brewing, i.e., making a fermented
malt beverage. Non-fermentable carbohydrates form the majority of
the dissolved solids in the final beer. This residue remains
because of the inability of malt amylases to hydrolyze the
alpha-1,6-linkages of the starch. The non-fermentable carbohydrates
contribute about 50 calories per 12 ounces of beer. an amylase, in
combination with a glucoamylase and optionally a pullulanase and/or
isoamylase, assist in converting the starch into dextrins and
fermentable sugars, lowering the residual non-fermentable
carbohydrates in the final beer.
[0530] The principal raw materials used in making these beverages
are water, hops and malt. In addition, adjuncts such as common corn
grits, refined corn grits, brewer's milled yeast, rice, sorghum,
refined corn starch, barley, barley starch, dehusked barley, wheat,
wheat starch, torrified cereal, cereal flakes, rye, oats, potato,
tapioca, and syrups, such as corn syrup, sugar cane syrup, inverted
sugar syrup, barley and/or wheat syrups, and the like may be used
as a source of starch.
[0531] For a number of reasons, the malt, which is produced
principally from selected varieties of barley, has the greatest
effect on the overall character and quality of the beer. First, the
malt is the primary flavoring agent in beer. Second, the malt
provides the major portion of the fermentable sugar. Third, the
malt provides the proteins, which will contribute to the body and
foam character of the beer. Fourth, the malt provides the necessary
enzymatic activity during mashing. Hops also contribute
significantly to beer quality, including flavoring. In particular,
hops (or hops constituents) add desirable bittering substances to
the beer. In addition, the hops act as protein precipitants,
establish preservative agents and aid in foam formation and
stabilization.
[0532] Grains, such as barley, oats, wheat, as well as plant
components, such as corn, hops, and rice, also are used for
brewing, both in industry and for home brewing. The components used
in brewing may be unmalted or may be malted, i.e., partially
germinated, resulting in an increase in the levels of enzymes,
including .alpha.-amylase. For successful brewing, adequate levels
of .alpha.-amylase enzyme activity are necessary to ensure the
appropriate levels of sugars for fermentation. an amylase, by
itself or in combination with another .alpha.-amylase(s),
accordingly may be added to the components used for brewing.
[0533] As used herein, the term "stock" means grains and plant
components that are crushed or broken. For example, barley used in
beer production is a grain that has been coarsely ground or crushed
to yield a consistency appropriate for producing a mash for
fermentation. As used herein, the term "stock" includes any of the
aforementioned types of plants and grains in crushed or coarsely
ground forms. The methods described herein may be used to determine
.alpha.-amylase activity levels in both flours and stock.
[0534] Processes for making beer are well known in the art. See,
e.g., Wolfgang Kunze (2004) "Technology Brewing and Malting,"
Research and Teaching Institute of Brewing, Berlin (VLB), 3rd
edition. Briefly, the process involves: (a) preparing a mash, (b)
filtering the mash to prepare a wort, and (c) fermenting the wort
to obtain a fermented beverage, such as beer. Typically, milled or
crushed malt is mixed with water and held for a period of time
under controlled temperatures to permit the enzymes present in the
malt to convert the starch present in the malt into fermentable
sugars. The mash is then transferred to a mash filter where the
liquid is separated from the grain residue. This sweet liquid is
called "wort," and the left over grain residue is called "spent
grain." The mash is typically subjected to an extraction, which
involves adding water to the mash in order to recover the residual
soluble extract from the spent grain. The wort is then boiled
vigorously to sterilizes the wort and help develop the color,
flavor and odor. Hops are added at some point during the boiling.
The wort is cooled and transferred to a fermentor.
[0535] The wort is then contacted in a fermentor with yeast. The
fermentor may be chilled to stop fermentation. The yeast
flocculates and is removed. Finally, the beer is cooled and stored
for a period of time, during which the beer clarifies and its
flavor develops, and any material that might impair the appearance,
flavor and shelf life of the beer settles out. The beer usually
contains from about 2% to about 10% v/v alcohol, although beer with
a higher alcohol content, e.g., 18% v/v, may be obtained. Prior to
packaging, the beer is carbonated and, optionally, filtered and
pasteurized.
[0536] The brewing composition comprising an amylase, in
combination with a glucoamylase and optionally a pullulanase and/or
isoamylase, may be added to the mash of step (a) above, i.e.,
during the preparation of the mash. Alternatively, or in addition,
the brewing composition may be added to the mash of step (b) above,
i.e., during the filtration of the mash. Alternatively, or in
addition, the brewing composition may be added to the wort of step
(c) above, i.e., during the fermenting of the wort.
[0537] A fermented beverage, such as a beer, can be produced by one
of the methods above. The fermented beverage can be a beer, such as
full malted beer, beer brewed under the "Reinheitsgebot," ale, IPA,
lager, bitter, Happoshu (second beer), third beer, dry beer, near
beer, light beer, low alcohol beer, low calorie beer, porter, bock
beer, stout, malt liquor, non-alcoholic beer, non-alcoholic malt
liquor and the like, but also alternative cereal and malt beverages
such as fruit flavored malt beverages, e.g., citrus flavored, such
as lemon-, orange-, lime-, or berry-flavored malt beverages, liquor
flavored malt beverages, e.g., vodka-, rum-, or tequila-flavored
malt liquor, or coffee flavored malt beverages, such as
caffeine-flavored malt liquor, and the like.
9. Reduction of Iodine-Positive Starch
[0538] Variant amylases may reduce the iodine-positive starch
(IPS), when used in a method of liquefaction and/or
saccharification. One source of IPS is from amylose that escapes
hydrolysis and/or from retrograded starch polymer. Starch
retrogradation occurs spontaneously in a starch paste, or gel on
ageing, because of the tendency of starch molecules to bind to one
another followed by an increase in crystallinity. Solutions of low
concentration become increasingly cloudy due to the progressive
association of starch molecules into larger articles. Spontaneous
precipitation takes place and the precipitated starch appears to be
reverting to its original condition of cold-water insolubility.
Pastes of higher concentration on cooling set to a gel, which on
ageing becomes steadily firmer due to the increasing association of
the starch molecules. This arises because of the strong tendency
for hydrogen bond formation between hydroxy groups on adjacent
starch molecules. See J. A. Radley, ed., Starch and its Derivatives
194-201 (Chapman and Hall, London (1968)).
[0539] The presence of IPS in saccharide liquor negatively affects
final product quality and represents a major issue with downstream
processing. IPS plugs or slows filtration system, and fouls the
carbon columns used for purification. When IPS reaches sufficiently
high levels, it may leak through the carbon columns and decrease
production efficiency. Additionally, it may results in hazy final
product upon storage, which is unacceptable for final product
quality. The amount of IPS can be reduced by isolating the
saccharification tank and blending the contents back. IPS
nevertheless will accumulate in carbon columns and filter systems,
among other things. The use of variant amylases is expected to
improve overall process performance by reducing the amount of
IPS.
[0540] All references cited herein are herein incorporated by
reference in their entirety for all purposes. In order to further
illustrate the compositions and methods, and advantages thereof,
the following specific examples are given with the understanding
that they are illustrative rather than limiting.
EXAMPLES
Example 1
Assays
[0541] Various assays used herein are set forth, below, for ease in
reading. Any deviations from the protocols in later Examples are
indicated in the relevant sections. In these experiments, a
spectrophotometer was used to measure the absorbance of the
products formed after the completion of the reactions.
A. Chelex Bead Treatment of Culture Supernatants
[0542] 96-well microtiter plates (MTPs) containing growing cultures
were removed from incubators and Enzyscreen lids were replaced with
disposable plastic sealers (Nunc cat. #236366; Rochester, N.Y.,
USA). Cells were separated from culture supernatant via
centrifugation (1118 RCF, 5 minutes). 150 .mu.L supernatant was
removed from each well and transferred to filter plates (Millipore
Multiscreen HTS, Billerica, Mass., USA) containing Chelex beads
prepared as described below. Plates were shaken vigorously for 5
minutes and supernatant from 3 replicate growth plates were
collected into a single deep-well microtiter plate (Axygen,
PDW-11-C) using a vacuum manifold device. Plates containing
supernatants were sealed and stored at 4.degree. C.
[0543] Chelex-100 beads, 200-400 mesh (BioRad, Hercules, Calif.,
USA) were washed twice with 2 bed-volumes of 1 M HCl followed by 5
bed-volumes of ultrapure water on a sintered glass filter
apparatus. 2 bed-volumes of 1 M KOH were used to wash the beads
followed by another 5 bed-volume wash with ultrapure water.
Filtered beads were transferred to a beaker and suspended with
enough ultrapure water to produce slurry capable of mixing. The pH
of the slurry was adjusted to 8-8.5 using HCl. The liquid was
removed and the beads were dried using a scintered glass filter. A
slurry of beads (40% w/v) was prepared in ultra pure water and its
pH was adjusted to 8.0 using KOH/HCl. A slurry having a constant
consistency was maintained by vigorous mixing. A bubble paddle
reservoir device (V&P Scientific, San Diego, Calif., USA) was
used to transfer 100 .mu.L of slurry to all wells of filter plates.
Liquid was removed using a vacuum manifold device.
B. Protein Purification
[0544] Bacillus strains expressing amylase variants were grown in
2.5 L flasks in cultivation medium (enriched semi-defined media
based on MOPs buffer, with urea as the major nitrogen source,
glucose as the main carbon source, and supplemented with 1% soytone
for robust cell growth) for 60-72 hours at 37.degree. C. or in 14 L
tanks using a fed batch fermentation process with a medium of corn
steep and soy flour supplemented with mineral salts and glucose as
carbon source for 100 hours at 36.degree. C. Following incubation,
the cells were separated from the fermentation medium by
centrifugation and the supernatants were concentrated by
ultra-filtration. Ammonium sulphate was added to the concentrate to
a final concentration of 0.5M. The proteins were purified using
hydrophobic interaction chromatography using a phenyl sepharose
column on the AKTA Explorer FPLC system (GE Healthcare). The column
was equilibrated with 50 mM HEPES, pH 8, with 2 mM CaCl.sub.2) and
0.5 M ammonium sulfate, and the proteins were eluted with 50 mM
HEPES, pH 8, with 2 mM CaCl.sub.2) and 50% propylene glycol. After
each HPLC run, liquid fractions associated with the peak of
interest were pooled, and absorbance measurements of the pooled
fractions were taken to estimate initial concentrations. Protein
concentration of concentrated samples was determined by averaging
the result from three different measurements: absorbance
measurements at 280 nm, SDS-PAGE densitometry of acid-treated
samples compared to a known standard, and by running the proteins
on an HPLC system and taking absorbance measurements at 215 nm and
280 nm.
C. Protein Determination Assay
[0545] Protein determination assays were performed using chelex
bead-treated culture supernatant from cultures grown in 96-well
micro-titer plates (MTPs) over 3 days at 37.degree. C. with shaking
at 300 rpm and 80% humidity. A fresh 96-well round-bottom MTP
containing 25 supernatant per well was used for the High
Performance Liquid Chromatography (HPLC) protein determination
method. Supernatants were diluted four fold into 25 mM sodium
acetate pH 5.5, and 10 .mu.L of each diluted sample was analyzed.
An Agilent 1200 (Hewlett Packard) HPLC equipped with a Poroshell
300SB-C8 (Agilent Technologies Santa Clara, Calif., USA) column was
used. Sample was bound to the column using 25 mM sodium acetate pH
5.5 and eluted over a gradient up to 70% acetonitrile. Absorbance
was measured at 220 nm, integrated using ChemStation software
(Agilent Technologies) and the protein concentration of samples was
determined based on a standard curve of purified CspAmy2-v1
protein.
D. Ceralpha .alpha.-Amylase Activity Assay
[0546] The Ceralpha .alpha.-amylase assay was performed using the
Ceralpha Kit (Megazyme, Wicklow, Ireland). The assay involves
incubating culture supernatant with a substrate mixture under
defined conditions, and the reaction is terminated (and color
developed) by the addition of borate buffer (200 mM Boric acid/NaOH
buffer, pH 10). The substrate is a mixture of the defined
oligosaccharide "nonreducing-end blocked p-nitrophenyl
maltoheptaoside" (BPNPG7) and excess levels of .alpha.-glucosidase
(which has no action on the native substrate due to the presence of
the "blocking group"). On hydrolysis of the oligosaccharide by
endoacting .alpha.-amylase, the excess quantities of
.alpha.-glucosidase present in the mixture give instantaneous and
quantitative hydrolysis of the p-nitrophenyl maltosaccharide
fragment to glucose and free p-nitrophenol. The absorbance at 405
nm was measured, which relates directly to the level of amylase in
the sample analyzed.
[0547] The equipment used for this assay included a Biomek FX Robot
(Beckman Coulter Brea, Calif., USA); a SpectraMAX MTP Reader (type
340-Molecular Devices, Sunnyvale, Calif., USA) and iEMS
incubator/shaker (Thermo Scientific, Rockford, Ill., USA). The
reagent and solutions used were: [0548] 1) p-nitrophenyl
maltoheptaoside (BPNPG7) substrate (Megazyme Ceralpha HR kit);
[0549] 2) 50 mM Malate buffer, 0.005% TWEEN.RTM. 80, pH 5.6 or 50
mM MOPS, 0.005% TWEEN.RTM. 80, pH 7 (dilution buffers); and [0550]
3) 200 mM Boric acid/NaOH buffer, pH 10 (STOP buffer).
[0551] A vial containing 54.5 mg BPNPG7 substrate was dissolved in
10 mL of MilliQ water and then diluted into 30 mL of dilution
buffer to make up 40 mL of the working substrate (1.36 mg/mL). The
amylase samples (fermentation supernatant) were diluted 40.times.
with dilution buffer. The assay was performed by adding 54, of
diluted amylase solution into the wells of a MTP followed by the
addition of 55 .mu.L of diluted BPNPG7 working substrate solution.
The solutions were mixed and the MTP was sealed with a plate seal
and placed in an incubator/shaker (iEMS--Thermo Scientific) for 4
minutes at 25.degree. C. The reaction was terminated by adding 70
.mu.L STOP buffer and the absorbance was read at wavelength 400 nm
in an MTP-Reader. A non-enzyme control was used to correct for
background absorbance values.
E. Thermostability Assay
[0552] The thermostability of CspAmy2-v1 and variants was measured
by determining the amylase activity using the Ceralpha
.alpha.-amylase assay. The equipment used for this assay included a
Biomek FX Robot (Beckman Coulter); a SpectraMAX MTP Reader (type
340-Molecular Devices), a Tetrad2DNA Engine PCR machine (Biorad),
and iEMS incubator/shaker (Thermo Scientific). The reagent
solutions used were (* not in all assays): [0553] 1) Heat stress
buffers [0554] a) 50 mM KOAc pH 4.5 (5 ppm CaCl.sub.2), 50 ppm
NaCl)*, [0555] b) 50 mM KOAc pH 5.0 (10 ppm CaCl.sub.2), 10 mM
NaCl) [0556] c) 50 mM KOAc pH 5.7 (5 ppm CaCl.sub.2), 50 ppm NaCl),
[0557] d) 50 mM KOAc pH 5.7 (no salt condition)*, [0558] 2)
p-nitrophenyl maltoheptaoside (BPNPG7) substrate (Megazyme Ceralpha
HR kit): [0559] 3) 50 mM Malate buffer, 0.005% TWEEN.RTM. 80, pH
5.6 (dilution buffer); and [0560] 4) 200 mM Boric acid/NaOH, pH 10
(STOP buffer). [0561] 5) Amylase culture supernatant: 1:10 master
dilution enzyme plates were diluted 1:10 in each of the four heat
stress buffers in a PCR plate
[0562] 5 .mu.L of the diluted enzyme samples were added to a
96-well PCR plate containing 55 .mu.L of diluted BPNPG7 working
substrate solution and the initial amylase activity of the samples
was determined using the Ceralpha .alpha.-amylase assay as
described in Section C. The samples were subjected to heat stress
for 3-6 minutes in a PCR thermocycler as follows: Buffers (a)
50.degree. C., (b) 59.degree.-60.degree. C., (c)
65.degree.-70.degree. C., and (d) 65.degree. C. The heat stressed
samples were cooled immediately to room temperature and 5 .mu.L
aliquots were assayed for amylase activity using the Ceralpha
.alpha.-amylase assay as described in Section C. For each variant,
the ratio of the initial and residual amylase activities was used
to calculate the thermostability as follows:
Thermostability=[t.sub.residual value]/[t.sub.initial value], so
the heat stability activity ratio was calculated based on enzyme
activity after heat incubation divided by enzyme activity before
heat incubation. For each sample (variants) the performance index
(PI) is calculated. The performance index for thermostability
stability is determined by comparing the thermostability of the
variant enzyme with that of a similarly treated reference
enzyme.
F. Starch Hydrolysis Assays (Corn Flour and Corn Starch Application
Assays)
[0563] Starch hydrolysis of corn flour and corn starch were used to
measure specific activity of CspAmy2-v1 and variants. Activity was
measured as reducing ends generated by the enzymatic breakdown of
corn flour or corn starch. The reducing ends generated during the
incubation with either substrate were quantified using a PAHBAH
(p-hydroxybenzoic acid hydrazide) assay. The equipment used for the
assay included a Biomek FX Robot (Beckman Coulter); a SpectraMAX
MTP Reader (type 340-Molecular Devices), a Tetrad2DNA Engine PCR
machine (Biorad), and iEMS incubator/shaker (Thermo Scientific),
and a Bubble Paddle Reservoir.
[0564] Azure Farms Organic Corn Flour (Norco, CA) was ground to a
fine powder using a consumer coffee grinder and then sifted to
obtain a <250 micron fraction. The sifted corn flour was washed
extensively with MilliQ water by repeated suspension and
centrifugation. Cargill Farms Organic Corn Starch material was also
washed extensively with MilliQ water by repeated suspension and
centrifugation.
[0565] Both corn flour and corn starch washed fractions were
suspended in MilliQ water containing 0.005% sodium azide as 20%
(w/w) stock solutions. The stock solutions were further diluted
with a 20.times. stock buffer solution to 10.9% w/v corn flour and
corn starch solutions (final buffer concentration: 55 mM KOAc, pH
5).
[0566] 55 .mu.L of the diluted corn flour and corn starch
substrates were added to PCR microtiter plates along with 5 .mu.L
of 1:10 diluted enzyme samples using a bubble paddle reservoir. The
plates were sealed and placed at 83.degree. C. for 5 minutes
followed by a ramp down to 45.degree. C. The starch hydrolysis
reaction was terminated by addition of 70 .mu.L 0.1 N NaOH. The
plates were sealed and centrifuged for 3 minutes at 1610 RCF. The
starch hydrolysis reaction products from both reactions were
analyzed by the PAHBAH assay as described below.
[0567] PAHBAH Assay:
[0568] Aliquots of 80 .mu.L of 0.5 N NaOH were added to all wells
of an empty PCR plate (a "PAHBAH reaction plate"), followed by 20
.mu.L of PAHBAH reagent (5% w/v p-hydroxybenzoic acid hydrazide
(Sigma # H9882, St. Louis, Mo.), dissolved in 0.5 N HCl). The
solutions were mixed by pipetting up and down. 20 .mu.L of the
starch hydrolysis reaction supernatants were added to each well of
the PAHBAH reaction plate. The plates were sealed and placed in a
thermocycler, programmed for 2 minutes at 95.degree. C. to develop
color, and then cooled to 20.degree. C. Samples of 80 .mu.L of the
developed PAHBAH reaction mixtures were transferred to a fresh
plate, and absorbance was measured at 450 nm in a
spectrophotometer.
G. CS-28 Rice Starch Microswatch Assay
[0569] The principle of this amylase assay is the liberation of an
orange dye due to the hydrolysis of rice starch incorporated in a
cotton microswatch. The absorbance at 488 nm of the wash liquid is
measured and this relates to the level of amylase activity in the
sample analyzed at the desired conditions (pH, temperature, and
buffer).
[0570] The equipment used for this assay included a Biomek FX Robot
(Beckman Coulter), a SpectraMAX MTP Reader (type 340-Molecular
Devices) and iEMS incubator/shaker (Thermo Scientific). The reagent
and solutions used were: [0571] 1)CS-28 Microswatches (rice starch,
colored); [0572] 2) 10 mM HEPES, 2 mM CaCl.sub.2), 0.005% TWEEN 80
buffer, pH 8.0, conductivity 1 mS/cm; [0573] 3) 25 mM CAPS, 2 mM
CaCl.sub.2), 0.005% TWEEN 80 buffer, pH 10.0; conductivity 5 mS/cm
(adjusted with 5M NaCl); and [0574] 4) 10 mM NaCl, 0.1 mM
CaCl.sub.2), 0.005% TWEEN 80. [0575] 5) 50 mM MOPS pH7.15, 0.1 mM
CaCl.sub.2).
[0576] CS-28 microswatches of 5.5 mm circular diameter were
provided by the Center for Testmaterials (CFT, Vlaardingen, The
Netherlands). Two microswatches were placed in each well of a
96-well Corning 9017 flat bottomed polystyrene MTP. The culture
supernatants were diluted eight fold in 50 mM MOPS pH7.15, 0.1 mM
CaCl.sub.2), and subsequently in 10 mM NaCl, 0.1 mM CaCl.sub.2),
0.005% TWEEN.RTM. 80 solution to approximately 1 ppm, final enzyme
concentration.
[0577] The incubator/shaker was set at the desired temperature,
25.degree. C. (ambient temperature) or 50.degree. C. 174 .mu.L or
177 .mu.L of either HEPES or CAPS buffer, respectively, was added
to each well of microswatch containing MTP and subsequently 6 .mu.L
or 3 .mu.L of diluted enzyme solution was added to each well
resulting in a total volume of 180 .mu.L/well. The MTP was sealed
with a plate seal and placed in the iEMS incubator/shaker and
incubated for 15 minutes at 1150 rpm at 25.degree. C. for cleaning
at pH 8, low conductivity (1 mS/cm), or 15 minutes at 1150 rpm at
50.degree. C. for cleaning at pH 10, high conductivity (5 mS/cm).
Following incubation under the appropriate conditions, 100 .mu.L of
solution from each well was transferred to a new MTP, and the
absorbance at 488 nm was measured using a MTP-spectrophotometer.
Controls containing two microswatches and buffer but no enzyme were
included for subtraction of background cleaning performance.
[0578] Each absorbance value was corrected by subtracting the blank
(obtained after incubation of microswatches in the absence of
enzyme), and the resulting absorbance provided a measure of the
hydrolytic activity. A performance index (PI) was calculated for
each sample.
[0579] For calculation of the wash performance indices (PI), the
Langmuir equation was used to fit the data based on the reference
enzyme control. Using the protein concentration of the variants,
the expected performance based on the curve-fit was calculated. The
observed performance was divided by the calculated performance.
This value was then divided by the performance of the reference
enzyme.
H. Detergent Stability Assay
[0580] The stability of the reference amylase and variants thereof
was determined by measuring their activity after incubation under
defined conditions, in the presence of a 10% detergent mixture
(commercially purchased Persil Color Gel detergent, Henkel
(Dusseldorf, Germany), purchased in 2011). The detergent was
heat-inactivated before use, and the initial and residual amylase
activities were determined using the Ceralpha .alpha.-amylase assay
as described in section C, above.
[0581] The equipment used for this assay included a Biomek FX Robot
(Beckman Coulter); a SpectraMAX MTP Reader (type 340-Molecular
Devices), a Tetrad2DNA Engine PCR machine (Biorad), and iEMS
incubator/shaker (Thermo Scientific). The reagent solutions used
were: [0582] 1) p-nitrophenyl maltoheptaoside (BPNPG7) substrate
(Megazyme Ceralpha HR kit): [0583] 2) Liquid detergent (Persil
color gel, enzyme inactivated by heating for 4 hrs at 90.degree.
C.); [0584] 3) 50 mM MOPS, 0.1 mM CaCl.sub.2), 0.005% TWEEN.RTM.80
buffer, pH 7 (dilution buffer); [0585] 4) 10% detergent solution
diluted in dilution buffer; [0586] 5) 200 mM Boric acid/NaOH
buffer, pH 10 (STOP buffer); and [0587] 6) Amylase culture
supernatants diluted eight fold in 50 mM MOPS pH 7.15, 0.1 mM
CaCl.sub.2) containing 0-100 .mu.g/mL protein.
[0588] 85 .mu.L of a 10% detergent solution was added to a 96-well
PCR plate and mixed with 15 .mu.L of the diluted culture
supernatant. A sample from the PCR plate was diluted 3.times. in
dilution buffer and a 5 .mu.L aliquot of this dilution was used to
determine initial amylase activity. The PCR plate was incubated in
a Tetrad PCR block at 80.5.degree. C. for 5 minutes. After
incubation, detergent-enzyme mix was diluted 3.times. in dilution
buffer and residual activity was measured. Initial (t.sub.initial)
and residual (t.sub.residual) amylase activity was determined by
the Ceralpha .alpha.-amylase assay as described above in Section C
using a 5 .mu.L sample.
[0589] For each variant, the ratio of the residual and initial
amylase activities was used to calculate the detergent stability as
follows: Detergent stability=[t.sub.residual value]/[t.sub.initial
value].
[0590] For each sample (variants) the performance index (PI) was
calculated. The performance index for detergent stability is
determined by comparing the detergent stability of the variant
enzyme with that of the similarly treated reference enzyme.
I. Performance Index
[0591] The performance index (PI) compares the performance or
stability of the variant and the reference enzyme at the same
protein concentration. In addition, the theoretical values can be
calculated, using the parameters of the Langmuir equation of the
standard enzyme. A performance index (PI) that is greater than 1
(PI>1) indicates improved performance by a variant as compared
to the reference enzyme, while a PI of 1 (PI=1) identifies a
variant that performs the same as the reference enzyme, and a PI
that is less than 1 (PI<1) identifies a variant that performs
worse than the reference enzyme.
Example 2
Generation of Bacillus Strains Expressing CspAmy2-v1
[0592] In this example, the construction of Bacillus subtilis
strains expressing CspAmy2-v1 (SEQ ID NO: 2) amylase and variants,
thereof, is described. CspAmy2-v1 amylase is a variant of CspAmy2
(SEQ ID NO: 1) amylase having a deletion of both R178 and G179
(i.e., .DELTA.RG). CspAmy2 is an amylase from a Cytophaga sp., for
which the nucleotide sequence was described by Chii-Ling et al.
(2002) Appl. Environ. Microbiol. 68: 3651-3654. CspAmy2-v1 was
described as having increased thermostability over the CspAmy2 by
Rong-Jen et al. (2003) Appl. Environ. Microbiol. 69: 2383-85.
[0593] A synthetic DNA fragment (SEQ ID NO: 7) encoding CspAmy2-v1
was produced by GeneArt AG (Regensburg, Germany) and served as
template DNA for the construction of Bacillus subtilis strains
expressing CspAmy2-v1 amylase and variants, thereof. The DNA
fragment includes a codon-modified nucleotide sequence encoding the
mature form of CspAmy2-v1 amylase adjacent to a sequence encoding
the LAT signal peptide (underlined):
TABLE-US-00008 ATGAAACAACAAAAACGGCTTTACGCCCGATTGCTGACGCTGTTATTTGC
GCTCATCTTCTTGCTGCCTCATTCTGCAGCTAGCGCAGCAGCGACAAACG
GAACAATGATGCAGTATTTCGAGTGGTATGTACCTAACGACGGCCAGCAA
TGGAACAGACTGAGAACAGATGCCCCTTACTTGTCATCTGTTGGTATTAC
AGCAGTATGGACACCGCCGGCTTATAAGGGCACGTCTCAAGCAGATGTGG
GGTACGGCCCGTACGATCTGTATGATTTAGGCGAGTTTAATCAAAAAGGT
ACAGTCAGAACGAAGTATGGCACAAAAGGAGAACTTAAATCTGCTGTTAA
CACGCTGCATTCAAATGGAATCCAAGTGTATGGTGATGTCGTGATGAATC
ATAAAGCAGGTGCTGATTATACAGAAAACGTAACGGCGGTGGAGGTGAAT
CCGTCTAATAGAAATCAGGAAACGAGCGGCGAATATAATATTCAGGCATG
GACAGGCTTCAACTTTCCGGGCAGAGGAACAACGTATTCTAACTTCAAAT
GGCAGTGGTTCCATTTTGATGGAACGGATTGGGACCAGAGCAGAAGCCTC
TCTAGAATCTTCAAATTCACGGGAAAGGCGTGGGACTGGGAGGTTTCTTC
AGAAAACGGAAATTATGACTATCTGATGTACGCGGACATTGATTATGACC
ATCCGGATGTCGTGAATGAAATGAAAAAGTGGGGCGTCTGGTATGCCAAC
GAAGTTGGGTTAGATGGATACAGACTTGACGCGGTCAAACATATTAAATT
TAGCTTTCTCAAAGACTGGGTGGATAACGCAAGAGCAGCGACGGGAAAAG
AAATGTTTACGGTTGGCGAATATTGGCAAAATGATTTAGGGGCCCTGAAT
AACTACCTGGCAAAGGTAAATTACAACCAATCTCTTTTTGATGCGCCGTT
GCATTACAACTTTTACGCTGCCTCAACAGGGGGTGGATATTACGATATGA
GAAATATTCTTAATAACACGTTAGTCGCAAGCAATCCGACAAAGGCTGTT
ACGTTAGTTGAGAATCATGACACACAGCCTGGACAATCACTGGAATCAAC
AGTCCAACCGTGGTTTAAACCGTTAGCCTACGCGTTTATTCTCACGAGAA
GCGGAGGCTATCCTTCTGTATTTTATGGAGATATGTACGGTACAAAAGGA
ACGACAACAAGAGAGATCCCTGCTCTTAAATCTAAAATCGAACCTTTGCT
TAAGGCTAGAAAAGACTATGCTTATGGAACACAGAGAGACTATATTGATA
ACCCGGATGTCATTGGCTGGACGAGAGAAGGGGACTCAACGAAAGCCAAG
AGCGGTCTGGCCACAGTGATTACAGATGGGCCGGGCGGTTCAAAAAGAAT
GTATGTTGGCACGAGCAATGCGGGTGAAATCTGGTATGATTTGACAGGGA
ATAGAACAGATAAAATCACGATTGGAAGCGATGGCTATGCAACATTTCCT
GTCAATGGGGGCTCAGTTTCAGTATGGGTGCAGCAA
[0594] The mature form of the CspAmy2-v1 polypeptide produced from
the pHPLT02-CspAmy2-v1 vector is shown, below, as SEQ ID NO: 2.
TABLE-US-00009 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRNQETSGEYNIQAWTGFNFPGRGTTY
SNFKWQWFHFDGTDWDQSRSLSRIFKFTGKAWDWEVSSENGNYDYLMYAD
IDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ
[0595] To express CspAmy2-v1, the CspAmy2-v1-encoding DNA fragment
was cloned into the pHPLT02 vector, a modified version of the pHPLT
vector (Solingen et al. (2001) Extremophiles 5:333-341) by GeneArt
and fused in-frame to the AmyL (LAT) signal peptide using the
unique NheI and XhoI restriction sites, resulting in plasmid
pHPLT02-CspAmy2-v1. The pHPLT expression vector contains the B.
licheniformis LAT promoter (Plat) and additional elements from
pUB110 (McKenzie et al. (1986) Plasmid, 15: 93-103) including a
replicase gene (reppUB), a neomycin/kanamycin resistance gene (neo)
and a bleomycin resistance marker (bleo). Site-directed mutagenesis
(Stratagene) was used to change the nucleotides 5'-TCA-3' of Serine
28 of the AmyL signal peptide to nucleotides 5'-AGC-3' in order to
introduce the unique NheI restriction site.
[0596] A suitable B. subtilis strain was transformed with
pHPLT02-CspAmy2-v1 plasmid DNA using a method known in the art (WO
02/14490). The B. subtilis transformants were selected on agar
plates containing heart infusion agar (Difco, Catalog No. 244400,
Lawrence, Kans., USA and 10 mg/L neomycin sulfate (Sigma, Catalog
No. N-1876; contains 732 .mu.g neomycin per mg, St. Louis, Mo.,
USA). Selective growth of B. subtilis transformants harboring the
pHPLT02-CspAmy2-v1 plasmid was performed in shake flasks at
37.degree. C. for .about.65h in MBD medium (enriched semi-defined
medium based on MOPs buffer, with urea as major nitrogen source,
glucose as the main carbon source, and supplemented with 1% soytone
for robust cell growth) containing 5 mM CaCl.sub.2) and 10 ppm
neomycin. Growth resulted in the production of secreted CspAmy2-v1
amylase with starch hydrolyzing activity.
Example 3
Generation of Bacillus Strains Expressing CspAmy2 Combinatorial
Variants
[0597] In this example, the construction of Bacillus subtilis and
Bacillus licheniformis strains expressing two combinatorial CspAmy2
variants (CspAmy2-v5 and CspAmy2-v6) is described. CspAmy2-v5 is a
variant of CspAmy2 with the mutations E187P, I203Y, G476K, and
lacking R178 and G179. CspAmy2-v6 is a variant of CspAmy2 with the
mutations E187P, I203Y, G476K, R458N, T459S, D460T, and lacking
R178 and G179. For expression of CspAmy2-v5 and CspAmy2-v6,
expression constructs were created by a combination of gene
synthesis and fusion PCR, using standard techniques.
[0598] The CspAmy2-v5 and CspAmy2-v6 expression constructs were
ligated into vector pICatH (described in, e.g., EP2428572 and U.S.
Pat. No. 7,968,691) and transformed into competent B. subtilis
cells (amyE negative) as known in the art (WO 2002/014490). In
these clones, the DNA fragments encoding CspAmy2-v5 and CspAmy2-v6
are fused in-frame to a sequence encoding the B. licheniformis
amylase (amyL) signal peptide which leads to secretion of the
amylase variants into the growth medium. Expression of CspAmy2-v5
and CspAmy2-v6 in these clones is driven by the B. licheniformis
amylase (amyL) promoter (FIGS. 1A and 1B).
[0599] B. subtilis transformants were selected on agar plates
containing heart infusion agar (Difco) and 10 mg/L neomycin
sulfate, 5 mg/L chloramphenicol and starch azure (Sigma). For each
construct, one transformant with starch hydrolyzing activity was
selected and the amylase expression constructs in pICatH were
sequence verified by DNA sequencing (BaseClear, the Netherlands).
The resulting plasmids, pICatH-CspAmy2-v5 and pICatH-CspAmy2-v6,
were isolated from the B. subtilis clones and transformed into B.
licheniformis cells (amyL and catH negative) and integrated into
the genome as described in EP2428572 and U.S. Pat. No. 7,968,691.
After excision of vector sequences, expression constructs were
amplified by subjecting the strains to a stepwise increase in
chloramphenicol concentration up to 50 .mu.g/ml. The B.
licheniformis strains showed halos on starch azure plates,
indicating secretion of active CspAmy2-v5 and CspAmy2-v6
amylase.
[0600] The amino acid sequence of the mature form of CspAmy2-v5 is
shown, below, as SEQ ID NO: 8:
TABLE-US-00010 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRNQETSGEYNIQAWTGFNFPGRGTTY
SNFKWQWFHFDGTDWDQSRSLSRIFKFTGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNKGSVSVWVQQ
[0601] The amino acid sequence of the mature form of CspAmy2-v6 is
shown, below, as SEQ ID NO: 9:
TABLE-US-00011 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRNQETSGEYNIQAWTGFNFPGRGTTY
SNFKWQWFHFDGTDWDQSRSLSRIFKFTGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNNSTKITIGSDGYATFPVNKGSVSVWVQQ
Example 4
Cleaning Performance of CspAmy2 Combinatorial Variants
[0602] The cleaning performance of purified CspAmy2-v5 and
CspAmy2-v6 was analyzed in a microswatch cleaning assay. CFT CS-28
rice starch on cotton swatches or EMPA 161 maize starch on cotton
cretonne (Center for Testmaterials, BV, Vlaardingen, Netherlands)
containing an indicator dye bound to the starch were punched to
form discs measuring 5.5 mm in diameter. Two discs were placed in
each well of 3 flat-bottom non-binding 96-well assay plates.
[0603] Both variants (i.e., CspAmy2-v5 and CspAmy2-v6), the parent
molecule (CspAmy2), and a commercially-available benchmark
detergent .alpha.-amylase, i.e., PURASTAR.RTM. (AmyL; B.
licheniformis .alpha.-amylase; Danisco US Inc.), were diluted to
0.5 mg/mL in dilution buffer (50 mM MOPS, pH 7.2, 0.005% Tween),
and then further diluted to 2 ppm in a microtiter plate. 200 .mu.L
of these samples were transferred into the first row of each of the
swatch plates. 100 .mu.L of HEPES buffer (25 mM HEPES, pH 8.0, with
2 mM CaCl.sub.2) and 0.005% Tween-80) was then added to each well
of the next 5 rows of the swatch plates. 100 .mu.L of the diluted
enzyme samples were then transferred from the first row into the
next row, mixed well, and serial dilutions were continued until
before the last row, which served as blanks. Once all rows
contained 100 .mu.L of solution, 100 .mu.L of buffer were added to
each well of the plate to result in final volumes of 200 .mu.L per
well, and final enzyme concentrations of 1, 0.5, 0.25, 0.125,
0.0625, and 0 ppm.
[0604] Plates were incubated at 25.degree. C. with agitation at
1150 rpm for 15 minutes. Enzyme performance was assessed by the
amount of color released into the wash liquor, which was quantified
spectrophotometrically at 488 nm by the transfer of 150 .mu.L of
the final wash solution to fresh medium-binding microtiter plates.
Triplicate reads were blank-subtracted and averaged. The results of
the soil removal assay are shown in FIG. 2. CspAmy2-v5 and
CspAmy2-v6 demonstrated similar performance to each other and are
significantly better than the parent molecule, CspAmy2, and much
better than PURASTAR.RTM..
Example 5
Thermostability Assessment of CspAmy2 Combinatorial Variants
A. Thermostability in Buffer
[0605] 0.5 mg/mL stocks of purified enzymes (i.e., CspAmy2-v5,
CspAmy2-v6, CspAmy2), PURASTAR.RTM. (Bacillus licheniformis
.alpha.-amylase), and ACE-QK (variant of Bacillus sp. TS-23
.alpha.-amylase described in US20120045817 and WO2010/115028) were
further diluted to 5 ppm in dilution buffer (50 mM MOPS, pH 7.2,
0.005% Tween). 50 .mu.L of each enzyme were added to each of 9
strips of PCR tubes and sealed. One "unstressed" sample of each
enzyme was incubated at room temperature throughout the duration of
the experiment. The other 8 samples were incubated in a
thermocycler at 45, 55, 65, 70, 75, 80, 85, or 90.degree. C. for 15
minutes. The samples were then transferred to microtiter plates in
triplicate, and .alpha.-amylase activity was for the unstressed and
stressed samples using the Ceralpha reagent (Megazyme, Inc.).
Residual activity was calculated by dividing the activity of each
amylase after the thermal stress by the activity of that unstressed
amylase. The results are shown in FIG. 3. CspAmy2-v5 and CspAmy2-v6
demonstrated increased thermostability at low calcium
concentrations compared to all other tested enzymes.
B. Thermostability in Buffer with Added Calcium
[0606] As above, 0.5 mg/mL stocks of purified enzymes were further
diluted to 5 ppm in dilution buffer, in this case supplemented with
5 mM calcium chloride (i.e., 50 mM MOPS, pH 7.2, 0.005% Tween, 5 mM
calcium chloride). The experiment was carried out as described
above. The results are shown in FIG. 4. CspAmy2-v5 and CspAmy2-v6
demonstrated similar thermostability compared to the other tested
enzymes.
C. Thermostability in Liquid Detergent
[0607] Commercial detergents EPSIL.TM. Perfect (McBride) and
OMO.TM. Color (Unilever) were heat inactivated at 90.degree. C. for
4 hours to eliminate existing enzyme activities. Following
inactivation, the activity CspAmy2-v5, CspAmy2-v6, CspAmy2,
PURASTAR.RTM., and ACE-QK in the heat-inactivated detergents was
measured using the Suc-AAPF-pNA and Ceralpha assays to ensure that
any protease and amylase activities, respectively, had been
abolished. 10% solutions of both of these liquid detergents were
made in water.
[0608] 100 .mu.L of each of the 0.5 mg/mL purified enzyme stocks at
were added to 400 .mu.L of the 10% detergent solutions. 50 .mu.L of
these samples were added to strips of PCR tubes and incubated at
the aforementioned temperatures for 15 minutes each, as described
above. When samples were removed from the thermocycler, and before
.alpha.-amylase activity was measured, an additional 1:20 dilution
was made in dilution buffer. As above, residual activity was
calculated by dividing the activity of each amylase after the
detergent and thermal stress by the activity of that unstressed
amylase. The results are shown in FIG. 5 (OMO.TM.) and FIG. 6
(EPSIL.TM.) CspAmy2-v5 and CspAmy2-v6 demonstrated superior
thermostability compared to the parent enzyme and other tested
enzymes.
Example 6
Generation of Bacillus Strains Expressing CspAmy2 Combinatorial
Variants
A. Design of Combinatorial Variants
[0609] Synthetic DNA encoding CspAmy2-v1 variants having various
combinations of substitutions at positions N126, Y150, F153, L171,
T180, E187, 1203, and S241 (referring to SEQ ID NO: 1) were
constructed by GeneArt and delivered as plasmids transformed in a
B. subtilis host, as described for CspAmy2-v5 and CspAmy2-v6 in
Example 2. With single substitutions at each of eight sites, 256
combinations were possible. The specific substitutions N126Y,
Y150H, F153W, L171N, T180H, E187P, I203Y, and S241Q, were selected,
and ten of the possible combinations were made and tested. The
names of the variants, and the amino acid residues present at each
of the eight positions, is shown in Table 2. For clarity, the full
names of the variants in the Table are CspAmy2-C16A-CspAmy2-C16J.
In some table and figure the names of the variants are abbreviated
but always clear from the description. Further variants of
CspAmy2-v1 additionally having the mutations E187P or S241Q were
made, and designated CspAmy2-v1-E187P or CspAmy2-v1-S241Q,
respectively.
TABLE-US-00012 TABLE 2 Combinations of mutations tested Name N126
Y150 F153 L171 T180 E187 I203 S241 C16A Y Y F L T P Y S C16B Y Y F
L T E Y Q C16C Y Y W L T P Y S C16D Y Y W L T E Y Q C16E Y Y W L H
P Y S C16F Y Y W L H E Y Q C16G Y H W N T P Y S C16H Y H W N T E Y
Q C16I Y H W N H P Y S C16J Y H W N H E Y Q
B. Construction of Bacillus Strains Expressing CspAmy2
Combinatorial Variants
[0610] The pHPLT02-CspAmy2-v1 plasmid DNA (encoding CspAmy2-v1, see
Example 2) served as template to produce the additional
combinatorial libraries at pre-selected sites in the mature region.
The pHPLT02 expression vector was derived from the pHPLT vector.
The pHPLT expression vector contains the B. licheniformis LAT
promoter (Plat) and additional elements from pUB110 (McKenzie et
al. (1986) Plasmid, 15: 93-103) including a replicase gene
(reppUB), a neomycin/kanamycin resistance gene (neo) and a
bleomycin resistance marker (bleo). GeneArt AG (Regensburg,
Germany) created combinatorial libraries at the positions described
using their standard protocols. The corresponding codons for each
site of interest were substituted with codons for at least one
non-wild-type amino acid. The codon-mutagenized pHPLT02-CspAmy2-v1
mixes were used to transform competent B. subtilis cells as known
in the art (WO 2002/014490) to generate the CspAmy2-v1
combinatorial libraries. Transformation mixes were plated on Heart
Infusion (HI) agar plates containing 10 mg/L neomycin sulfate. For
each library, single bacterial colonies were picked and grown in
TSB (tryptone and soy-based broth) liquid medium with 10 mg/ml
neomycin selection for subsequent DNA isolation and gene sequence
analysis. Variants were generated and identified as members of this
combinatorial library. Selective growth of the variants was
performed in 96-well MTPs at 37.degree. C. for 68 hours in MBD
medium (enriched semi-defined medium based on MOPs buffer, with
urea as major nitrogen source, glucose as the main carbon source,
and supplemented with 1% soytone for robust cell growth).
[0611] The amino acid sequence of mature CspAmy2-16A is shown,
below, as SEQ ID NO: 10:
TABLE-US-00013 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTY
SNFKWQWFHFDGTDWDQSRSLSRIFKFTGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ
[0612] The amino acid sequence of mature CspAmy2-16B is shown,
below, as SEQ ID NO: 11:
TABLE-US-00014 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTY
SNFKWQWFHFDGTDWDQSRSLSRIFKFTGKAWDWEVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFQFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ
[0613] The amino acid sequence of mature CspAmy2-16C is shown,
below, as SEQ ID NO: 12:
TABLE-US-00015 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTY
SNWKWQWFHFDGTDWDQSRSLSRIFKFTGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ
[0614] The amino acid sequence of mature CspAmy2-16D is shown,
below, as SEQ ID NO: 13:
TABLE-US-00016 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTY
SNWKWQWFHFDGTDWDQSRSLSRIFKFTGKAWDWEVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFQFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ
[0615] The amino acid sequence of the mature CspAmy2-16E is shown,
below, as SEQ ID NO: 14:
TABLE-US-00017 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTY
SNWKWQWFHFDGTDWDQSRSLSRIFKFHGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ
[0616] The amino acid sequence of mature CspAmy2-16F is shown,
below, as SEQ ID NO: 15:
TABLE-US-00018 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTY
SNWKWQWFHFDGTDWDQSRSLSRIFKFHGKAWDWEVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFQFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ
[0617] The amino acid sequence of mature CspAmy2-16G is shown,
below, as SEQ ID NO: 16:
TABLE-US-00019 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTH
SNWKWQWFHFDGTDWDQSRSNSRIFKFTGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ
[0618] The amino acid sequence of mature CspAmy2-16H is shown,
below, as SEQ ID NO: 17:
TABLE-US-00020 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTH
SNWKWQWFHFDGTDWDQSRSNSRIFKFTGKAWDWEVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFQFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ
[0619] The amino acid sequence of mature CspAmy2-16I is shown,
below, as SEQ ID NO: 18:
TABLE-US-00021 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTH
SNWKWQWFHFDGTDWDQSRSNSRIFKFHGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ
[0620] The amino acid sequence of mature CspAmy2-16J is shown,
below, as SEQ ID NO: 19:
TABLE-US-00022 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTH
SNWKWQWFHFDGTDWDQSRSNSRIFKFHGKAWDWEVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFQFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ
[0621] The amino acid sequence of mature CspAmy2-v1-E187P is shown,
below, as SEQ ID NO: 20:
TABLE-US-00023 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRNQETSGEYNIQAWTGFNFPGRGTTY
SNFKWQWFHFDGTDWDQSRSLSRIFKFTGKAWDWPVSSENGNYDYLMYAD
IDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ
[0622] The amino acid sequence of mature CspAmy2-v1-S241Q is shown,
below, as SEQ ID NO: 21:
TABLE-US-00024 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRNQETSGEYNIQAWTGFNFPGRGTTY
SNFKWQWFHFDGTDWDQSRSLSRIFKFTGKAWDWEVSSENGNYDYLMYAD
IDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFQFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ
Example 7
Thermostability Assessment of CspAmy2 Variants
[0623] Combination variants CspAmy2-C16A-CspAmy2-C16J from Example
5 (i.e., "C16A"-"C16G" or "16A-16G," respectively) were tested for
thermal stability under the following conditions:
[0624] 1. 50 mM potassium acetate, pH 5.7, 0.125 mM CaCl.sub.2),
2.2 mM NaCl, 85.degree. C.
[0625] 2. 50 mM potassium acetate, pH 5.0, 0.125 mM CaCl.sub.2),
2.2 mM NaCl, 70.degree. C.
[0626] 3. 50 mM potassium acetate, pH 4.5, 0.125 mM CaCl.sub.2),
2.2 mM NaCl, 65.degree. C.
[0627] Stock solutions of each variant were prepared by diluting
purified variants to a final protein concentration of 1 mg/mL in
milli-Q water, then each variant was further diluted (200-fold) in
each of the above buffers (final enzyme dose is 5 .mu.g/mL). The
diluted enzyme solutions were pre-heated to appropriate temperature
for two minutes and then cooled on ice to disrupt any protein
aggregates. 50 .mu.L of each enzyme solution was transferred to 0.2
mL PCR strip tubes, which were heated to the appropriate
temperature (based on buffer pH) and allowed to incubate over a
two-hour period. The samples were then placed in an ice-water bath
to end the heat-stress period.
[0628] Once all time points were collected for each buffer,
residual activity was determined using the Ceralpha assay, as
described in Example 1. Two independent inactivation time-course
experiments were performed for each variant. Plots of residual
activity vs. time were modeled with a single exponential decay
equation to determine a rate constant (k) for decay. The half-life
of decay was defined as ln(2)/k. These experiments were performed
in duplicate for each variant.
[0629] The performance index (PI) for each variant was defined as
the ratio of the variant half-life to the half-life of a reference
parent molecule. For variants C16A, C16C, C16E, C16G, and C16I, the
reference molecule was CspAmy2-v1-E187P. For variants C16B, C16D,
C16F, C16H, and C16J, the reference molecule was CspAmy2-v1-S241Q.
The relative half-lives and performance indexes are shown in the
Table in FIG. 7. Other experiments demonstrated that the stability
of C16D was similar to that of STAINZYME.RTM., while the stability
of C16F was greater than that of STAINZYME.RTM. (not shown). The
relative performance of the combination variants at pH 4.5 and
65.degree. C., pH 5.0 at 70.degree. C., and pH 5.7 ay 85.degree.
C., is shown graphically in FIGS. 8-10, respectively. All C16A-J
variants were more stable than their respective parent
molecules.
Example 8
Viscosity Reduction Using CspAmy2 Variants
[0630] Combination variants CspAmy2-C16A-CspAmy2-C16J from Example
5 were tested for their ability to reduce the viscosity of a starch
solution, as a measure of starch hydrolysis activity. Viscosity
experiments were performed using a Rapid Visco Analyser (Newport
Scientific; herein "RVA") with 38 mm.times.68 mm plain washed cans
(Part No. AA0384001) and a double-skirted paddle (Part No.
NS101783). Data acquisition and analysis was performed with
Thermocline for Windows (version 3.11; (Newport Scientific)).
Immediately before each run, 33 g of 25% dry solids (ds) corn flour
slurry was prepared in an RVA can as follows: 9.17 g corn flour
(11.8% moisture content) was weighed out on an analytical balance
and mixed with 23.83 g deionized water. Sample pH was adjusted with
0.0828 ml 1N sulfuric acid (for pH 5.8) or 0.285 ml 1N sulfuric
acid (for pH 5.0). Enzyme was added at appropriate dose, and the
can was placed in the viscometer. All runs were 10 minutes in
length, with a temperature ramp to 85.degree. C. over 80 seconds
followed by a temperature hold at 85.degree. C. for the remainder
of the run. Each enzyme was assessed at three doses. Dose responses
were calculated for two parts of the viscosity curve. Peak
viscosity is defined as the maximum viscosity reached during the
experiment, and final viscosity is defined as the viscosity reading
at the end of the experiment. Because viscosity has a reciprocal
relationship with enzyme dose, dose response curves were linearized
by plotting 1/viscosity (also called fluidity) vs. enzyme dose.
Slopes of these curves were used as a quantitative measure of the
specific activity of each variant. The performance index (PI) for
each variant is defined as the ratio of the variant specific
activity to the specific activity of a reference parent molecule.
For variants CspAmy2-C16A, CspAmy2-C16C, CspAmy2-C16E,
CspAmy2-C16G, and CspAmy2-C16I, the reference is CspAmy2-v1-E187P.
For variants CspAmy2-C16B, CspAmy2-C16D, CspAmy2-C16F,
CspAmy2-C16H, and CspAmy2-C16J, the reference is CspAmy2-v1-S241Q.
Viscosity reduction performance is shown in Tables 3 and 4.
TABLE-US-00025 TABLE 3 Improvements in viscosity reduction at pH
5.8 Peak Final Fluidity/mg Fluidity/mg enzyme Peak PI enzyme Final
PI C16A 0.0044 1.10 0.17 1.99 C16B 0.0047 1.18 0.17 1.47 C16C
0.0048 1.20 0.17 1.96 C16D 0.0049 1.22 0.18 1.53 C16E 0.0043 1.08
0.18 2.12 C16F 0.0044 1.10 0.18 1.56 C16G 0.0051 1.28 0.17 1.99
C16H 0.0045 1.11 0.17 1.44 C16I 0.0047 1.19 0.19 2.16 C16J 0.0050
1.25 0.19 1.65 E187P 0.0040 1.00 0.09 1.00 S241Q 0.0040 1.00 0.12
1.00
TABLE-US-00026 TABLE 4 Improvements in viscosity reduction at pH
5.0 Peak Final Fluidity/mg Fluidity/mg enzyme Peak PI enzyme Final
PI C16A 0.0180 1.27 0.17 1.96 C16B 0.0065 1.27 0.14 1.80 C16C
0.0061 1.24 0.19 2.08 C16D 0.0062 1.20 0.16 1.97 C16E 0.0065 1.33
0.19 2.16 C16F 0.0061 1.18 0.17 2.16 C16G 0.0072 1.46 0.19 2.10
C16H 0.0063 1.23 0.16 1.98 C16I 0.0067 1.35 0.20 2.23 C16J 0.0069
1.35 0.19 2.36 E187P 0.0049 1.00 0.09 1.00 S241Q 0.0051 1.00 0.08
1.00
Example 9
Cleaning Performance and Detergent Stability of CspAmy2
Variants
A. Cleaning Performance of CspAmy2 Variants
[0631] The cleaning performance of combination variants
CspAmy2-C16A-CspAmy2-C16J from Example 5 was analyzed in a
microswatch cleaning assay using CFT CS-28 rice starch on cotton
swatches. The assay was performed using culture supernatants.
Protein concentration for the supernatants was quantified using
HPLC. The equipment used for this assay included a Biomek FX Robot
(Beckman Coulter), a SpectraMAX MTP Reader (type 340-Molecular
Devices) and iEMS incubator/shaker (Thermo Scientific). The reagent
and solutions used were: [0632] 1)CS-28 Microswatches (rice starch,
colored); [0633] 2) 10 mM HEPES, 2 mM CaCl.sub.2), 0.005% TWEEN 80
buffer, pH 8.0, conductivity 1 mS/cm; [0634] 3) 50 mM MOPS pH7.15,
0.005% TWEEN 80.
[0635] CS-28 microswatches of 5.5 mm circular diameter were
provided by the Center for Testmaterials (CFT, Vlaardingen, The
Netherlands). Two microswatches were placed in each well of a
96-well Corning 9017 flat bottomed polystyrene MTP. The culture
supernatants were diluted twenty-fold in 50 mM MOPS pH7.15, 0.005%
TWEEN 80.
[0636] The incubator/shaker was set at 25.degree. C. (ambient
temperature). 178.5 .mu.L of HEPES buffer, respectively, was added
to each well of microswatch containing MTP and subsequently 1.5
.mu.L of diluted enzyme solution was added to each well resulting
in a total volume of 180 .mu.L/well. The MTP was sealed with a
plate seal and placed in the iEMS incubator/shaker and incubated
for 15 minutes at 1150 rpm at 25.degree. C. Following incubation
under the appropriate conditions, 100 .mu.L of solution from each
well was transferred to a new MTP, and the absorbance at 488 nm was
measured using a MTP-spectrophotometer. Controls containing two
microswatches and buffer but no enzyme were included for
subtraction of background cleaning performance.
[0637] Each absorbance value was corrected by subtracting the blank
(obtained after incubation of microswatches in the absence of
enzyme), and the resulting absorbance provided a measure of the
hydrolytic activity. A performance index (PI) was calculated for
each sample compared to CspAmy2-v1, assuming a linear response in
the range of the assay used. The results are shown in Table
4.1.
B. Detergent Stability of CspAmy2 Variants
[0638] The detergent stability of the additional CspAmy2 variants
was determined by measuring their activity after incubation under
defined conditions, in the presence of a 10% detergent mixture
(commercially purchased Persil Color Gel detergent, Henkel
(Dusseldorf, Germany), purchased in 2011). The detergent was
heat-inactivated before use, and the initial and residual amylase
activities were determined using the Ceralpha .alpha.-amylase
assay. The equipment used for this assay included a Biomek FX Robot
(Beckman Coulter); a SpectraMAX MTP Reader (type 340-Molecular
Devices), a Tetrad2DNA Engine PCR machine (Biorad), and iEMS
incubator/shaker (Thermo Scientific). The reagent solutions used
were as follows: [0639] 1) p-nitrophenyl maltoheptaoside (BPNPG7)
substrate (Megazyme Ceralpha HR kit): [0640] 2) Liquid detergent
(Persil color gel, enzyme inactivated by heating for 4 hrs at
90.degree. C.); [0641] 3) 50 mM MOPS, 0.1 mM CaCl.sub.2), 0.005%
TWEEN.RTM.80 buffer, pH 7 (dilution buffer); [0642] 4) 10%
detergent solution diluted in dilution buffer; [0643] 5) 200 mM
Boric acid/NaOH buffer, pH 10 (STOP buffer) [0644] 6) Amylase
culture supernatants diluted eight fold in 50 mM MOPS pH7.15, 0.1
mM CaCl.sub.2) containing 0-100 .mu.g/mL protein
[0645] 80 .mu.L of a 10% detergent solution was added to a 96-well
PCR plate and mixed with 20 .mu.L of the diluted culture
supernatant. A sample from the PCR plate was diluted 10.times. in
dilution buffer and a 5 .mu.L aliquot of this dilution was used to
determine initial amylase activity. The PCR plate was incubated in
a Tetrad PCR block at 80.5.degree. C. for 5 minutes. After
incubation, detergent-enzyme mix was diluted 10.times. in dilution
buffer and residual activity was measured. Initial (t.sub.initial)
and residual (t.sub.residual) amylase activity was determined in
triplicate by the Ceralpha .alpha.-amylase assay using 5 .mu.L
samples.
[0646] For each variant, the ratio of the residual and initial
amylase activities was used to calculate the detergent stability as
follows: Detergent stability=[t.sub.residual value]/[t.sub.initial
value]. For each sample (variants) the performance index (PI) was
calculated. The performance index for detergent stability is
determined by comparing the detergent stability of the variant
enzyme with that of the similarly treated CspAmy2-v1 enzyme. The
results are shown in Table 5.
TABLE-US-00027 TABLE 5 Cleaning performance and stability of
CspAmy2 variants Performance Index Variant Cleaning Stability
CspAmy2-v1 1 1 CspAmy2-C16B 1.02 2.51 CspAmy2-C16D 1.32 2.37
CspAmy2-C16F 1.25 2.41 CspAmy2-C16H 1.2 1.17 CspAmy2-C16J 1.06
0.83
Example 10
Generation of Bacillus Strains Expressing CspAmy2 Combinatorial
Variants
A. Design of Combinatorial Variants
[0647] Synthetic DNA encoding two additional CspAmy2 variants were
constructed by GeneArt and delivered as plasmids transformed in a
B. subtilis host, as described for CspAmy2-v5 and CspAmy2-v6 in
Example 2. CspAmy2 v171 is a variant of CspAmy2 having the
mutations T180D, E187P, I203Y, G476K, and lacking R178 and G179.
CspAmy2 v172 is a variant of CspAmy2 having the mutations N126Y,
T180D, E187P, I203Y, G476K, and lacking R178 and G179.
[0648] The amino acid sequence of mature CspAmy2-v171 is shown,
below, as SEQ ID NO: 22:
TABLE-US-00028 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRNQETSGEYNIQAWTGFNFPGRGTTY
SNFKWQWFHFDGTDWDQSRSLSRIFKFDGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNKGSVSVWVQQ
[0649] The amino acid sequence of mature CspAmy2-v172 is shown,
below, as SEQ ID NO: 23:
TABLE-US-00029 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTY
SNFKWQWFHFDGTDWDQSRSLSRIFKFDGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNKGSVSVWVQQ
Example 11
Thermostability Assessment of CspAmy2 Combinatorial Variants
[0650] The cleaning detergent stability of purified CspAmy2-v171
and CspAmy2-v172 was compared to that of CspAmy2-v5, and ACE-QK, as
described in Example 5. The results are shown in FIG. 11. The
stability of CspAmy2-v172 was greater than all other tested
enzymes.
Example 12
Cleaning Benefit of a CspAmy2 Combinatorial Variant in Hand
Dishwashing
[0651] The cleaning benefit of a CspAmy2 variant was compared to
that of a commercially-available .alpha.-amylase [STAINZYME.RTM.
(Novozymes A/S)] in a hand dishwashing application. In an assay
intended to simulate hand washing, 0.01% (wt/wt) active enzyme
(CspAmy2-v6, STAINZYME.RTM., or no enzyme as a control) was added
to European Dreft laundry detergent (1 g/L in water; Procter &
Gamble, Cincinnati, Ohio, USA) and incubated in the presence of
DM77 starch monitor (i.e., ADW melamine tiles from Center for Test
Materials CFT BV in Vlaardingen, the Netherlands) at 40.degree. C.
with agitation in Laundr-o-meter. The results are shown in FIG. 12.
CspAmy2-v5 was far superior to STAINZYME.RTM. in cleaning
performance in terms of the rate of cleaning observed and the end
point reached. Importantly, the rate of cleaning achieved by
CspAmy2-v5 was so rapid that benefits were observed within a period
of time relevant to hand dishwahing applications (i.e., about 30
seconds). These results indicate that CspAmy2 variants have
potential as additives for hand dishwashing compositions.
Example 13
Cleaning Benefit of a CspAmy2 Combinatorial Variant in Automatic
Dishwashing
[0652] The cleaning benefit of a CspAmy2 variant was compared to
that of two benchmark .alpha.-amylases (i.e., STAINZYME.RTM. and
POWERASE.RTM.) in an automatic dishwashing (ADW) application. The
cleaning assays were performed in a standard Miele 6382 dishwasher
using a normal cycle (50.degree. C. and 60 minutes) with water
having a hardness of 21 GH and 37.5 FH. 20 g of ADW powder
detergent was used for each dishwashing cycle. The detergent was
either WfK Type B or Type C (Testgewebe GmbH, Bruggen,
Deutschland), which are described in Tables A and B, respectively
of FIG. 13.
[0653] The test samples were either pasta or mixed-starch stained
dishes. The pasta samples were prepared by mixing 150 g of strained
pasta cooked in 17 GH water with 200 mL distilled water in a
blender for 5 minutes to obtain a chewing gum-like suspension.
About 3 g of the suspension was brushed onto the surface of each
dish to be included in the cleaning assay, allowed to dry for 24
hours, baked onto the dishes at 120.degree. C. for 2 hours, and
allowed to cool. Cleaning performance was evaluated by adding
iodine to the washed dishes and using a photo scale rating of 1-10.
The mixed starch samples were prepared by mixing 26 g each of
potato, corn, rice, and wheat starch in about 4 L g 16 GH water and
heating to 95.degree. C. for 10 minutes. After cooling to room
temperature, about 30 mL of the mixture was applied to the surface
of each dish to be included in the cleaning assay, allowed to dry
on the surface of the dishes for 48 hours at room temperature,
baked onto the dishes at 80.degree. C. for 1 hour, and allowed to
cool. Cleaning performance was determined by weighing the dishes
before and after the cleaning assay to determine the amount of
starch removed.
[0654] FIGS. 14 and 15 show the cleaning performance of CspAmy2-v6
(squares) compared to POWERASE.RTM. (diamonds), dosed at 0, 1, 2,
4, or 8 ppm in about 2.5 cents/kg (ct/kg) WfK B detergent against
the mixed starch stain (FIG. 14) and the pasta stain (FIG. 15).
CspAmy2-v6 clearly outperformed POWERASE.RTM., particularly against
the mixed starch stain. FIGS. 16 and 17 show the cleaning
performance of CspAmy2-v6 (squares) compared to STAINZYME.RTM.
(circles), dosed at 0, 1, 2, 4, or 8 ppm in about 2.5 ct/kg WfK B
detergent against the mixed starch stain (FIG. 16) and the pasta
stain (FIG. 17). CspAmy2-v6 clearly outperformed STAINZYME.RTM.,
especially at low doses. FIGS. 18 and 19 show the cleaning
performance of CspAmy2-v6 (squares) compared to POWERASE.RTM.
(diamonds), dosed at 0, 1, 2, 4, or 8 ppm in about 2.5 ct/kg WfK C
detergent against the mixed starch stain (FIG. 18) and the pasta
stain (FIG. 19). CspAmy2-v6 clearly outperformed POWERASE.RTM.
against both stains.
Example 14
Site Evaluation Library Screen of C18P and Identification of
Activity-Enhancing Mutations
[0655] Site evaluation libraries (SELs) were constructed and
screened at 283 of 485 positions in variant CspAmy2-C18P (i.e.,
CspAmy2 with the mutations N126Y, F153W, T180D, I203Y, and S241Q,
and lacking R178 and G179 (referring to SEQ ID NO: 1 for numbering)
to identify additional variants with enhanced activity on one or
more of the following substrates: corn amylopectin, swelled corn
starch, granular corn starch, and corn starch-stained
microswatches. Mutations were discovered throughout the molecule
that improved activity. A subset of these variants was re-tested at
different dose responses to confirm enhanced activity.
[0656] The amino acid sequence of the mature CspAmy2-C18P amylase
polypeptide is shown, below, as SEQ ID NO: 24 (the substitutions
are underlined):
TABLE-US-00030 AATNGTMMQY FEWYVPNDGQ QWNRLRTDAP YLSSVGITAV
WTPPAYKGTS QADVGYGPYD LYDLGEFNQK GTVRTKYGTK GELKSAVNTL HSNGIQVYGD
VVMNHKAGAD YTENVTAVEV NPSNRYQETS GEYNIQAWTG FNFPGRGTTY SNWKWQWFHF
DGTDWDQSRS LSRIFKFDGK AWDWEVSSEN GNYDYLMYAD YDYDHPDVVN EMKKWGVWYA
NEVGLDGYRL DAVKHIKFQF LKDWVDNARA ATGKEMFTVG EYWQNDLGAL NNYLAKVNYN
QSLFDAPLHY NFYAASTGGG YYDMRNILNN TLVASNPTKA VTLVENHDTQ PGQSLESTVQ
PWFKPLAYAF ILTRSGGYPS VFYGDMYGTK GTTTREIPAL KSKIEPLLKA RKDYAYGTQR
DYIDNPDVIG WTREGDSTKA KSGLATVITD GPGGSKRMYV GTSNAGEIWY DLTGNRTDKI
TIGSDGYATF PVNGGSVSVW VQQ
Corn Amylopectin Assay
[0657] Hydrolysis of soluble corn amylopectin was used to measure
specific activity of amylase variants. Activity was measured as
reducing ends generated by the enzymatic hydrolysis of amylopectin
polymers as quantified using a bicinchoninic acid (BCA) assay. The
equipment used for the assay included a Biomek FX Robot (Beckman
Coulter), a SpectraMAX MTP Reader (type 340-Molecular Devices), and
a Tetrad2DNA Engine PCR machine (Biorad).
[0658] Purified corn amylopectin (MP Biomedicals, LLC, cat.
#195048) was solubilized by boiling for 5 minutes while stirring as
a 1.5% (w/w) suspension in water. The material was allowed to cool
to room temperature and water and concentrated stock solutions were
added to obtain a final amylopectin substrate with 1.25% (w/w)
amylopectin, 6.25 ppm calcium, 62.5 ppm sodium, 62.5 mM potassium
acetate (pH 5.0), and 0.005% (v/v) Tween 80.
[0659] 40 .mu.l of the amylopectin substrate was added to 3 PCR
microtiter plates. 10 .mu.l of culture supernatant diluted 1:2000
in water/0.005% tween 80 was added to plates followed by through
mixing by up and down pipetting. The plates were sealed and placed
at 70.degree. C. for 5 minutes followed by a ramp down to
25.degree. C. Amylopectin hydrolysis was terminated by immediate
addition/mixing of 10 .mu.L 0.5 N NaOH.
[0660] The starch hydrolysis reaction products were analyzed by the
BCA assay. Briefly, a reagent provided in a commercial kit (Pierce
Chemical, cat. no. 23225) was prepared as per manufacturer's
instructions. 90 .mu.l was aliquoted to PCR plates followed by 10
.mu.l of the terminated enzyme reaction described above. After
through mixing of components the plates were sealed and placed in a
thermocycler, programmed for 3 minutes at 95.degree. C. to develop
color, and then cooled to 30.degree. C. Samples of 70 .mu.L of the
developed BCA reaction mixtures were transferred to a fresh plate
and absorbance was measured at 562 nm in a spectrophotometer. Data
acquired for three replicate plates were averaged.
Corn Starch Assay
[0661] Cargill Corn Starch material (flour or starch) was washed
extensively with MilliQ water by repeated suspension and
centrifugation prior to use in the assay. The washed corn starch
was suspended in MilliQ water containing 0.005% sodium azide to
obtain 20% (w/w) stock solutions, which were further diluted with a
20.times. stock buffer solution to 10.9% w/v corn flour and corn
starch solutions (final buffer concentration of 55 mM KOAc, pH
5).
[0662] 55 .mu.L of the diluted corn flour or corn starch substrates
were added to PCR microtiter plates along with 5 .mu.L of 1:10
diluted enzyme samples using a bubble paddle reservoir. The plates
were sealed and placed at 86.degree. C. for 5 minutes followed by a
ramp down to 45.degree. C. The starch hydrolysis reaction was
terminated by addition of 70 .mu.L 0.1 N NaOH. The plates were
sealed and centrifuged for 3 minutes at 1,610 RCF. The starch
hydrolysis reaction products were analyzed by the BCA assay as
described above.
Swelled Corn Starch (SCS) Assay
[0663] The SCS assay measures alpha-amylase activity on hydrated
but intact ("swelled") starch granules. The overall number of
enzymatic turnovers is assessed using the BCA reducing sugar assay,
while the tendency of the enzyme to release large starch fragments
into solution is assessed using iodine staining.
[0664] 2% (w/w) swelled corn starch substrate was prepared by
suspending 2 g of corn starch (Cargill Farms Organic Corn Starch)
in 90 g of MilliQ water and heating in a submerged water bath set
to 80.degree. C. for 1 hour with regular swirling. After overnight
cooling at room temperature, the volume was brought up to 100 mL
with the addition of potassium acetate buffer, calcium chloride,
sodium chloride and Tween-80 to give final concentrations of 50 mM
KOAc (pH5.5), 0.125 mM CaCl.sub.2), 2 mM NaCl, and 0.005%
Tween-80.
[0665] 55 .mu.L of swelled corn starch substrate was dispensed into
NUNC V-bottom polystyrene microtiter plates using a bubble paddle
reservoir. The reaction was initiated by adding 5 .mu.L of
12.times. enzyme solution to the plate to give a final enzyme
concentration of -0.03 ppm in reaction. The reaction plate was
sealed with a Nunc seal and immediately placed in an iEMS
incubator. Incubation occurred for 10 min at 25.degree. C., or for
4 min at 60.degree. C., while shaking at 1,150 RPM. After
incubation, the reaction was terminated with the addition of 70
.mu.L of 0.1 N NaOH. The plates were sealed and spun for 3 min at
3,000 RPM. Swelled corn starch reactions were done as a single
plate, but assayed in triplicate for subsequent Iodine and BCA
assays.
[0666] The BCA assay is described above. For the iodine reaction,
95 .mu.L of Lugol's reagent (freshly diluted by 12-fold in water;
Sigma-Aldrich L6146-1L) was added to 96-well Costar 9017 microtiter
plates. 5 .mu.L of supernatant sample was added to the plates and
mixed six times by pipetting up and down. The plates were then
shaken on a table top microtiter plate mixer for 1 minute at speed
6-7. Absorbance was read at 530 nm using a SpectraMax M5
spectrophotometer.
CS-26 Corn Starch Microswatch Assay
[0667] The assays were performed as described, above, except that
170 .mu.l of potassium acetate buffer was added to each well of
microswatch-containing MTP and subsequently 10 .mu.L of diluted
enzyme solution was added to each well resulting in a total volume
of 180 .mu.L/well. The MTP was sealed and incubated for 20 minutes
at 1,150 rpm at 25.degree. C.
Viscosity Analysis
[0668] Viscosity experiments were performed using a Rapid Visco
Analyser (Newport Scientific) with 38 mm.times.68 mm plain washed
cans (Part No. AA0384001) and a double-skirted paddle (Part no.
NS101783). Data acquisition and analysis was performed with
Thermocline for Windows (version 3.11). Immediately before each
run, 33 g of 25% dry solids (ds) corn flour slurry was prepared in
an RVA can as follows: 9.17 g corn flour (11.8% moisture content)
was weighed out on an analytical balance and mixed with 23.83 g
deionized water. The sample pH was adjusted with 0.0828 mL 1N
sulfuric acid (for pH 5.7) or 0.285 mL 1N sulfuric acid (for pH
5.2). Enzyme was added at appropriate dose, and the can was placed
in the viscometer. All runs were 10 minutes in length, with a
temperature ramp to 85.degree. C. or 95.degree. C. over 80 seconds
followed by a temperature hold at 85.degree. C. or 95.degree. C.
for the remainder of the run.
Initial Screening Results
Mutations resulting in variants with expression greater than 250
ug/mL and at least 110% of the performance of CspAmy2-C18P in any
one of the indicated assays in each assay were classified as
performance-enhancing mutations. CspAmy2-C18P SEL variants with
mutations at positions 6, 7, 8, 11, 14, 15, 20, 21, 23, 26, 27, 28,
37, 38, 39, 40, 42, 45, 46, 48, 49, 50, 51, 52, 53, 54, 58, 61, 62,
68, 70, 71, 72, 73, 79, 80, 81, 82, 84, 85, 87, 88, 89, 92, 93, 94,
95, 96, 97, 98, 101, 108, 111, 112, 113, 114, 115, 116, 117, 118,
120, 122, 123, 124, 126, 127, 129, 130, 131, 132, 133, 134, 136,
137, 138, 140, 142, 143, 144, 147, 148, 149, 150, 151, 152, 153,
154, 155, 156, 158, 159, 165, 167, 168, 170, 171, 172, 175, 176,
177, 180, 181, 182, 187, 190, 191, 193, 199, 200, 201, 203, 206,
208, 210, 211, 212, 214, 215, 216, 219, 221, 223, 225, 226, 227,
235, 238, 239, 240, 241, 242, 243, 245, 246, 247, 248, 249, 250,
252, 253, 254, 256, 257, 258, 260, 261, 262, 266, 267, 268, 269,
270, 271, 273, 276, 277, 279, 280, 282, 284, 285, 286, 288, 296,
299, 300, 301, 302, 303, 304, 307, 308, 310, 311, 312, 313, 316,
317, 318, 320, 321, 325, 327, 335, 338, 342, 348, 349, 352, 356,
357, 360, 362, 363, 368, 369, 377, 381, 382, 383, 384, 385, 388,
390, 392, 394, 395, 396, 397, 398, 400, 401, 402, 403, 404, 405,
407, 408, 410, 414, 415, 416, 418, 419, 420, 421, 422, 423, 424,
426, 428, 429, 430, 431, 434, 435, 436, 439, 441, 442, 444, 445,
446, 447, 448, 449, 450, 451, 454, 455, 457, 460, 461, 462, 463,
464, 465, 466, 467, 469, 470, 471, 473, 474, 475, 476, 477, 479,
480, 481, 482, 483, and 484 met this criteria. Specific
performance-enhancing mutations included T6A, T6D, T6E, T6G, T6K,
T6M, T6N, T6Q, T6S, M7A, M7V, MBC, MBF, M8I, M8L, M8Y, F11V, Y14A,
Y14I, Y14Q, Y14T, Y14V, V15C, V15D, V15I, V15N, V15T, Q20A, Q20C,
Q20D, Q20H, Q20K, Q20M, Q2ON, Q20R, Q205, Q20Y, Q21F, Q21W, N23A,
N23C, N23D, N23E, N23H, N23K, N23Q, N23R, N23S, N23T, N23V, R26C,
R26E, R26G, R26K, R26M, R26S, R26T, T27A, T27C, T27D, T27E, T27F,
T27H, T27I, T27K, T27L, T27M, T27N, T27Q, T27R, T27S, T27V, T27Y,
D28A, D28C, D28T, I37F, I37V, T38D, T38N, A39L, V40A, V40C, V40D,
V40G, V40H, V401, V40K, V40M, V40P, V40Q, V40R, V405, V40T, V40W,
V40Y, T42A, T42C, T421, T42M, T42V, A45C, A45G, Y46F, G48A, T49I,
S50A, S50C, S50E, S50G, S50K, S50M, S50N, S50Q, 550R, S50T, S50Y,
Q51C, Q51D, Q51E, Q51S, Q51V, A52C, A52D, A52E, A52F, A52G, A52H,
A52K, A52L, A52M, A52R, A52S, A52T, A52V, A52Y, D53E, V54N, V54T,
P58A, P58C, P58H, P581, P58S, P58T, P58V, L61M, L61V, Y62A, Y62C,
Y62D, Y62F, Y62G, Y62H, Y62I, Y62K, Y62L, Y62M, Y62N, Y62P, Y62Q,
Y62R, Y62S, Y62V, N68C, N68D, N68E, N68F, N68H, N68L, N68P, N68Q,
N68R, N68S, N68V, N68W, N68Y, K70R, G71A, G71C, G71D, G71E, G71K,
G71R, G71S, T72G, T72S, V73S, T79F, T791, T79L, T79M, T79N, T79S,
T79Y, K80A, K80C, K80D, K80F, K80H, K80I, K80M, K80N, K80Q, K80R,
K80S, K80T, K80V, K80Y, G81A, G81D, G81E, G81F, G81H, G81I, G81K,
G81N, G81P, G81R, G81S, G81T, E82A, E82D, E82M, E82Q, K84A, K84C,
K84E, K84I, K84Q, K84R, K84S, K84T, K84Y, S85A, S85C, S85D, S85E,
S85G, S85H, 5851, S85L, S85M, S85N, S85Q, S85R, S85T, S85V, S85Y,
V871, V87T, N88C, N88D, N88E, N88G, N88H, N88I, N88K, N88L, N88M,
N88Q, N88R, N88S, N88T, N88V, N88W, N88Y, T89A, T89D, T89E, T89F,
T89H, T891, T89K, T89L, T89M, T89N, T89Q, T89R, T89S, T89Y, S92A,
S92C, S92D, S92E, S92F, S92G, S92H, S92L, S92M, S92N, S92Q, S92R,
S92T, S92W, S92Y, N93A, N93C, N93E, N93F, N93H, N93I, N93K, N93L,
N93Q, N93S, N93T, N93Y, G94A, G94C, G94N, I95M, Q96A, Q96E, Q96H,
Q961, Q96K, Q96L, Q96M, Q96N, Q96R, Q96V, Q96Y, V971, V97T, Y98F,
Y981, Y98L, Y98V, V101C, V101T, G108A, G108S, Y111D, Y111E, Y111L,
Y111N, Y111S, Y111T, Y111V, T112A, T112F, T112H, T1121, T112K,
T112L, T112M, T112N, T112P, T112R, T112V, T112W, E113D, E113N,
E113Q, E113T, N114A, N114C, N114D, N114E, N114F, N114G, N114H,
N114I, N114L, N114P, N114Q, N114R, N114S, N114T, N114V, N114W,
N114Y, V115A, V115I, T116A, T116C, T116D, T116G, T116H, T116I,
T116K, T116N, T116P, T116Q, T116R, T116S, A117C, A117I, A117S,
A117V, V118A, V118C, V118E, V118F, V118H, V118I, V118L, V118M,
V118N, V118Q, V118R, V118S, V118W, V118Y, V120A, V120C, V1201,
V120M, V120T, P122A, P122D, P122E, P122G, P122H, P122N, P122R,
P122S, P122T, P122W, S123A, S123C, S123G, S123K, S123L, N124A,
N124D, N124F, N124G, N124L, N124R, N124S, N124V, Y126A, Y126C,
Y126D, Y126E, Y126G, Y126H, Y1261, Y126K, Y126L, Y126M, Y126N,
Y126Q, Y126R, Y126S, Y126T, Y126V, Y126W, Q127A, Q127C, Q127D,
Q127E, Q127F, Q127H, Q1271, Q127K, Q127L, Q127M, Q127N, Q127R,
Q127S, Q127T, Q127V, Q127W, Q127Y, T129A, T129C, T129D, T129E,
T129F, T129G, T129I, T129K, T129L, T129M, T129N, T129Q, T129R,
T129S, T129V, T129W, T129Y, S130A, S130G, 51301, S130K, S130L,
S130M, S130N, S130P, S130R, S130T, S130V, S130W, G131A, G131C,
G131D, G131F, G131H, G131I, G131K, G131L, G131M, G131P, G131Q,
G131V, G131W, G131Y, E132A, E132C, E132D, E132F, E132G, E132H,
E132I, E132K, E132L, E132M, E132N, E132P, E132Q, E132R, E132V,
E132W, Y133A, Y133D, Y133E, Y133G, Y133K, Y133L, Y133M, Y133R,
Y133S, Y133T, Y133W, N134A, N134D, N134F, N134G, N134H, N134I,
N134K, N134L, N134M, N134R, N134S, N134V, N134W, N134Y, Q136A,
Q136C, Q136D, Q136E, Q136F, Q136H, Q136K, Q136L, Q136M, Q136N,
Q136R, Q136S, Q136T, Q136V, Q136W, Q136Y, A137S, A137T, A137V,
W138F, W138Y, G140A, G140M, N142F, N142K, N142L, N142M, N142P,
N142V, N142W, N142Y, F143H, F143Y, P144C, P144D, P144E, P144G,
P144H, P144I, P144K, P144L, P144M, P144N, P144Q, P144S, P144T,
P144Y, G147A, G147C, G147H, G147K, G147L, G147M, G147N, G147Q,
G147R, T148A, T148D, T148E, T148F, T1481, T148K, T148L, T148R,
T148S, T148V, T148W, T149A, T149C, T149D, T149E, T149F, T149H,
T149K, T149L, T149M, T149N, T149Q, T149V, T149W, T149Y, Y150H,
S151A, S151E, S151F, S151H, S151I, S151K, S151L, S151M, S151Q,
S151R, S151V, N152A, N152C, N152D, N152E, N152G, N152H, N152K,
N152M, N152P, N152Q, N152R, N152S, N152T, W153F, W153H, W153Q,
W153R, W153T, W153Y, K154A, K154C, K154D, K154E, K154G, K1541,
K154L, K154M, K154N, K154R, K154T, K154Y, W155P, Q156A, Q156D,
Q156E, Q156F, Q156G, Q156H, Q1561, Q156L, Q156M, Q156N, Q156R,
Q156S, Q156Y, F158C, F158D, F158K, F158L, F158M, F158N, F158P,
F158Q, F158R, F158S, F158V, H159M, H159Y, W165C, W165D, W165E,
W165F, W165H, W1651, W165K, W165L, W165M, W165Q, W165R, W165T,
W165Y, Q167A, Q167D, Q167E, Q167G, Q167H, Q167K, Q167M, Q167N,
Q167P, Q167S, Q167T, Q167V, S168A, S168D, S168F, S168H, 51681,
S168K, S168M, S168N, S168Q, S168R, S168T, S168V, S168W, S168Y,
S170A, S170C, S170D, S170E, S170F, S170L, S170M, S170N, S170Q,
S170R, S170T, L171A, L171C, L171F, L171G, L171H, L171I, L171N,
L171Q, L171R, L171T, L171V, L171W, L171Y, S172A, S172D, S172H,
S172R, S172T, F175M, F175Y, K1761, K176T, F177L, F177V, F177W,
D180A, D180C, D180F, D180G, D180H, D1801, D180L, D180M, D180N,
D180Q, D180R, D180S, D180T, D180V, D180W, D180Y, G181A, G181C,
G181D, G181E, G181F, G181H, G181K, G181L, G181M, G181N, G181Q,
G181R, G181S, G181T, G181V, G181Y, K182A, K182P, E187D, E1871,
E187K, E187M, E187N, E187P, E187R, E187S, E187T, E187V, E187Y,
S190A, S190C, S190D, S190F, S190L, S190N, S190P, S190Q, E191A,
E191C, E191F, E191G, E191H, E1911, E191K, E191N, E191Q, E191R,
E191S, E191T, E191W, E191Y, G193A, G193C, G193D, G193E, G193F,
G193H, G193K, G193L, G193M, G193N, G193Q, G193R, G193S, G193T,
G193V, G193W, M199L, Y200F, A201M, Y2031, Y203L, Y203V, D206A,
D206E, D206G, D206H, D206M, D206N, D206Q, D206R, D206S, D206T,
P208A, P208E, P208F, P2081, P208K, P208L, P208T, P208V, P208Y,
V210A, V210E, V210H, V210K, V210N, V210Q, V210R, V210S, V210T,
V211A, V211E, V211H, V2111, V211Q, V211R, N212E, N212F, N212G,
N212L, N212M, N212R, N212V, M2141, M214L, K215C, K215E, K215F,
K215M, K215N, K215R, K215Y, K216F, V2191, V219T, Y221F, Y2211,
Y221L, N223C, N223E, N223I, N223K, N223Q, N223R, N223 S, N223T,
N223V, N223W, N223Y, V225A, V2251, V225L, V225M, G226D, G226M,
G226Q, G226R, G226S, L227F, L227I, L227W, L227Y, V235A, I238A,
I238L, I238M, K239D, K239E, K239P, K239Q, K239R, K239S, K239T,
F240K, F240L, F240M, F240Q, F240R, Q241A, Q241C, Q241D, Q241E,
Q241G, Q241H, Q241K, Q241L, Q241M, Q241N, Q241P, Q241R, Q241S,
Q241T, Q241V, Q241W, Q241Y, F242V, L243C, L243Y, D245A, D245C,
D245E, D245G, D245L, D245M, D245N, W246F, V2471, V247L, D248E,
D248H, D248N, D248T, D248V, N249A, N249E, N249G, N249H, N249Q,
N249Y, A250M, A250S, A250V, A252C, A252D, A252E, A252G, A252H,
A2521, A252K, A252L, A252M, A252N, A252Q, A252S, A252V, A252W,
A252Y, A253E, A2531, A253K, A253L, A253M, A253Q, A253S, A253T,
A253V, A253Y, T254F, T254K, T254S, K256A, K256M, K256N, K256S,
E257Q, E257S, M258L, T260A, T260C, T260S, T260V, V261I, V261W,
G262A, Q266A, Q266D, Q266E, Q266H, Q2661, Q266M, Q266N, Q266S,
Q266T, Q266V, Q266Y, N267H, N267I, N267Q, N267R, N267S, N267T,
N267V, N267Y, D268G, D268N, L269C, L269D, L269I, L269K, L269Q,
L269S, L269T, L269Y, G270A, G270D, G270E, G270F, G270H, G270I,
G270L, G270M, G270Q, G270T, G270V, G270Y, A271C, A271D, A271E,
A271H, A271K, A271M, A271Q, A271R, A271S, A271T, A271V, A271Y,
N273C, N273G, N273H, N273I, N273K, N273R, L2761, L276M, A277C,
A277D, A277E, A277F, A277G, A277I, A277K, A277L, A277M, A277N,
A277Q, A277R, A277S, A277T, A277V, A277W, A277Y, V279C, V279T,
N280A, N282S, N282T, S284T, S284Y, L285A, L285C, L2851, L285V,
F286M, A288C, A296C, A296D, A296E, A296F, A296G, A296H, A2961,
A296L, A296M, A296N, A296Q, A296R, A296S, A296V, A296W, A296Y,
T299A, T299D, T299E, T299F, T299G, T299K, T299L, T299M, T299R,
T299S, T299V, T299W, G300A, G300C, G300D, G300E, G300F, G300H,
G300K, G300M, G300Q, G300R, G300V, G300W, G301A, G301C, G301D,
G301E, G301F, G301H, G301K, G301L, G301M, G301Q, G301R, G301S,
G301T, G301V, G301W, G301Y, G302S, Y303A, Y303C, Y303D, Y303E,
Y303F, Y303G, Y303H, Y3031, Y303K, Y303L, Y303M, Y303N, Y303Q,
Y303R, Y303S, Y303T, Y303V, Y303W, Y304F, Y304K, Y304W, R307A,
R307C, R307E, R307G, R307H, R307K, R307M, R307N, R307Q, R307S,
R307T, N308C, N308D, N308E, N308G, N308L, N308M, N308T, N308V,
L310A, L310C, L310D, L310E, L310H, L310I, L310M, L310P, L310W,
L310Y, N311C, N311E, N311G, N311H, N311K, N311Q, N311R, N311S,
N311V, N311W, N311Y, N312D, N312F, N312G, N312H, N312K, N312Q,
N312R, T313A, T313S, A316D, A316E, A316G, A316H, A316K, A316Q,
A316R, A316Y, S317C, S317D, S317G, S317H, S317K, S317L, S317N,
S317Q, S317R, S317T, S317W, S317Y, N318A, N318C, N318F, N318G,
N318I, N318K, N318L, N318M, N318Q, N318R, N318S, N318T, N318V,
N318W, T320A, T320C, T320D, T320E, T320G, T320H, T320I, T320K,
T320N, T320P, T320Q, T320R, T320V, T320W, T320Y, K321C, K321F,
K321H, K321N, K321S, K321Y, L325A, L325C, L325F, L3251, L325M,
L325Q, L325V, E327D, E327L, Q335C, Q335E, E338A, E338D, E338F,
E338G, E338H, E338I, E338K, E338P, E338Q, E338R, E338T, E338V,
E338Y, Q342A, Q342C, Q342G, Q342L, Q342M, Q342R, Q342S, Q342T,
Q342V, Q342W, L348A, L348C, L348H, L3481, L348M, L348Q, L348S,
L348T, A349G, A349R, F3521, F352L, F352M, F352T, F352V, R356Q,
S357A, S357C, S357D, S357E, S357F, S357H, 53571, S357K, S357L,
S357N, S357Q, S357T, S357V, S357W, S357Y, Y360F, Y3601, Y360L,
Y360M, Y360V, S362A, S362C, S362E, 53621, S362T, S362V, V363I,
V363L, M368F, M368I, M368L, M368Y, Y369A, Y369E, Y369I, Y369L,
Y369N, Y369V, R377A, R377C, R377D, R377E, R377F, R377G, R377H,
R3771, R377K, R377L, R377N, R377Q, R377S, R377T, R377V, R377W,
R377Y, A381C, A381E, A381G, A381H, A381K, A381L, A381M, A381N,
A381P, A381Q, A381V, A381W, A381Y, L382F, L382H, L382Q, L382S,
K383A, K383C, K383D, K383E, K383H, K3831, K383L, K383M, K383N,
K383Q, K383R, K383S, K383W, K383Y, S384A, S384C, S384D, S384F,
S384G, S384H, S3841, S384L, S384N, S384R, S384V, S384Y, K385A,
K385D, K385E, K385F, K385G, K385H, K385L, K385M, K385Q, K385R,
K385T, K385V, K385Y, P388A, P388C, P388D, P388I, P388L, P388N,
P388R, P388S, P388T, P388V, L390M, L390V, A392C, A392S, K394A,
K394C, K394E, K394F, K394G, K394H, K3941, K394L, K394Q, K394R,
K394S, K394T, K394V, K394W, K394Y, D395A, D395E, D395N, D395S,
D395T, Y396A, Y396D, Y396F, Y396K, Y396M, Y396N, Y396Q, Y396T,
Y396V, Y396W, A397D, A397E, A397H, A397K, A397M, A397N, A397Q,
A397V, Y398A, Y398C, Y398F, Y398H, Y3981, Y398L, Y398W, T400A,
T400C, T400D, T400F, T400G, T400I, T400K, T400L, T400M, T400N,
T400Q, T400R, T400W, T400Y, Q401A, Q401C, Q401F, Q401H, Q401I,
Q401L, Q401M, Q401N, R402C, R402D, R402F, R402K, R402L, R402M,
R402N, R402Q, R402S, R402W, D403E, D403N, D403S, Y404E, Y404G,
Y404K, Y404M, Y404N, Y404R, Y404W, I405C, I405F, I405L, I405M,
I405V, N407A, N407C, N407D, N407E, N407G, N407K, N407M, N407Q,
N407R, P408A, P408C, P408D, P4081, P408K, P408L, P408M, P408N,
P408Q, P408V, P408W, V410A, V410C, V410D, V410E, V410F, V410H,
V4101, V410L, V410M, V410N, V410Q, V410S, V410T, T414A, T4141,
T414S, T414V, R415M, E416C, E416D, E416F, E416H, E4161, E416K,
E416L, E416M, E416N, E416Q, E416R, E416T, E416V, E416W, E416Y,
D418A, D418E, D418G, D418H, D4181, D418K, D418L, D418M, D418N,
D418Q, D418S, D418T, D418V, D418W, S419A, S419C, S419E, S419G,
S419L, S419M, S419N, S419R, S419V, S419W, S419Y, T420A, T420C,
T420D, T420E, T420G, T420H, T420I, T420K, T420M, T420P, T420S,
T420V, T420W, T420Y, K421A, K421D, K421E, K421H, K4211, K421L,
K421M, K421N, K421P, K421Q, K421R, K421T, K421V, K421W, K421Y,
A422C, A422D, A422E, A422F, A422G, A4221, A422L, A422N, A422P,
A422Q, A422R, A422S, A422Y, K423A, K423D, K423E, K423F, K423H,
K4231, K423L, K423M, K423N, K423Q, K423R, K423S, K423T, K423V,
K423W, K423Y, S424A, S424C, S424G, S424K, S424N, S424Q, S424R,
S424T, L426S, L426T, L426V, T428G, T428V, V429A, V429C, V4291,
V429L, I430C, I430G, I430L, I430M, I430Q, I430V, T431A, T431C,
T431S, P434A, P434C, P434D, P434E, P434F, P434H, P4341, P434K,
P434L, P434M, P434N, P434Q, P434R, P434S, P434V, P434Y, G435A,
G435C, G435D, G435E, G435F, G435H, G4351, G435K, G435M, G435N,
G435P, G435Q, G435R, G435S, G435T, G435W, G436F, G4361, G436M,
G436N, G436Q, G436S, G436V, R439A, R439D, R439G, R439H, R439K,
R439M, R439N, R439P, R439Q, R439S, R439V, R439W, R439Y, Y441A,
Y441C, Y441D, Y441F, Y441G, Y441H, Y441K, Y441L, Y441M, Y441N,
Y441P, Y441R, Y441S, Y441T, Y441W, V442A, V442C, V4421, V442T,
T444C, T444D, T444E, T444F, T444G, T444H, T4441, T444K, T444L,
T444M, T444N, T444P, T444R, T444S, T444W, S445A, S445C, S445E,
S445G, S445H, S445K, S445L, S445M, S445N, S445T, S445V, N446A,
N446C, N446H, N446K, A447C, A447D, A447F, A447H, A447L, A447M,
A447N, A447Q, A447R, A447S, A447Y, G448A, G448C, G448D, G448E,
G448H, G448K, G448L, G448M, G448N, G448Q, G448R, G448S, G448T,
G448W, E449D, E449H, E449K, E449T, I450A, I450C, I450D, I450E,
I450G, I450K, I450L, I450M, I450N, I450Q, I450S, I450T, I450W,
I450Y, W451Y, L454A, L4541, L454K, L454M, L454W, T455A, T4551,
T455L, T455S, N457H, N457K, N457R, N457T, N457V, N457Y, D460A,
D460E, D460G, D460M, D460N, D460Q, D460S, D460V, K461C, K461H,
K461L, K461M, K461N, K461Q, K461T, K461Y, I462A, I462L, I462M,
I462Q, I462T, I462V, T463D, T463E, T463H, T463P, T463Q, T463R,
T463V, T463Y, I464P, I464T, G465A, G465C, G465D, G465E, G465K,
G465L, G465M, G465N, G465Q, G465W, G465Y, S466A, S466C, S466D,
S466G, S466H, S466K, S466L, S466M, S466N, S466T, S466W, S466Y,
D467E, D467G, D467L, Y469A, Y469D, Y469E, Y4691, Y469M, Y469N,
Y469R, Y469S, Y469T, Y469V, Y469W, A470S, A470V, T471A, T471C,
T471E, T471G, T471H, T4711, T471L, T471M, T471N, T471S, T471V,
T471W, P473C, P473D, P473E, P473G, P4731, P473K, P473L, P473R,
P473T, P473W, V474A, V474C, V474L, V474S, N475A, N475C, N475E,
N475F, N475H, N475K, N475L, N475M, N475P, N475Q, N475R, N475S,
N475T, N475V, G476A, G476D, G476E, G476F, G476H, G4761, G476L,
G476M, G476N, G476P, G476Q, G476R, G476S, G476T, G476V, G476W,
G476Y, G477A, G477D, G477F, G477H, G4771, G477K, G477L, G477M,
G477Q, G477S, G477T, G477V, G477W, G477Y, V479C, V479D, V479E,
V479F, V479H, V4791, V479N, V479P, V479Y, S480A, S480C, S480H,
V481A, V481C, V481N, W482Y, V483A, V483G, V4831, V483K, V483L,
V483M, V483R, V483Y, Q484A, Q484C, Q484F, Q484G, Q484H, Q484K,
Q484L, Q484M, Q484P, Q484R, Q484T, and Q484Y.
[0669] Dose Response Screening Results
[0670] A subset of the variants with performance-enhancing
mutations were tested more rigorously to demonstrate improved
activity relative to wild type at multiple protein concentrations
(i.e., improved specific activity). FIG. 20 shows examples of
variants with improved hydrolysis of corn starch at high
temperature. FIG. 21 shows examples of variants with improved
hydrolysis of amylopectin from corn. FIG. 22 shows examples of
variants with improved generation of reducing sugars from starch.
FIG. 23 shows examples of variants with improved release of iodine
staining material from starch. In FIGS. 20-23, the legends adjacent
to the graphs indicate the mutations present in addition to those
present to the control variant, CspAmy2-C18P. FIG. 24 shows
examples of variants that demonstrate improved reduction of corn
slurry viscosity compared to the CspAmy2-C18P control. CspAmy2-C16F
is also included for comparison.
Example 15
Pair-Wise Combinations of Mutations at Positions 476 and 477
[0671] Site evaluation libraries were constructed and screened to
determined the effect of pair-wise combinations of mutations at
positions 476 and 477 in variant CspAmy2-C16F (CspAmy2 with the
mutations N126Y, F153W, T180H, I203Y, and S241Q, and lacking R178
and G179). The matrix in FIG. 25 shows the PI values for
CspAmy2-C16F position 476/477 variants compared to a CspAmy2-C16F
control (with a PI value set at 1) in a CS-26 corn starch
microswatch assay, as described, above. The amino acid residues at
positions 476 and 477 are indicated on the left-side and top of the
matrix, respectively. A "wild-type" revertant, i.e., G476G/G477G
had an experimentally obtained PI score of 1.01, suggesting that
the assay produces very reliable results. The absence of a number
at a position in the matrix indicates poor expression of the
particular variant or that the variant was not present among those
tested. The matrix in FIG. 26 shows the PI values for the same
variants in an amylose hydrolysis assay, as described, above.
Again, a "wild-type" revertant, i.e., G476G/G477G had an
experimentally obtained PI score corresponding to that of the
control.
[0672] Remarkably, the results suggest that almost any combination
of residues at positions 476 and 477, other the glycine pair that
occurs in naturally in CspAmy2 (and in many .alpha.-amylases),
increases the performance of the .alpha.-amylase in terms of
improving the hydrolysis of insoluble amylopectin and improving the
release of iodine staining material from hydrated starch. Without
being bound to a theory, it is believed that the adjacent GG
residues at the C-terminus of .alpha.-amylases contribute to
starch-binding. While binding tightly to a substrate may be
desirable in nature where substrate is limiting, in industrial
applications it may be more desirable for the enzyme to release
from one starch molecule after hydrolyzing it and find a different
starch molecules to hydrolize, rather than remaining associated
with the first starch molecule and hydrolysing it processively.
Example 16
Additional C18P-Based Variants with Superior Performance in
Secondary Liquifaction
[0673] Site evaluation libraries (SELs) were constructed and
screened to determined the effect of mutations at positions E132,
Q167, A277, and T400 in CspAmy2-C18P. The residues present at these
positions in the best tested variants are shown in Table 6.
TABLE-US-00031 TABLE 6 Combinations of mutations tested Name E132
A277 Q167 T400 C16F E A Q T C25F H F Q T C25B H F E T C25A H F E
K
[0674] The relative performance of CspAmy2-C25A, B, and F in a
liquefaction assay compared to C18F is shown in FIG. 27. Mutations
at positions 132 and 277 increase performance. Additional benefit
is observed from mutations at positions 167. In further
experiments, it was shown that C25B demonstrated superior
liquefaction performance to C16F at pH 5.2 and 5.8, and with or
without additional calcium. Variants CspAmy2-C25A, B, and F all
outperformed variant CspAmy2-C18P (not shown).
[0675] The amino acid sequence of the mature CspAmy2-C25A amylase
polypeptide is shown, below, as SEQ ID NO: 25 [the relevant
substitutions are underlined]:
TABLE-US-00032 AATNGTMMQY FEWYVPNDGQ QWNRLRTDAP YLSSVGITAV
WTPPAYKGTS QADVGYGPYD LYDLGEFNQK GTVRTKYGTK GELKSAVNTL HSNGIQVYGD
VVMNHKAGAD YTENVTAVEV NPSNRYQETS GHYNIQAWTG FNFPGRGTTY SNWKWQWFHF
DGTDWDESRS LSRIFKFDGK AWDWEVSSEN GNYDYLMYAD YDYDHPDVVN EMKKWGVWYA
NEVGLDGYRL DAVKHIKFQF LKDWVDNARA ATGKEMFTVG EYWQNDLGAL NNYLFKVNYN
QSLFDAPLHY NFYAASTGGG YYDMRNILNN TLVASNPTKA VTLVENHDTQ PGQSLESTVQ
PWFKPLAYAF ILTRSGGYPS VFYGDMYGTK GTTTREIPAL KSKIEPLLKA RKDYAYGKQR
DYIDNPDVIG WTREGDSTKA KSGLATVITD GPGGSKRMYV GTSNAGEIWY DLTGNRTDKI
TIGSDGYATF PVNGGSVSVW VQQ
[0676] The amino acid sequence of the mature CspAmy2-C25B amylase
polypeptide is shown, below, as SEQ ID NO: 26 [the relevant
substitutions are underlined]:
TABLE-US-00033 AATNGTMMQY FEWYVPNDGQ QWNRLRTDAP YLSSVGITAV
WTPPAYKGTS QADVGYGPYD LYDLGEFNQK GTVRTKYGTK GELKSAVNTL HSNGIQVYGD
VVMNHKAGAD YTENVTAVEV NPSNRYQETS GHYNIQAWTG FNFPGRGTTY SNWKWQWFHF
DGTDWDESRS LSRIFKFDGK AWDWEVSSEN GNYDYLMYAD YDYDHPDVVN EMKKWGVWYA
NEVGLDGYRL DAVKHIKFQF LKDWVDNARA ATGKEMFTVG EYWQNDLGAL NNYLFKVNYN
QSLFDAPLHY NFYAASTGGG YYDMRNILNN TLVASNPTKA VTLVENHDTQ PGQSLESTVQ
PWFKPLAYAF ILTRSGGYPS VFYGDMYGTK GTTTREIPAL KSKIEPLLKA RKDYAYGTQR
DYIDNPDVIG WTREGDSTKA KSGLATVITD GPGGSKRMYV GTSNAGEIWY DLTGNRTDKI
TIGSDGYATF PVNGGSVSVW VQQ
[0677] The amino acid sequence of the mature CspAmy2-C25F amylase
polypeptide is shown, below, as SEQ ID NO: 27 [the relevant
substitutions are underlined]:
TABLE-US-00034 AATNGTMMQY FEWYVPNDGQ QWNRLRTDAP YLSSVGITAV
WTPPAYKGTS QADVGYGPYD LYDLGEFNQK GTVRTKYGTK GELKSAVNTL HSNGIQVYGD
VVMNHKAGAD YTENVTAVEV NPSNRYQETS GHYNIQAWTG FNFPGRGTTY SNWKWQWFHF
DGTDWDQSRS LSRIFKFDGK AWDWEVSSEN GNYDYLMYAD YDYDHPDVVN EMKKWGVWYA
NEVGLDGYRL DAVKHIKFQF LKDWVDNARA ATGKEMFTVG EYWQNDLGAL NNYLFKVNYN
QSLFDAPLHY NFYAASTGGG YYDMRNILNN TLVASNPTKA VTLVENHDTQ PGQSLESTVQ
PWFKPLAYAF ILTRSGGYPS VFYGDMYGTK GTTTREIPAL KSKIEPLLKA RKDYAYGTQR
DYIDNPDVIG WTREGDSTKA KSGLATVITD GPGGSKRMYV GTSNAGEIWY DLTGNRTDKI
TIGSDGYATF PVNGGSVSVW VQQ
Example 17
Additional CspAmy2-v5-Based Variants with Superior Performance in
Cleaning Applications
[0678] Additional CspAmy2-v5-based variants were made in an effort
to further improve performance in cleaning applications. The
variants and the mutations are shown in the Table 7. CspAmy2-v171
and CspAmy2-172 were previously described in Example 11. All
variants included deletions at positions R178 and G179, indicated
by "del (R178, G179)."
TABLE-US-00035 TABLE 7 Additional CspAmy2-v5-based variants Variant
Mutations CspAmy2-v5 del (R178, G179) + E187P + I203Y + G476K
CspAmy2-v171 del (R178, G179) + T180D + E187P + I203Y + G476K
CspAmy2-v172 del (R178, G179) + N126Y + T180D + E187P + I203Y +
G476K CspAmy2-v179 del (R178, G179) + N126Y + T180D + E187P + I203Y
+ Y303D + G476T + G477E CspAmy2-v180 del (R178, G179) + N126Y +
T180D + E187P + I203Y + Y303D + N475E + G477Q CspAmy2-v181 del
(R178, G179) + N126Y + T180D + E187P + I203Y + Y303R + N475E +
G476T + G477R CspAmy2-v186 del (R178, G179) + T38N + N88H + N126Y +
T129I + N134M + F153W + L171R + T180D + E187P + I203Y + G476K +
G477E CspAmy2-v191 del (R178, G179) + N126Y + E132H + T180D + E187P
+ I203Y + Y303D + G476T + G477E
[0679] The cleaning performance of the purified variants was
analyzed in a microswatch cleaning assay performed essentially as
described above. CFT CS-28 swatches were punched to form discs
measuring 5.5 mm in diameter. Two discs were placed in each well of
3 each flat-bottom, non-binding 96-well assay plates. The CspAmy2
variants, STAINZYME.RTM., and ACE-QK were each diluted to 0.5 mg/mL
in dilution buffer (50 mM MOPS (pH 7.2) and 0.005% Tween), and then
further diluted to 18 ppm in a microtiter plate. Several dilutions
were made in the microtiter plates, down to 0.27 ppm. 10 .mu.L of
each of these samples were added to the three swatch plates, and
170 .mu.L of HEPES buffer (25 mM HEPES, pH 8.0 with 2 mM
CaCl.sub.2) and 0.005% Tween-80) was added to each well for a final
volume of 180 .mu.L. The final enzyme concentrations ranged from 1
ppm down to 0.015 ppm. The plates were incubated at 25.degree. C.
with agitation at 1150 rpm for 15 minutes. Enzyme performance was
judged by the amount of color released into the wash liquor. Color
release was quantified spectrophotometrically at 488 nm by the
transfer of 140 .mu.L of the final wash solution to fresh
medium-binding microtiter plates, and triplicate reads were
blank-subtracted and averaged.
[0680] The results of the microswatch cleaning assays performed at
0.015 ppm enzyme are shown in FIG. 28. Variants CspAmy2-v179, v186,
and v191 all demonstrated superior cleaning performance compared to
CspAmy2-v5, and all CspAmy2 combinatorial variants were far
superior to STAINZYME.RTM.. The results of the cleaning assays
performed at all enzyme concentrations are shown in Table 8. All
CspAmy2 demonstrated superior cleaning performance at lower
concentrations compared to STAINZYME.RTM..
TABLE-US-00036 TABLE 8 Results of the cleaning assays performed
using CspAmy2 variants Enzyme concentration (ppm) Enzyme 0.015
0.032 0.045 0.063 0.090 0.125 0.25 0.5 1.0 CspAmy2-v179 0.25 0.31
0.32 0.35 0.35 0.37 0.37 0.36 0.38 CspAmy2-v186 0.28 0.33 0.34 0.35
0.38 0.38 0.39 0.39 0.39 CspAmy2-v191 0.22 0.30 0.31 0.35 0.37 0.38
0.39 0.39 0.40 CspAmy2-v5 0.19 0.26 0.30 0.34 0.35 0.37 0.38 0.39
0.38 STAINZYME .RTM. 0.07 0.12 0.14 0.19 0.19 0.25 0.30 0.34 0.38
ACE-QK 0.30 0.33 0.36 0.34 0.35 0.36 0.36 0.38 0.38
[0681] Thermal stability assays were performed essentially as
described. Stocks of CspAmy2 variants, STAINZYME.RTM., and ACE-QK
at 0.5 mg/mL were diluted to 5 ppm, 10 ppm, or 1 ppm, respectively,
in dilution buffer (50 mM MOPS (pH 7.2) and 0.005% Tween) to
account for their relative specific activities on the soluble
substrate. 50 .mu.L of each enzyme were added to each of 12 wells
of PCR tubes and sealed. The "unstressed" samples were incubated at
room temperature throughout the duration of the experiment. The
other samples were incubated in a thermocycler in a gradient from
77.degree. C. to 97.degree. C. for 15 minutes. Samples were
transferred to microtiter plates in triplicate, and alpha-amylase
activity was measured on all unstressed and stressed samples using
the Ceralpha reagent (Megazyme, Inc.). Residual activity was
calculated by dividing the activity of each amylase after the
thermal stress by the activity of that unstressed amylase.
[0682] The results of the thermostability assay are shown in FIG.
29. Variants CspAmy2-v186 and v191 both demonstrated superior
thermal stability compared to CspAmy2-v5. All the CspAmy2 variants
demonstrated superior thermal stability compared to STAINZYME.RTM.
and ACE-QK.
[0683] The in-detergent storage stability of the CspAmy2 variants
was tested in a several commercial detergents, i.e., TIDE.RTM.
regular HDL and TIDE.RTM. PODS.TM. (Procter & Gamble) for the
USA market, ARIEL.TM. HDL (Procter & Gamble) and OMO Color HDL
(Unilever) for the European market, and OMO.TM. (Unilever) and
LIBY.TM. HDL (Liby) for the Chinese market. All detergents were
heat inactivated at 90.degree. C. for 4 hours to eliminate existing
enzyme activities. Enzyme activity in the heat inactivated
detergents was measured using the Suc-AAPF-pNA and Ceralpha assays
for measuring protease and amylase activity, respectively.
[0684] To prepare the stability samples, 2% w/w protease
(PURAFECT.RTM. Prime HA, DuPont Industrial Biosciences) and 0.5%
w/w amylase were added to each detergent sample and mixed. Samples
were stored in a CO2 incubator (Sanyo) at 37.degree. C. for 28
days. Aliquots were taken from each reaction sample at various time
points, diluted in 50 mM MOPS (pH 7.15) buffer with 1% BSA added,
and alpha-amylase activity was measured using the Ceralpha
substrate (Megazyme, Inc). The activity for each sample was
determined using an Arena 20XT Photometric Analyzer (Thermo
Scientific) using a calibrated standard. The remaining activity
after incubation for 28 days was reported as a percent of the total
activity determined at time zero.
[0685] The amount of residual activity of the CspAmy2 variants
compared to STAINZYME.RTM. and ACE-QK are shown in FIGS. 30-35.
CspAmy2-v179 was particularly stable compared to other tested
variants and the controls.
[0686] The amino acid sequence of mature CspAmy2-v179 is shown,
below, as SEQ ID NO: 28:
TABLE-US-00037 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTY
SNFKWQWFHFDGTDWDQSRSLSRIFKFDGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
DYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNTESVSVWVQQ
[0687] The amino acid sequence of mature CspAmy2-v180 is shown,
below, as SEQ ID NO: 29:
TABLE-US-00038 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTY
SNFKWQWFHFDGTDWDQSRSLSRIFKFDGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
DYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVEGQSVSVWVQQ
[0688] The amino acid sequence of mature CspAmy2-v181 is shown,
below, as SEQ ID NO: 30:
TABLE-US-00039 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGEYNIQAWTGFNFPGRGTTY
SNFKWQWFHFDGTDWDQSRSLSRIFKFDGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
DYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVETRSVSVWVQQ
[0689] The amino acid sequence of mature CspAmy2-v186 is shown,
below, as SEQ ID NO: 31:
TABLE-US-00040 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGINAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVHTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQEISGEYMIQAWTGFNFPGRGTTY
SNWKWQWFHFDGTDWDQSRSRSRIFKFDGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
YYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNKESVSVWVQQ
[0690] The amino acid sequence of mature CspAmy2-v191 is shown,
below, as SEQ ID NO: 32:
TABLE-US-00041 AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTS
QADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGD
VVMNHKAGADYTENVTAVEVNPSNRYQETSGHYNIQAWTGFNFPGRGTTY
SNFKWQWFHFDGTDWDQSRSLSRIFKFDGKAWDWPVSSENGNYDYLMYAD
YDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARA
ATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGG
DYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAF
ILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQR
DYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWY
DLTGNRTDKITIGSDGYATFPVNTESVSVWVQQ
Example 18
Combinatorial Variants of PcuAmy1
[0691] To determine whether equivalent combinatorial mutations
resulted in similar performance gains in a different
.alpha.-amylase molecule, equivalent mutations were made in an
.alpha.-amylase from Paenibacillus curdlanolyticus (i.e., PcuAmy1).
The amino acid sequence of the mature of PcuAmy1 polypeptide is
shown, below (SEQ ID NO: 3):
TABLE-US-00042 ADNGTIMQYFEWYLPNDGAHWNRLNNDAQNLKNVGITAVWIPPAYKGGSS
ADVGYGVYDTYDLGEFNQKGTVRTKYGTKSELISAVNNLHAKGIAVYGDV
VLNHRMNADATELVDAVEVDPNNRNVETTSTYQIQAWTQYDFPGRGNTYS
SFKWRWYHFDGVDWDQSRGLNRIYKLRGDGKDWDWEVDSEYGNYDYLMGA
DLDFNHPDVVNETKTWGKWFVNTVNLDGVRLDAVKHIKFDFMRDWVNNVR
STTGKNLFAVGEYWHYDVNKLNSYITKTNGTMSLFDVPLHFRFYDASNGG
GGYDMRNLLNNTLMSSNPMKAVTFVENHDTQPTQALQSTVQSWFKPLAYA
TILTREQGYPCVFYGDYYGTSDGKISSYKPIMDKLLNARKVYAYGTQRDY
FDHPDIVGWTREGDAAHAGSGLATLITDGPGGSKWMYVGTSKAGQVWTDK
TGNRSGTVTIDANGWGNFWVNGGSVSVWAK
[0692] Mutations were made at positions N125, F152, R177, G178,
E186, G472, and G473 (using SEQ ID NO: 3 for numbering),
corresponding to mutations at positions N126, F153, R178, G179,
E187P, G476, and G477, respectively in CspAmy2 (SEQ ID NO: 1). A
further mutation was introduced at position N205. Consistent with
previous nomenclature "del (R177, G178)" refers to deletions, in
this case at positions R177 and G178. In addition to the
aforementioned mutations, the PcuAmy1 variants further included
mutations at position T333, A335, and Q337E. These mutations,
particularly at T333, impart protease resistance to PcuAmy1 but do
not affect performance (see Example 19). The variants are shown in
Table 9.
TABLE-US-00043 TABLE 9 Combinatorial variants of PcuAmy1 amylase
Variant Mutations PcuAmy1-v1A del (R177, G178) + N125Y + E186P +
T333G + A335S + Q337E + G472K PcuAmy1-v6 del (R177, G178) + N125Y +
F152W + E186P + T333G + A335S + Q337E + G472K PcuAmy1-v8 del (R177,
G178) + N125Y + F152W + E186P + T333G + A335S + Q337E + G472R +
G473R PcuAmy1-v16 del (R177, G178) + N125Y + F152W + E186P + N205D
+ T333G + A335S + Q337E + G472K
[0693] The codon-optimized nucleotide sequence of the PcuAmy1 gene
is set forth as SEQ ID NO: 33:
TABLE-US-00044 GCCGACAACGGCACAATCATGCAGTATTTCGAGTGGTACCTGCCGAACGA
CGGAGCGCACTGGAACAGACTTAATAACGACGCACAAAACCTGAAAAATG
TGGGCATCACGGCAGTGTGGATTCCTCCGGCATACAAGGGCGGCAGCTCA
GCAGATGTTGGCTACGGAGTTTACGATACATACGACCTGGGCGAGTTCAA
TCAGAAAGGCACGGTCAGAACAAAGTACGGAACGAAGAGCGAACTGATTT
CAGCGGTCAACAATCTTCACGCAAAGGGCATTGCGGTTTACGGCGACGTG
GTCCTGAACCATAGAATGAATGCGGATGCAACGGAGCTTGTGGATGCGGT
TGAGGTGGATCCGAACAACAGAAACGTCGAGACGACAAGCACGTATCAGA
TCCAGGCATGGACGCAATACGATTTCCCGGGCAGAGGCAACACGTACAGC
AGCTTTAAATGGAGATGGTATCACTTCGACGGCGTCGACTGGGACCAGAG
CAGAGGCCTGAACAGAATCTATAAGCTGAGAGGCGATGGCAAGGATTGGG
ACTGGGAGGTCGACAGCGAGTACGGCAACTACGATTACCTGATGGGAGCG
GACCTGGACTTCAACCACCCGGATGTGGTTAACGAAACAAAGACATGGGG
CAAATGGTTTGTGAACACGGTGAACCTGGATGGCGTCAGACTGGACGCGG
TTAAGCACATCAAGTTCGACTTCATGAGAGACTGGGTGAACAACGTGAGA
AGCACGACGGGCAAGAACCTTTTCGCAGTTGGCGAGTATTGGCACTACGA
CGTGAACAAACTGAACAGCTACATCACGAAGACGAATGGCACGATGAGCC
TGTTCGACGTGCCGCTGCACTTTAGATTTTATGATGCAAGCAACGGCGGA
GGCGGCTACGACATGAGAAACCTGCTGAATAACACGCTGATGAGCAGCAA
CCCGATGAAGGCGGTTACATTCGTTGAGAACCATGACACACAACCGACGC
AGGCCCTGCAATCAACGGTCCAAAGCTGGTTTAAGCCGCTTGCGTATGCT
ACAATCCTGACGAGAGAGCAAGGCTACCCGTGCGTTTTCTACGGCGACTA
TTATGGAACAAGCGACGGCAAAATTAGCAGCTACAAGCCGATCATGGATA
AGCTTCTTAACGCGAGAAAGGTGTACGCCTACGGCACGCAGAGAGATTAC
TTCGATCATCCGGACATCGTTGGCTGGACAAGAGAAGGCGATGCAGCACA
TGCTGGCTCAGGACTGGCAACGCTTATCACAGATGGCCCTGGCGGAAGCA
AGTGGATGTATGTTGGAACGTCAAAGGCAGGCCAGGTCTGGACGGATAAA
ACAGGAAACAGAAGCGGAACGGTGACGATTGATGCCAATGGCTGGGGAAA
CTTTTGGGTTAATGGCGGATCAGTTAGCGTTTGGGCAAAATAA
[0694] The amino acid sequence of the mature of PcuAmy1-v1A
polypeptide is shown below as SEQ ID NO: 34:
TABLE-US-00045 ADNGTIMQYFEWYLPNDGAHWNRLNNDAQNLKNVGITAVWIPPAYKGGSS
ADVGYGVYDTYDLGEFNQKGTVRTKYGTKSELISAVNNLHAKGIAVYGDV
VLNHRMNADATELVDAVEVDPNNRYVETTSTYQIQAWTQYDFPGRGNTYS
SFKWRWYHFDGVDWDQSRGLNRIYKLDGKDWDWPVDSEYGNYDYLMGADL
DFNHPDVVNETKTWGKWFVNTVNLDGVRLDAVKHIKFDFMRDWVNNVRST
TGKNLFAVGEYWHYDVNKLNSYITKTNGTMSLFDVPLHFRFYDASNGGGG
YDMRNLLNNTLMSSNPMKAVTFVENHDTQPGQSLESTVQSWFKPLAYATI
LTREQGYPCVFYGDYYGTSDGKISSYKPIMDKLLNARKVYAYGTQRDYFD
HPDIVGWTREGDAAHAGSGLATLITDGPGGSKWMYVGTSKAGQVWTDKTG
NRSGTVTIDANGWGNFWVNKGSVSVWAK
[0695] The amino acid sequence of the mature of PcuAmy1-v6
polypeptide is shown below as SEQ ID NO: 35:
TABLE-US-00046 ADNGTIMQYFEWYLPNDGAHWNRLNNDAQNLKNVGITAVWIPPAYKGGSS
ADVGYGVYDTYDLGEFNQKGTVRTKYGTKSELISAVNNLHAKGIAVYGDV
VLNHRMNADATELVDAVEVDPNNRYVETTSTYQIQAWTQYDFPGRGNTYS
SWKWRWYHFDGVDWDQSRGLNRIYKLDGKDWDWPVDSEYGNYDYLMGADL
DFNHPDVVNETKTWGKWFVNTVNLDGVRLDAVKHIKFDFMRDWVNNVRST
TGKNLFAVGEYWHYDVNKLNSYITKTNGTMSLFDVPLHFRFYDASNGGGG
YDMRNLLNNTLMSSNPMKAVTFVENHDTQPGQSLESTVQSWFKPLAYATI
LTREQGYPCVFYGDYYGTSDGKISSYKPIMDKLLNARKVYAYGTQRDYFD
HPDIVGWTREGDAAHAGSGLATLITDGPGGSKWMYVGTSKAGQVWTDKTG
NRSGTVTIDANGWGNFWVNKGSVSVWAK
[0696] The amino acid sequence of the mature of PcuAmy1-v8
polypeptide is shown below as SEQ ID NO: 36:
TABLE-US-00047 ADNGTIMQYFEWYLPNDGAHWNRLNNDAQNLKNVGITAVWIPPAYKGGSS
ADVGYGVYDTYDLGEFNQKGTVRTKYGTKSELISAVNNLHAKGIAVYGDV
VLNHRMNADATELVDAVEVDPNNRYVETTSTYQIQAWTQYDFPGRGNTYS
SWKWRWYHFDGVDWDQSRGLNRIYKLDGKDWDWPVDSEYGNYDYLMGADL
DFNHPDVVNETKTWGKWFVNTVNLDGVRLDAVKHIKFDFMRDWVNNVRST
TGKNLFAVGEYWHYDVNKLNSYITKTNGTMSLFDVPLHFRFYDASNGGGG
YDMRNLLNNTLMSSNPMKAVTFVENHDTQPGQSLESTVQSWFKPLAYATI
LTREQGYPCVFYGDYYGTSDGKISSYKPIMDKLLNARKVYAYGTQRDYFD
HPDIVGWTREGDAAHAGSGLATLITDGPGGSKWMYVGTSKAGQVWTDKTG
NRSGTVTIDANGWGNFWVNRRSVSVWAK
[0697] The amino acid sequence of the mature of PcuAmy1-v16
polypeptide is shown below as SEQ ID NO: 37:
TABLE-US-00048 ADNGTIMQYFEWYLPNDGAHWNRLNNDAQNLKNVGITAVWIPPAYKGGSS
ADVGYGVYDTYDLGEFNQKGTVRTKYGTKSELISAVNNLHAKGIAVYGDV
VLNHRMNADATELVDAVEVDPNNRYVETTSTYQIQAWTQYDFPGRGNTYS
SWKWRWYHFDGVDWDQSRGLNRIYKLDGKDWDWPVDSEYGNYDYLMGADL
DFDHPDVVNETKTWGKWFVNTVNLDGVRLDAVKHIKFDFMRDWVNNVRST
TGKNLFAVGEYWHYDVNKLNSYITKTNGTMSLFDVPLHFRFYDASNGGGG
YDMRNLLNNTLMSSNPMKAVTFVENHDTQPGQSLESTVQSWFKPLAYATI
LTREQGYPCVFYGDYYGTSDGKISSYKPIMDKLLNARKVYAYGTQRDYFD
HPDIVGWTREGDAAHAGSGLATLITDGPGGSKWMYVGTSKAGQVWTDKTG
NRSGTVTIDANGWGNFWVNKGSVSVWAK
[0698] The cleaning performance of PcuAmy1-v1, PcuAmy1-v6, and
PcuAmy1-v16, compared to STAINZYME.RTM. and ACE-QK is shown in FIG.
36. The microswatch assay was performed as described in Example 17.
PcuAmy1-v6 and PcuAmy1-v16 outperformed PcuAmy1-v1 and
STAINZYME.RTM. at low doses (e.g., 0.1 ppm enzyme or less).
[0699] The thermal stability of PcuAmy1-v1, PcuAmy1-v6, and
PcuAmy1-v16, compared to STAINZYME.RTM. is shown in FIG. 37. The
assays were performed as described in Example 17. PcuAmy1-v16 were
more thermostable than the other tested molecules using the same
detergents and enzyme doses.
Example 19
Combinatorial Variants in BASE
[0700] As in Example 18, to determine whether equivalent
combinatorial mutations resulted in similar performance gains in a
different .alpha.-amylase molecule, equivalent mutations were made
in an .alpha.-amylase derived from Bacillus sp. TS-23. The amino
acid sequence of BASE, a C-terminal-truncated version of the
Bacillus sp. TS-23 .alpha.-amylase (see, e.g., US20120045817 and
WO2010/115028), is shown below as SEQ ID NO: 5:
TABLE-US-00049 NTAPINETMMQYFEWDLPNDGTLWTKVKNEAANLSSLGITALWLPPAYKG
TSQSDVGYGVYDLYDLGEFNQKGTIRTKYGTKTQYIQAIQAAKAAGMQVY
ADVVFNHKAGADGTEFVDAVEVDPSNRNQETSGTYQIQAWTKFDFPGRGN
TYSSFKWRWYHFDGTDWDESRKLNRIYKFRSTGKAWDWEVDTENGNYDYL
MFADLDMDHPEVVTELKNWGTWYVNTTNIDGFRLDAVKHIKYSFFPDWLT
YVRNQTGKNLFAVGEFWSYDVNKLHNYITKTNGSMSLFDAPLHNNFYTAS
KSSGYFDMRYLLNNTLMKDQPSLAVTLVDNHDTQPGQSLQSWVEPWFKPL
AYAFILTRQEGYPCVFYGDYYGIPKYNIPGLKSKIDPLLIARRDYAYGTQ
RDYIDHQDIIGWTREGIDTKPNSGLAALITDGPGGSKWMYVGKKHAGKVF
YDLTGNRSDTVTINADGWGEFKVNGGSVSIWVAK
[0701] The codon-modified nucleic acid sequence encoding the mature
form of BASE (AmyTS23t), is set forth as SEQ ID NO: 38:
TABLE-US-00050 TCTGCAGCT TCAGCAAAC ACCGCGCCG ATTAACGAA ACCATGATG
CAGTATTTC GAATGGGAT CTGCCGAAC GATGGCACC CTGTGGACC AAAGTGAAA
AACGAAGCG GCGAACCTG AGCAGCCTG GGCATTACC GCGCTGTGG CTGCCGCCG
GCATATAAA GGCACCAGC CAGAGCGAT GTGGGCTAT GGCGTGTAT GATCTGTAC
GATCTGGGC GAATTTAAC CAGAAAGGC ACCATTCGT ACCAAATAT GGCACCAAA
ACCCAGTAT ATTCAGGCG ATCCAGGCG GCGAAAGCG GCGGGTATG CAGGTGTAT
GCGGATGTG GTGTTTAAC CATAAAGCG GGTGCGGAT GGCACCGAA TTTGTGGAT
GCGGTGGAA GTGGATCCG AGCAACCGT AACCAGGAA ACCAGCGGC ACCTATCAG
ATTCAGGCG TGGACCAAA TTTGATTTT CCCGGCCGT GGCAACACC TATAGCAGC
TTTAAATGG CGCTGGTAT CATTTTGAT GGCACCGAT TGGGATGAA AGCCGTAAA
CTGAACCGC ATCTATAAA TTTCGTAGC ACCGGCAAA GCGTGGGAT TGGGAAGTG
GATACCGAA AACGGCAAC TATGATTAC CTGATGTTC GCAGACCTG GATATGGAT
CATCCGGAA GTGGTGACC GAACTGAAA AACTGGGGC ACCTGGTAT GTGAACACC
ACCAACATT GATGGCTTT CGTCTGGAT GCGGTGAAA CACATCAAA TACAGCTTT
TTTCCGGAT TGGCTGACC TATGTGCGT AACCAGACC GGCAAAAAC CTGTTTGCG
GTGGGCGAA TTTTGGAGC TATGATGTG AACAAACTG CACAACTAC ATCACCAAA
ACCAACGGC AGCATGAGC CTGTTTGAT GCGCCGCTG CATAACAAC TTTTATACC
GCGAGCAAA AGCAGCGGC TATTTTGAT ATGCGTTAT CTGCTGAAC AACACCCTG
ATGAAAGAT CAGCCGAGC CTGGCCGTG ACCCTGGTG GATAACCAT GATACCCAG
CCGGGCCAG AGCCTGCAA AGCTGGGTG GAACCGTGG TTTAAACCG CTGGCCTAC
GCGTTTATT CTGACCCGT CAAGAGGGC TATCCGTGC GTTTTTTAT GGCGATTAT
TACGGCATC CCGAAATAT AACATTCCG GGCCTGAAA AGCAAAATT GATCCGCTG
CTGATTGCG CGTCGTGAT TATGCGTAT GGCACCCAG CGTGATTAT ATTGATCAC
CAGGATATT ATTGGCTGG ACCCGTGAA GGCATTGAT ACCAAACCG AACAGCGGC
CTGGCCGCG CTGATTACC GATGGCCCG GGTGGCAGC AAATGGATG TATGTGGGC
AAAAAACAT GCGGGCAAA GTGTTTTAT GATCTGACC GGCAACCGT AGCGATACC
GTGACCATT AACGCGGAT GGCTGGGGT GAGTTTAAA GTGAACGGC GGCAGCGTG
AGCATTTGG GTGGCGAAA TAAGTTAAC AGA
[0702] Mutations were made at positions N128, T134, F155, T182,
R180, S181, E189, and G475 were made in BASE (using SEQ ID NO: 5
for numbering), which corresponds to mutations at positions N126,
E132, F153, R178, G179, E187P, and G476, respectively in CspAmy2
(SEQ ID NO: 1). The variants are shown in Table 10.
TABLE-US-00051 TABLE 10 Combinatorial variants of BASE amylase
Variant Mutations BASE-V28 del (R180, G181) + N128Y + E189P + G475R
BASE-V29 del (R180, G181) + F155W + E189P + G475R BASE-V30 del
(R180, G181) + T134E + T182H + E189P + G475R BASE-V31 del (R180,
G181) + N128Y + T134E + T182H + E189P + G475R BASE-V32 del (R180,
G181) + N128Y + F155W + E189P + G475R BASE-V33 del (R180, G181) +
T134E + F155W + T182H + E189P + G475R BASE-V34 del (R180, G181) +
N128Y + T134E + F155W + T182H + E189P + G475R BASE-V35 del (R180,
G181) + N128Y + T134H + F155W + T182D + E189P + G475R BASE-V36 del
(R180, G181) + N128Y + T134E + F155W + T182G + E189P + G457R ACE-QK
del (R180, G181) + S243Q + G475K
[0703] The amino acid sequence of BASE-V28 is shown below as SEQ ID
NO: 39:
TABLE-US-00052 NTAPINETMMQYFEWDLPNDGTLWTKVKNEAANLSSLGITALWLPPAYKG
TSQSDVGYGVYDLYDLGEFNQKGTIRTKYGTKTQYIQAIQAAKAAGMQVY
ADVVFNHKAGADGTEFVDAVEVDPSNRYQETSGTYQIQAWTKFDFPGRGN
TYSSFKWRWYHFDGTDWDESRKLNRIYKFTGKAWDWPVDTENGNYDYLMF
ADLDMDHPEVVTELKNWGTWYVNTTNIDGFRLDAVKHIKYSFFPDWLTYV
RNQTGKNLFAVGEFWSYDVNKLHNYITKTNGSMSLFDAPLHNNFYTASKS
SGYFDMRYLLNNTLMKDQPSLAVTLVDNHDTQPGQSLQSWVEPWFKPLAY
AFILTRQEGYPCVFYGDYYGIPKYNIPGLKSKIDPLLIARRDYAYGTQRD
YIDHQDIIGWTREGIDTKPNSGLAALITDGPGGSKWMYVGKKHAGKVFYD
LTGNRSDTVTINADGWGEFKVNRGSVSIWVAK
[0704] The amino acid sequence of BASE-V29 is shown below as SEQ ID
NO: 40:
TABLE-US-00053 NTAPINETMMQYFEWDLPNDGTLWTKVKNEAANLSSLGITALWLPPAYKG
TSQSDVGYGVYDLYDLGEFNQKGTIRTKYGTKTQYIQAIQAAKAAGMQVY
ADVVFNHKAGADGTEFVDAVEVDPSNRNQETSGTYQIQAWTKFDFPGRGN
TYSSWKWRWYHFDGTDWDESRKLNRIYKFTGKAWDWPVDTENGNYDYLMF
ADLDMDHPEVVTELKNWGTWYVNTTNIDGFRLDAVKHIKYSFFPDWLTYV
RNQTGKNLFAVGEFWSYDVNKLHNYITKTNGSMSLFDAPLHNNFYTASKS
SGYFDMRYLLNNTLMKDQPSLAVTLVDNHDTQPGQSLQSWVEPWFKPLAY
AFILTRQEGYPCVFYGDYYGIPKYNIPGLKSKIDPLLIARRDYAYGTQRD
YIDHQDIIGWTREGIDTKPNSGLAALITDGPGGSKWMYVGKKHAGKVFYD
LTGNRSDTVTINADGWGEFKVNRGSVSIWVAK
[0705] The amino acid sequence of BASE-V30 is shown below as SEQ ID
NO: 41:
TABLE-US-00054 NTAPINETMMQYFEWDLPNDGTLWTKVKNEAANLSSLGITALWLPPAYKG
TSQSDVGYGVYDLYDLGEFNQKGTIRTKYGTKTQYIQAIQAAKAAGMQVY
ADVVFNHKAGADGTEFVDAVEVDPSNRNQETSGEYQIQAWTKFDFPGRGN
TYSSFKWRWYHFDGTDWDESRKLNRIYKFHGKAWDWPVDTENGNYDYLMF
ADLDMDHPEVVTELKNWGTWYVNTTNIDGFRLDAVKHIKYSFFPDWLTYV
RNQTGKNLFAVGEFWSYDVNKLHNYITKTNGSMSLFDAPLHNNFYTASKS
SGYFDMRYLLNNTLMKDQPSLAVTLVDNHDTQPGQSLQSWVEPWFKPLAY
AFILTRQEGYPCVFYGDYYGIPKYNIPGLKSKIDPLLIARRDYAYGTQRD
YIDHQDIIGWTREGIDTKPNSGLAALITDGPGGSKWMYVGKKHAGKVFYD
LTGNRSDTVTINADGWGEFKVNRGSVSIWVAK
[0706] The amino acid sequence of BASE-V31 is shown below as SEQ ID
NO: 42:
TABLE-US-00055 NTAPINETMMQYFEWDLPNDGTLWTKVKNEAANLSSLGITALWLPPAYKG
TSQSDVGYGVYDLYDLGEFNQKGTIRTKYGTKTQYIQAIQAAKAAGMQVY
ADVVFNHKAGADGTEFVDAVEVDPSNRYQETSGEYQIQAWTKFDFPGRGN
TYSSFKWRWYHFDGTDWDESRKLNRIYKFHGKAWDWPVDTENGNYDYLMF
ADLDMDHPEVVTELKNWGTWYVNTTNIDGFRLDAVKHIKYSFFPDWLTYV
RNQTGKNLFAVGEFWSYDVNKLHNYITKTNGSMSLFDAPLHNNFYTASKS
SGYFDMRYLLNNTLMKDQPSLAVTLVDNHDTQPGQSLQSWVEPWFKPLAY
AFILTRQEGYPCVFYGDYYGIPKYNIPGLKSKIDPLLIARRDYAYGTQRD
YIDHQDIIGWTREGIDTKPNSGLAALITDGPGGSKWMYVGKKHAGKVFYD
LTGNRSDTVTINADGWGEFKVNRGSVSIWVAK
[0707] The amino acid sequence of BASE-V32 is shown below as SEQ ID
NO: 43:
TABLE-US-00056 NTAPINETMMQYFEWDLPNDGTLWTKVKNEAANLSSLGITALWLPPAYKG
TSQSDVGYGVYDLYDLGEFNQKGTIRTKYGTKTQYIQAIQAAKAAGMQVY
ADVVFNHKAGADGTEFVDAVEVDPSNRYQETSGTYQIQAWTKFDFPGRGN
TYSSWKWRWYHFDGTDWDESRKLNRIYKFTGKAWDWPVDTENGNYDYLMF
ADLDMDHPEVVTELKNWGTWYVNTTNIDGFRLDAVKHIKYSFFPDWLTYV
RNQTGKNLFAVGEFWSYDVNKLHNYITKTNGSMSLFDAPLHNNFYTASKS
SGYFDMRYLLNNTLMKDQPSLAVTLVDNHDTQPGQSLQSWVEPWFKPLAY
AFILTRQEGYPCVFYGDYYGIPKYNIPGLKSKIDPLLIARRDYAYGTQRD
YIDHQDIIGWTREGIDTKPNSGLAALITDGPGGSKWMYVGKKHAGKVFYD
LTGNRSDTVTINADGWGEFKVNRGSVSIWVAK
[0708] The amino acid sequence of BASE-V33 is shown below as SEQ ID
NO: 44:
TABLE-US-00057 NTAPINETMMQYFEWDLPNDGTLWTKVKNEAANLSSLGITALWLPPAYKG
TSQSDVGYGVYDLYDLGEFNQKGTIRTKYGTKTQYIQAIQAAKAAGMQVY
ADVVFNHKAGADGTEFVDAVEVDPSNRNQETSGEYQIQAWTKFDFPGRGN
TYSSWKWRWYHFDGTDWDESRKLNRIYKFHGKAWDWPVDTENGNYDYLMF
ADLDMDHPEVVTELKNWGTWYVNTTNIDGFRLDAVKHIKYSFFPDWLTYV
RNQTGKNLFAVGEFWSYDVNKLHNYITKTNGSMSLFDAPLHNNFYTASKS
SGYFDMRYLLNNTLMKDQPSLAVTLVDNHDTQPGQSLQSWVEPWFKPLAY
AFILTRQEGYPCVFYGDYYGIPKYNIPGLKSKIDPLLIARRDYAYGTQRD
YIDHQDIIGWTREGIDTKPNSGLAALITDGPGGSKWMYVGKKHAGKVFYD
LTGNRSDTVTINADGWGEFKVNRGSVSIWVAK
[0709] The amino acid sequence of BASE-V34 is shown below as SEQ ID
NO: 45:
TABLE-US-00058 NTAPINETMMQYFEWDLPNDGTLWTKVKNEAANLSSLGITALWLPPAYKG
TSQSDVGYGVYDLYDLGEFNQKGTIRTKYGTKTQYIQAIQAAKAAGMQVY
ADVVFNHKAGADGTEFVDAVEVDPSNRYQETSGEYQIQAWTKFDFPGRGN
TYSSWKWRWYHFDGTDWDESRKLNRIYKFHGKAWDWPVDTENGNYDYLMF
ADLDMDHPEVVTELKNWGTWYVNTTNIDGFRLDAVKHIKYSFFPDWLTYV
RNQTGKNLFAVGEFWSYDVNKLHNYITKTNGSMSLFDAPLHNNFYTASKS
SGYFDMRYLLNNTLMKDQPSLAVTLVDNHDTQPGQSLQSWVEPWFKPLAY
AFILTRQEGYPCVFYGDYYGIPKYNIPGLKSKIDPLLIARRDYAYGTQRD
YIDHQDIIGWTREGIDTKPNSGLAALITDGPGGSKWMYVGKKHAGKVFYD
LTGNRSDTVTINADGWGEFKVNRGSVSIWVAK
[0710] The amino acid sequence of BASE-V35 is shown below as SEQ ID
NO: 46:
TABLE-US-00059 NTAPINETMMQYFEWDLPNDGTLWTKVKNEAANLSSLGITALWLPPAYKG
TSQSDVGYGVYDLYDLGEFNQKGTIRTKYGTKTQYIQAIQAAKAAGMQVY
ADVVFNHKAGADGTEFVDAVEVDPSNRYQETSGHYQIQAWTKFDFPGRGN
TYSSWKWRWYHFDGTDWDESRKLNRIYKFDGKAWDWPVDTENGNYDYLMF
ADLDMDHPEVVTELKNWGTWYVNTTNIDGFRLDAVKHIKYSFFPDWLTYV
RNQTGKNLFAVGEFWSYDVNKLHNYITKTNGSMSLFDAPLHNNFYTASKS
SGYFDMRYLLNNTLMKDQPSLAVTLVDNHDTQPGQSLQSWVEPWFKPLAY
AFILTRQEGYPCVFYGDYYGIPKYNIPGLKSKIDPLLIARRDYAYGTQRD
YIDHQDIIGWTREGIDTKPNSGLAALITDGPGGSKWMYVGKKHAGKVFYD
LTGNRSDTVTINADGWGEFKVNRGSVSIWVAK
[0711] The amino acid sequence of BASE-V36 is shown below as SEQ ID
NO: 47:
TABLE-US-00060 NTAPINETMMQYFEWDLPNDGTLWTKVKNEAANLSSLGITALWLPPAYKG
TSQSDVGYGVYDLYDLGEFNQKGTIRTKYGTKTQYIQAIQAAKAAGMQVY
ADVVFNHKAGADGTEFVDAVEVDPSNRYQETSGEYQIQAWTKFDFPGRGN
TYSSWKWRWYHFDGTDWDESRKLNRIYKFGGKAWDWPVDTENGNYDYLMF
ADLDMDHPEVVTELKNWGTWYVNTTNIDGFRLDAVKHIKYSFFPDWLTYV
RNQTGKNLFAVGEFWSYDVNKLHNYITKTNGSMSLFDAPLHNNFYTASKS
SGYFDMRYLLNNTLMKDQPSLAVTLVDNHDTQPGQSLQSWVEPWFKPLAY
AFILTRQEGYPCVFYGDYYGIPKYNIPGLKSKIDPLLIARRDYAYGTQRD
YIDHQDIIGWTREGIDTKPNSGLAALITDGPGGSKWMYVGKKHAGKVFYD
LTGNRSDTVTINADGWGEFKVNRGSVSIWVAK
[0712] The thermal stability of BASE-V28, V29, V30, V31, V32, V33,
V34, and V35, compared to ACE-QK (e.g., US20120045817 and
WO2010/115028), is shown in FIG. 38. All the BASE variants were
more stable than ACE-QK, although BASE-V28 was only marginally more
stable.
Example 19
Interactions Between Residues in RG-Deletion Molecules
[0713] A structural interaction between residues 132 and 180
(referring to SEQ ID NO: 1 for numbering) explains the increased
stability of some of the variants. Details of the crystal structure
of CspAmy2-v1 are shown in FIGS. 39-42. As shown in FIG. 39, the
naturally occurring glutamate side chain at position 132 is
positioned towards the side chain of the naturally-occurring
threonine at position 180. The distance of 5.4 Angstroms, however,
is too great for the formation of any stabilizing interaction. As
shown in FIG. 40, a T180H variant (e.g., CspAmy2-vC16C) has the
histidine imidazole NH group in proximity to the E132 glutamate
carboxylate. The distance of 3.2 Angstroms allows the formation of
a stabilizing hydrogen bond. At pH of roughly 4.5 to 7.0, a
favorable charge interaction (i.e., salt bridge) is also likely
between these residues. Referring to, e.g., Examples 6 and 7 and
FIGS. 8-10, the observations that CspAmy2-C16E is more stable than
CspAmy2-C16C, CspAmy2-C16F is more stable than CspAmy2-C16D,
CspAmy2-C16I is more stable than CspAmy2-C16G, and CspAmy2-C16J is
more stable than CspAmy2-C16H, which pairs of variants differ only
by the presence or absence of the mutation T180H, supports this
hypothesis. Similarly, referring to, e.g., Examples 17 and FIG. 29,
the observation that CspAmy2-v191 is more stable than CspAmy2-v179,
which pair of variants differ only by the presence or absence of
the mutation E132H, supports this hypothesis.
[0714] As shown in FIG. 41, an aspartic acid at position 180 may
also be capable of hydrogen bonding with the glutamate at position
132, although hydrogen bonding may be overwhelmed by unfavorable
like charge interactions. However, the presence of histidine at
position 132, in combination with an aspartate at position 180,
restores the possibility for a favorable interaction created by a
T180D mutation (FIG. 42). The stabilizing effect of the E132H
mutation in CspAmy2-v191 and CspAmy2-C25A, B, and F, which all have
a T180D mutation, supports this hypothesis (e.g., Example 16 and
FIG. 27).
[0715] In BASE, position E132 corresponds to position T134 and
position T180 corresponds to position T182. The observations that
BASE-V31 is more stable than BASE-V28 and BASE-V33 is more stable
than BASE-V29 further supports this hypothesis in the context of a
different .alpha.-amylase. In both cases, the mutations T134E and
T180H appear to work together to enable the formation of a
stabilizing interaction, likely a salt bridge. Similarly, V34 is
more stable than V36, because of the stabilizing interaction
between the glutamate and histidine in V34, which does not occur
between the glutamate and glycine in V36.
[0716] Although the position 132-180 interaction was demonstrated
using an "RG" deletion, it can fully be expected to work in the
context of an adjacent "DG" or TG'' deletion. It will be
appreciated that the conserved amino acid sequence motif
X.sub.1G/S.sub.1X.sub.2G.sub.2 (SEQ ID NO: 48), as exemplified by
RGTG (SEQ ID NO: 49) in CspAmy2 .alpha.-amylase (SEQ ID NO: 1), is
adjacent to the calcium-binding loop in .alpha.-amylases. X1 is
typically arginine. G/Si is most often glycine but is serine in the
case of BASE. X2 varies but is commonly aspartate or threonine. G2
is highly conserved. Deleting the TG in CspAmy2 .alpha.-amylase,
instead of RG, would mean that the remaining residues would be RG
rather than TG. Although the arginine would be derived from
position 178 as opposed to the threonine, which is derived from
position 180, the three-dimensional structure of the resulting
variant is indistinguishable from one having an RG deletion plus an
R to G substitution, and stabilizing the resulting molecule is
simply a matter of selecting a suitable residue at position 132 to
form a stabilizing interaction with whatever residue is remaining
at the equivalent position in the X.sub.1G/S.sub.1X.sub.2G.sub.2
motif, whether it originally corresponded to position 180 or
position 178 in the patent molecule (using SEQ ID NO: 1 for
numbering). For convenience, this residue may be referred to as the
remaining non-G residue in the aforementioned motif.
[0717] Therefore, in general, if position 132 is negatively charged
(i.e., D or E), then the remaining non-G residue should be
positively charged (i.e., H, R, or K). If position 132 is
positively charged (i.e., H, R, or K), then the remaining non-G
residue should be negatively charged (i.e., D or E).
Example 19
Proteolytic Cleavage of PcuAmy1 and Variants
[0718] Incubation of wild-type PcuAmy1 amylase, or the PcuAmy-v1
variant, with subtilisin proteases leads to cleavage of the
proteins, as observed when the reaction products are subjected to
SDS/PAGE electrophoresis. FIG. 43 is an image of an SDS/PAGE gel
showing the cleavage of 20 .mu.g of PcuAmy1-v1 in the presence of
increasing amounts of GG36 protease, (from 0 to 40 .mu.g as
indicated above the gel). The letters on the right side of the gel
indicate (A) intact full-length PcuAmy1-v1, (B) a first cleavage
product of PcuAmy1-v1, (C) GG36 protease, (D) a contaminant in the
GG36 protein preparation, and (E) a second cleavage product of
PcuAmy1-v1. The main degradation products observed after incubation
of PcuAmy-v1 amylase with a subtilisin protease have a molecular
weight of about 38 and 16 kDa (B and E, respectively). The amount
of proteolytic degradation is dependent on the concentration of
protease used. This makes PcuAmy1 amylase suboptimal for inclusion
in enzyme detergent formulations that contain commonly-used
subtilisin proteases.
[0719] A sample of PcuAmy-v1 protein was incubated with GG36
protease (Bacillus lentis subtilisin) and the reaction products
were analyzed by mass spectroscopy. The results were consistent
with hydrolysis occurring between residues Q334 and L336 (not
shown).
[0720] To determine whether protease-stable variants of PcuAmy
could be engineered, PcuAmy1-v3 and further variants 3A to 3L were
constructed and tested. PcuAmy1-v3 is a variant of PcuAmy1 with the
mutations E186P, G472K and lacking R177 and G178 (using SEQ ID NO:
3 for numbering). The substitution E186P and the deletions at R177
and G178 increase the detergent stability of PcuAmy1. The
substitution G472K improves cleaning performance. None of these
mutations has any effect on protease sensitivity (data not shown).
Therefore, including these mutations in variants made to explore
the effect of other mutations on protease stability does not
interfere with the results.
[0721] The mature form of PcuAmy1-v3 is shown, below, as (SEQ ID
NO: 50):
TABLE-US-00061 ADNGTIMQYFEWYLPNDGAHWNRLNNDAQNLKNVGITAVWIPPAYKGGSS
ADVGYGVYDTYDLGEFNQKGTVRTKYGTKSELISAVNNLHAKGIAVYGDV
VLNHRMNADATELVDAVEVDPNNRNVETTSTYQIQAWTQYDFPGRGNTYS
SFKWRWYHFDGVDWDQSRGLNRIYKLDGKDWDWPVDSEYGNYDYLMGADL
DFNHPDVVNETKTWGKWFVNTVNLDGVRLDAVKHIKFDFMRDWVNNVRST
TGKNLFAVGEYWHYDVNKLNSYITKTNGTMSLFDVPLHFRFYDASNGGGG
YDMRNLLNNTLMSSNPMKAVTFVENHDTQPTQALQSTVQSWFKPLAYATI
LTREQGYPCVFYGDYYGTSDGKISSYKPIMDKLLNARKVYAYGTQRDYFD
HPDIVGWTREGDAAHAGSGLATLITDGPGGSKWMYVGTSKAGQVWTDKTG
NRSGTVTIDANGWGNFWVNKGSVSVWAK
[0722] To identify the reason for PcuAmy1 protease sensitivity, the
amino acid sequence of PcuAmy1 was compared to that of other CAZy
Family GH-13 amylases which show protease-resistance, such as
PURASTAR.RTM. ST (B. licheniformis amylase or AmyL), SPEZYME.RTM.
XTRA (Geobacillus stearothermophilus amylase or AmyS), ACE-QK
(WO2010/115021), and STAINZYME.RTM. (Novozymes). Based on observed
differences in sequence, PcuAmy1 variants PcuAmy1-v3A to
PcuAmy1-v3L (i.e., 3A-3L) were designed and tested for protease
resistance. The mutations present in each variant are listed in
Table 11. They were introduced into PcuAmy1-v3 (SEQ ID NO: 49),
using standard methods, many of which are described above.
TABLE-US-00062 TABLE 11 List of mutations introduced in PcuAmy1-v3
resulting in variants 3A to 3L Position wt 3A 3B 3C 3D 3E 3F 3G 3H
3I 3J 3K 3L 319 M T 333 T G G G G G G 335 A S S S S S S 337 Q E E E
E E 339 T W 341 Q E 342 S P P P P T 351 T F F W
[0723] The 12 PcuAmy1 variants were expressed in B. subtilis as
described, above. 40 filtered supernatant from each PcuAmy1 variant
culture broth was incubated with 100 .mu.g GG36 protease for 6
hours at room temperature, and subsequently analyzed for remaining
amylase activity using the Megazyme Ceralpha substrate assay
(Megazyme International Ireland, Co. Wicklow, Ireland). Residual
activity after protease incubation was compared to the amylase
activity of each sample after incubation with buffer alone. The
results are shown in FIG. 44. A subset of the samples was also
analyzed by SDS-PAGE (FIG. 45), with the protein standard,
SEEBLUE.RTM. Plus2 (Invitrogen). The commercially available
amylases were included for comparison.
[0724] PcuAmy1 variants 3A, 3B, 3C, 3D, and 3L maintained >70%
of their enzymatic activity after incubation with GG36 protease.
PcuAmy1 variants 3J and 3K maintained >65% of their enzymatic
activity after incubation with GG36 protease. PcuAmy1 variants 3E,
3F, 3G, 3H, and 3I did not show an appreciable increase of
stability compared to the wild-type enzyme. Samples of the 3B, 3C,
3D and 3L incubations were analyzed by SDS/PAGE and showed
significant reduction in degradation products when incubated with
GG36 protease, confirming that the increase in residual amylase
activity was due to decreased proteolytic cleavage.
[0725] These results of the small-scale experiment indicate that
the introduction of a mutation at position T333 significantly
reduces the proteolytic cleavage of PcuAmy1, and that additional
mutations at A335, Q337, and 5342 further reduce proteolytic
cleavage. The T351W mutation but not the T351F mutation, also
appears to reduce the proteolytic cleavage of PcuAmy1.
[0726] To better characterize the relative contributions of these
mutations to protease resistance, the following additional variants
were made:
[0727] PcuAmy1-v10: del (R177, G178)+E186P+T333G+Q337E+G472K
[0728] PcuAmy1-v11: del (R177, G178)+E186P+T333G+A335S+G472K
[0729] PcuAmy1-v12: del (R177, G178)+E186P+A335S+Q337E+G472K
[0730] PcuAmy1-v13: del (R177,
G178)+E186P+T333G+A335S+Q337E+T351W+G472K
[0731] Variants PcuAmy1-v10, PcuAmy1-v11, and PcuAmy1-v12 include
pair-wise combination of mutations at positions T333, A335, and
Q337. PcuAmy1-v3-v13 includes mutations at all the aforementioned
positions and includes the additional mutation T351W. These
variants were compared in a large scale detergent stability assay
to the following previously-described variants:
[0732] PcuAmy1-v3B: del (R177,
G178)+E186P+T333G+A335S+Q337E+G472K
[0733] PcuAmy1-v3L: del (R177,
G178)+E186P+T333G+A335S+Q337E+S342T+G472K
[0734] The commercial detergents Total Color (MIFA Ag Frenkendorf,
Switzerland) and Omo (Unilever, London, UK) were heat inactivated
at 90.degree. C. for 4 hours to eliminate existing enzyme
activities. Enzyme activity in the heat inactivated detergents was
measured using the Suc-AAPF-pNA and Ceralpha assays for measuring
protease and amylase activity, respectively. To prepare the
stability samples, 2% w/w protease (PURAFECT.RTM. Prime 4000L,
Danisco US Inc.) and 0.5% w/w amylase were added to each detergent
sample and mixed. Samples were stored in a CO2 incubator (Sanyo) at
37.degree. C. for 14 days. Aliquots were taken from each reaction
sample at various time points, diluted in 50 mM MOPS, pH 7.15
buffer with 1% BSA added, and alpha-amylase activity was measured
using the Ceralpha substrate (Megazyme, Inc). The activity for each
sample was determined using a Arena 20XT Photometric Analyzer
(Thermo Scientific) using a calibrated standard. The remaining
activity at each time point was reported as a percent (%) of the
total activity determined at time zero.
[0735] The results of the detergent stability assays performed in
MIFA Total (MIFA Ag Frenkendorf, Switzerland) and Unilever OMO
(Unilever, London, UK) are shown in FIGS. 46-50. FIGS. 46 and 48
show the residual activity of PcuAmy1 variants over time in MIFA
Total and Unilever Omo, respectively, supplemented with FNA
protease. FIGS. 47 and 49 summarize the data for the 3 and 14 day
time points. PcuAmy1 variants that include the T333 mutation, i.e.,
3B, 3L, v10, v13, and to a lesser degree, v11 were the most stable.
Variants that did not include the T333 mutation, i.e., V1 and v12,
were the least stable. As evidenced by v10 and v11, the presence of
a mutation at Q337E further improves stability.
[0736] The cleaning performance of purified PcuAmy1-v3B and
PcuAmy1-v3L was analyzed in a microswatch cleaning assay. CFT CS-28
rice starch on cotton swatches (Center for Testmaterials, BV,
Vlaardingen, Netherlands) containing an indicator dye bound to the
starch were punched to form discs measuring 5.5 mm in diameter. Two
discs were placed in each well of three flat-bottom non-binding
96-well assay plates.
[0737] Both enzymes and two commercial amylase products:
PURASTAR.RTM. ST (alpha-amylase from Bacillus licheniformis; DuPont
Industrial Biosciences, Palo Alto, Calif., USA), and STAINZYME.RTM.
(Novozymes, Copenhagen, Denmark) were diluted to 0.5 mg/mL in
dilution buffer (50 mM MOPS, pH 7.2, 0.005% Tween), and then
further diluted to 2 ppm in a microtiter plate. 200 .mu.L of these
samples were transferred into the first row of each of three swatch
plates. 100 .mu.L of HEPES buffer (25 mM HEPES, pH 8.0 with 2 mM
CaCl.sub.2) and 0.005% Tween-80) was then added to each well of the
next five rows of the swatch plates, and serial dilutions were made
to result in final enzyme concentrations of 2, 1, 0.5, 0.25, and
0.125 ppm as well as a row of blank (buffer only) wells with 200
.mu.L in every well. Plates were incubated at 25.degree. C. with
agitation at 1150 rpm for 15 minutes. The wash liquor was
transferred to fresh microtiter plates and enzyme performance was
judged by the amount of color released into the wash liquor. Color
release was quantified spectrophotometrically at 488 nm, and
triplicate reads were blank-subtracted and averaged.
[0738] The results are shown in FIG. 50. Both PcuAmy1-v3B and
PcuAmy1-v3L demonstrated excellent cleaning performance.
[0739] Commercial detergent Persil Universal Gel Gold (Henkel,
Dusseldorf, Germany) was heat inactivated at 90.degree. C. for 4
hours to eliminate existing enzyme activities. Following
inactivation, enzyme activity in the heat inactivated detergents
was measured using the Suc-AAPF-pNA substrate-based and Ceralpha
assay (Megazyme, Wicklow, Ireland) to ensure that any protease and
amylase activities, respectively, had been abolished. A 10%
solution of detergent was then made in water.
[0740] 100 .mu.L of each of the enzyme stocks (at 0.5 mg/mL) were
added to 400 .mu.L of the 10% detergent solutions. The enzymes
tested were PURASTAR.RTM., STAINZYME.RTM., PcuAmy1-v3B and
PcuAmy-v3L. 50 .mu.L of enzyme stock solution was added to PCR
tubes and incubated at either 60, 70, 80, or 90.degree. C. for 15
minutes. Prior to incubation, 10 .mu.L was removed and incubated at
room temperature throughout the duration of the experiment to serve
as the "unstressed" samples. Following incubation, an additional
1:10 dilution of each sample was made in dilution buffer. Samples
were then transferred to microtiter plates in triplicate, and
alpha-amylase activity was measured on all unstressed and stressed
samples using the Ceralpha assay. Residual activity was calculated
by dividing the activity of each amylase after the thermal stress
by the activity of the unstressed amylase.
[0741] The results are shown in FIGS. 51 and 52. PcuAmy1-v3B and
PcuAmy-v3L demonstrated similar thermostability compared to
STAINZYME.RTM., and significantly better stability than
PURASTAR.RTM..
[0742] Although the foregoing compositions and methods have been
described in some detail by way of illustration and examples for
purposes of clarity of understanding, it will be apparent to those
skilled in the art that certain changes and modifications may be
made. Therefore, the description should not be construed as
limiting the scope of the invention, which is delineated by the
appended claims.
[0743] All publications, patents, and patent applications cited
herein are hereby incorporated by reference in their entireties for
all purposes and to the same extent as if each individual
publication, patent, or patent application were specifically and
individually indicated to be so incorporated by reference.
Sequence CWU 1
1
501485PRTArtificial SequenceSynthetic amino acid sequence of the
mature form of the CspAmy2 alpha-amylase polypeptide 1Ala Ala Thr
Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10 15Asn Asp
Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu 20 25 30Ser
Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys Gly 35 40
45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu
50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr
Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His Ser Asn
Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly
Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val Asn Pro
Ser Asn Arg Asn Gln Glu 115 120 125Thr Ser Gly Glu Tyr Asn Ile Gln
Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr Thr Tyr
Ser Asn Phe Lys Trp Gln Trp Phe His Phe145 150 155 160Asp Gly Thr
Asp Trp Asp Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys 165 170 175Phe
Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn 180 185
190Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro
195 200 205Asp Val Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala
Asn Glu 210 215 220Val Gly Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys
His Ile Lys Phe225 230 235 240Ser Phe Leu Lys Asp Trp Val Asp Asn
Ala Arg Ala Ala Thr Gly Lys 245 250 255Glu Met Phe Thr Val Gly Glu
Tyr Trp Gln Asn Asp Leu Gly Ala Leu 260 265 270Asn Asn Tyr Leu Ala
Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala 275 280 285Pro Leu His
Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Tyr Tyr 290 295 300Asp
Met Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr305 310
315 320Lys Ala Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly Gln
Ser 325 330 335Leu Glu Ser Thr Val Gln Pro Trp Phe Lys Pro Leu Ala
Tyr Ala Phe 340 345 350Ile Leu Thr Arg Ser Gly Gly Tyr Pro Ser Val
Phe Tyr Gly Asp Met 355 360 365Tyr Gly Thr Lys Gly Thr Thr Thr Arg
Glu Ile Pro Ala Leu Lys Ser 370 375 380Lys Ile Glu Pro Leu Leu Lys
Ala Arg Lys Asp Tyr Ala Tyr Gly Thr385 390 395 400Gln Arg Asp Tyr
Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu 405 410 415Gly Asp
Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp 420 425
430Gly Pro Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly
435 440 445Glu Ile Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile
Thr Ile 450 455 460Gly Ser Asp Gly Tyr Ala Thr Phe Pro Val Asn Gly
Gly Ser Val Ser465 470 475 480Val Trp Val Gln Gln
4852483PRTArtificial SequenceSynthetic amino acid sequence of the
mature CspAmy2-v1 a-amylase polypeptide 2Ala Ala Thr Asn Gly Thr
Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10 15Asn Asp Gly Gln Gln
Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu 20 25 30Ser Ser Val Gly
Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser Gln
Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu 50 55 60Gly Glu
Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75
80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln
85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp Tyr
Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg
Asn Gln Glu 115 120 125Thr Ser Gly Glu Tyr Asn Ile Gln Ala Trp Thr
Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr Thr Tyr Ser Asn Phe
Lys Trp Gln Trp Phe His Phe145 150 155 160Asp Gly Thr Asp Trp Asp
Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys 165 170 175Phe Thr Gly Lys
Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr Asp
Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200
205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala Asn Glu Val Gly
210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys His Ile Lys Phe
Ser Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn Ala Arg Ala Ala
Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu Tyr Trp Gln Asn
Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala Lys Val Asn Tyr
Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His Tyr Asn Phe Tyr
Ala Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met 290 295 300Arg Asn Ile
Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys Ala305 310 315
320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser Val Phe Tyr Gly
Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr Arg Glu Ile Pro
Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu Lys Ala Arg Lys
Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp Tyr Ile Asp Asn
Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Thr Lys
Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro 420 425 430Gly
Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly Glu Ile 435 440
445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser
450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Asn Gly Gly Ser Val Ser
Val Trp465 470 475 480Val Gln Gln3480PRTArtificial
SequenceSynthetic amino acid sequence of the mature form of the
PcuAmy1 alpha-amylase polypeptide 3Ala Asp Asn Gly Thr Ile Met Gln
Tyr Phe Glu Trp Tyr Leu Pro Asn1 5 10 15Asp Gly Ala His Trp Asn Arg
Leu Asn Asn Asp Ala Gln Asn Leu Lys 20 25 30Asn Val Gly Ile Thr Ala
Val Trp Ile Pro Pro Ala Tyr Lys Gly Gly 35 40 45Ser Ser Ala Asp Val
Gly Tyr Gly Val Tyr Asp Thr Tyr Asp Leu Gly 50 55 60Glu Phe Asn Gln
Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser65 70 75 80Glu Leu
Ile Ser Ala Val Asn Asn Leu His Ala Lys Gly Ile Ala Val 85 90 95Tyr
Gly Asp Val Val Leu Asn His Arg Met Asn Ala Asp Ala Thr Glu 100 105
110Leu Val Asp Ala Val Glu Val Asp Pro Asn Asn Arg Asn Val Glu Thr
115 120 125Thr Ser Thr Tyr Gln Ile Gln Ala Trp Thr Gln Tyr Asp Phe
Pro Gly 130 135 140Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp
Tyr His Phe Asp145 150 155 160Gly Val Asp Trp Asp Gln Ser Arg Gly
Leu Asn Arg Ile Tyr Lys Leu 165 170 175Arg Gly Asp Gly Lys Asp Trp
Asp Trp Glu Val Asp Ser Glu Tyr Gly 180 185 190Asn Tyr Asp Tyr Leu
Met Gly Ala Asp Leu Asp Phe Asn His Pro Asp 195 200 205Val Val Asn
Glu Thr Lys Thr Trp Gly Lys Trp Phe Val Asn Thr Val 210 215 220Asn
Leu Asp Gly Val Arg Leu Asp Ala Val Lys His Ile Lys Phe Asp225 230
235 240Phe Met Arg Asp Trp Val Asn Asn Val Arg Ser Thr Thr Gly Lys
Asn 245 250 255Leu Phe Ala Val Gly Glu Tyr Trp His Tyr Asp Val Asn
Lys Leu Asn 260 265 270Ser Tyr Ile Thr Lys Thr Asn Gly Thr Met Ser
Leu Phe Asp Val Pro 275 280 285Leu His Phe Arg Phe Tyr Asp Ala Ser
Asn Gly Gly Gly Gly Tyr Asp 290 295 300Met Arg Asn Leu Leu Asn Asn
Thr Leu Met Ser Ser Asn Pro Met Lys305 310 315 320Ala Val Thr Phe
Val Glu Asn His Asp Thr Gln Pro Thr Gln Ala Leu 325 330 335Gln Ser
Thr Val Gln Ser Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile 340 345
350Leu Thr Arg Glu Gln Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr Tyr
355 360 365Gly Thr Ser Asp Gly Lys Ile Ser Ser Tyr Lys Pro Ile Met
Asp Lys 370 375 380Leu Leu Asn Ala Arg Lys Val Tyr Ala Tyr Gly Thr
Gln Arg Asp Tyr385 390 395 400Phe Asp His Pro Asp Ile Val Gly Trp
Thr Arg Glu Gly Asp Ala Ala 405 410 415His Ala Gly Ser Gly Leu Ala
Thr Leu Ile Thr Asp Gly Pro Gly Gly 420 425 430Ser Lys Trp Met Tyr
Val Gly Thr Ser Lys Ala Gly Gln Val Trp Thr 435 440 445Asp Lys Thr
Gly Asn Arg Ser Gly Thr Val Thr Ile Asp Ala Asn Gly 450 455 460Trp
Gly Asn Phe Trp Val Asn Gly Gly Ser Val Ser Val Trp Ala Lys465 470
475 4804478PRTArtificial SequenceSynthetic amino acid sequence of a
variant form of PcuAmy1 alpha-amylase having a deletion of both
R177 and R178 4Ala Asp Asn Gly Thr Ile Met Gln Tyr Phe Glu Trp Tyr
Leu Pro Asn1 5 10 15Asp Gly Ala His Trp Asn Arg Leu Asn Asn Asp Ala
Gln Asn Leu Lys 20 25 30Asn Val Gly Ile Thr Ala Val Trp Ile Pro Pro
Ala Tyr Lys Gly Gly 35 40 45Ser Ser Ala Asp Val Gly Tyr Gly Val Tyr
Asp Thr Tyr Asp Leu Gly 50 55 60Glu Phe Asn Gln Lys Gly Thr Val Arg
Thr Lys Tyr Gly Thr Lys Ser65 70 75 80Glu Leu Ile Ser Ala Val Asn
Asn Leu His Ala Lys Gly Ile Ala Val 85 90 95Tyr Gly Asp Val Val Leu
Asn His Arg Met Asn Ala Asp Ala Thr Glu 100 105 110Leu Val Asp Ala
Val Glu Val Asp Pro Asn Asn Arg Asn Val Glu Thr 115 120 125Thr Ser
Thr Tyr Gln Ile Gln Ala Trp Thr Gln Tyr Asp Phe Pro Gly 130 135
140Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His Phe
Asp145 150 155 160Gly Val Asp Trp Asp Gln Ser Arg Gly Leu Asn Arg
Ile Tyr Lys Leu 165 170 175Asp Gly Lys Asp Trp Asp Trp Glu Val Asp
Ser Glu Tyr Gly Asn Tyr 180 185 190Asp Tyr Leu Met Gly Ala Asp Leu
Asp Phe Asn His Pro Asp Val Val 195 200 205Asn Glu Thr Lys Thr Trp
Gly Lys Trp Phe Val Asn Thr Val Asn Leu 210 215 220Asp Gly Val Arg
Leu Asp Ala Val Lys His Ile Lys Phe Asp Phe Met225 230 235 240Arg
Asp Trp Val Asn Asn Val Arg Ser Thr Thr Gly Lys Asn Leu Phe 245 250
255Ala Val Gly Glu Tyr Trp His Tyr Asp Val Asn Lys Leu Asn Ser Tyr
260 265 270Ile Thr Lys Thr Asn Gly Thr Met Ser Leu Phe Asp Val Pro
Leu His 275 280 285Phe Arg Phe Tyr Asp Ala Ser Asn Gly Gly Gly Gly
Tyr Asp Met Arg 290 295 300Asn Leu Leu Asn Asn Thr Leu Met Ser Ser
Asn Pro Met Lys Ala Val305 310 315 320Thr Phe Val Glu Asn His Asp
Thr Gln Pro Thr Gln Ala Leu Gln Ser 325 330 335Thr Val Gln Ser Trp
Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu Thr 340 345 350Arg Glu Gln
Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr Tyr Gly Thr 355 360 365Ser
Asp Gly Lys Ile Ser Ser Tyr Lys Pro Ile Met Asp Lys Leu Leu 370 375
380Asn Ala Arg Lys Val Tyr Ala Tyr Gly Thr Gln Arg Asp Tyr Phe
Asp385 390 395 400His Pro Asp Ile Val Gly Trp Thr Arg Glu Gly Asp
Ala Ala His Ala 405 410 415Gly Ser Gly Leu Ala Thr Leu Ile Thr Asp
Gly Pro Gly Gly Ser Lys 420 425 430Trp Met Tyr Val Gly Thr Ser Lys
Ala Gly Gln Val Trp Thr Asp Lys 435 440 445Thr Gly Asn Arg Ser Gly
Thr Val Thr Ile Asp Ala Asn Gly Trp Gly 450 455 460Asn Phe Trp Val
Asn Gly Gly Ser Val Ser Val Trp Ala Lys465 470 4755484PRTArtificial
SequenceSynthetic amino acid sequence of a C-terminal- truncated
version of the Bacillus sp. TS-23 alpha-amylase 5Asn Thr Ala Pro
Ile Asn Glu Thr Met Met Gln Tyr Phe Glu Trp Asp1 5 10 15Leu Pro Asn
Asp Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Ala 20 25 30Asn Leu
Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr 35 40 45Lys
Gly Thr Ser Gln Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr 50 55
60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly65
70 75 80Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala Lys Ala Ala
Gly 85 90 95Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly
Ala Asp 100 105 110Gly Thr Glu Phe Val Asp Ala Val Glu Val Asp Pro
Ser Asn Arg Asn 115 120 125Gln Glu Thr Ser Gly Thr Tyr Gln Ile Gln
Ala Trp Thr Lys Phe Asp 130 135 140Phe Pro Gly Arg Gly Asn Thr Tyr
Ser Ser Phe Lys Trp Arg Trp Tyr145 150 155 160His Phe Asp Gly Thr
Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile 165 170 175Tyr Lys Phe
Arg Ser Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr 180 185 190Glu
Asn Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp 195 200
205His Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr Val
210 215 220Asn Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys
His Ile225 230 235 240Lys Tyr Ser Phe Phe Pro Asp Trp Leu Thr Tyr
Val Arg Asn Gln Thr 245 250 255Gly Lys Asn Leu Phe Ala Val Gly Glu
Phe Trp Ser Tyr Asp Val Asn 260 265 270Lys Leu His Asn Tyr Ile Thr
Lys Thr Asn Gly Ser Met Ser Leu Phe 275 280 285Asp Ala Pro Leu His
Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser Gly 290 295 300Tyr Phe Asp
Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln305 310 315
320Pro Ser Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly
325 330 335Gln Ser Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu
Ala Tyr 340 345 350Ala Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys
Val Phe Tyr Gly 355 360 365Asp Tyr Tyr Gly Ile Pro Lys Tyr Asn Ile
Pro Gly Leu Lys Ser Lys 370 375 380Ile Asp Pro Leu Leu Ile Ala Arg
Arg Asp Tyr Ala Tyr Gly Thr Gln385 390 395 400Arg Asp Tyr Ile Asp
His Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly 405 410 415Ile Asp Thr
Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly 420 425 430Pro
Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys 435 440
445Val Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn
450 455
460Ala Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser
Ile465 470 475 480Trp Val Ala Lys6482PRTArtificial
SequenceSynthetic amino acid sequence of a variant form of BASE
alpha-amylase having a deletion of both R180 and S181 6Asn Thr Ala
Pro Ile Asn Glu Thr Met Met Gln Tyr Phe Glu Trp Asp1 5 10 15Leu Pro
Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Ala 20 25 30Asn
Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr 35 40
45Lys Gly Thr Ser Gln Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr
50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr
Gly65 70 75 80Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala Lys
Ala Ala Gly 85 90 95Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys
Ala Gly Ala Asp 100 105 110Gly Thr Glu Phe Val Asp Ala Val Glu Val
Asp Pro Ser Asn Arg Asn 115 120 125Gln Glu Thr Ser Gly Thr Tyr Gln
Ile Gln Ala Trp Thr Lys Phe Asp 130 135 140Phe Pro Gly Arg Gly Asn
Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr145 150 155 160His Phe Asp
Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile 165 170 175Tyr
Lys Phe Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn 180 185
190Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp His Pro
195 200 205Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr Val
Asn Thr 210 215 220Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys
His Ile Lys Tyr225 230 235 240Ser Phe Phe Pro Asp Trp Leu Thr Tyr
Val Arg Asn Gln Thr Gly Lys 245 250 255Asn Leu Phe Ala Val Gly Glu
Phe Trp Ser Tyr Asp Val Asn Lys Leu 260 265 270His Asn Tyr Ile Thr
Lys Thr Asn Gly Ser Met Ser Leu Phe Asp Ala 275 280 285Pro Leu His
Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser Gly Tyr Phe 290 295 300Asp
Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro Ser305 310
315 320Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln
Ser 325 330 335Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala
Tyr Ala Phe 340 345 350Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val
Phe Tyr Gly Asp Tyr 355 360 365Tyr Gly Ile Pro Lys Tyr Asn Ile Pro
Gly Leu Lys Ser Lys Ile Asp 370 375 380Pro Leu Leu Ile Ala Arg Arg
Asp Tyr Ala Tyr Gly Thr Gln Arg Asp385 390 395 400Tyr Ile Asp His
Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile Asp 405 410 415Thr Lys
Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly 420 425
430Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys Val Phe
435 440 445Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn
Ala Asp 450 455 460Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val
Ser Ile Trp Val465 470 475 480Ala Lys71536DNAArtificial
SequenceSynthetic DNA fragment encoding CspAmy2-v1 7atgaaacaac
aaaaacggct ttacgcccga ttgctgacgc tgttatttgc gctcatcttc 60ttgctgcctc
attctgcagc tagcgcagca gcgacaaacg gaacaatgat gcagtatttc
120gagtggtatg tacctaacga cggccagcaa tggaacagac tgagaacaga
tgccccttac 180ttgtcatctg ttggtattac agcagtatgg acaccgccgg
cttataaggg cacgtctcaa 240gcagatgtgg ggtacggccc gtacgatctg
tatgatttag gcgagtttaa tcaaaaaggt 300acagtcagaa cgaagtatgg
cacaaaagga gaacttaaat ctgctgttaa cacgctgcat 360tcaaatggaa
tccaagtgta tggtgatgtc gtgatgaatc ataaagcagg tgctgattat
420acagaaaacg taacggcggt ggaggtgaat ccgtctaata gaaatcagga
aacgagcggc 480gaatataata ttcaggcatg gacaggcttc aactttccgg
gcagaggaac aacgtattct 540aacttcaaat ggcagtggtt ccattttgat
ggaacggatt gggaccagag cagaagcctc 600tctagaatct tcaaattcac
gggaaaggcg tgggactggg aggtttcttc agaaaacgga 660aattatgact
atctgatgta cgcggacatt gattatgacc atccggatgt cgtgaatgaa
720atgaaaaagt ggggcgtctg gtatgccaac gaagttgggt tagatggata
cagacttgac 780gcggtcaaac atattaaatt tagctttctc aaagactggg
tggataacgc aagagcagcg 840acgggaaaag aaatgtttac ggttggcgaa
tattggcaaa atgatttagg ggccctgaat 900aactacctgg caaaggtaaa
ttacaaccaa tctctttttg atgcgccgtt gcattacaac 960ttttacgctg
cctcaacagg gggtggatat tacgatatga gaaatattct taataacacg
1020ttagtcgcaa gcaatccgac aaaggctgtt acgttagttg agaatcatga
cacacagcct 1080ggacaatcac tggaatcaac agtccaaccg tggtttaaac
cgttagccta cgcgtttatt 1140ctcacgagaa gcggaggcta tccttctgta
ttttatggag atatgtacgg tacaaaagga 1200acgacaacaa gagagatccc
tgctcttaaa tctaaaatcg aacctttgct taaggctaga 1260aaagactatg
cttatggaac acagagagac tatattgata acccggatgt cattggctgg
1320acgagagaag gggactcaac gaaagccaag agcggtctgg ccacagtgat
tacagatggg 1380ccgggcggtt caaaaagaat gtatgttggc acgagcaatg
cgggtgaaat ctggtatgat 1440ttgacaggga atagaacaga taaaatcacg
attggaagcg atggctatgc aacatttcct 1500gtcaatgggg gctcagtttc
agtatgggtg cagcaa 15368483PRTArtificial SequenceSynthetic amino
acid sequence of the mature form of CspAmy2-v5 8Ala Ala Thr Asn Gly
Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10 15Asn Asp Gly Gln
Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu 20 25 30Ser Ser Val
Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser
Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu 50 55 60Gly
Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75
80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln
85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp Tyr
Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg
Asn Gln Glu 115 120 125Thr Ser Gly Glu Tyr Asn Ile Gln Ala Trp Thr
Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr Thr Tyr Ser Asn Phe
Lys Trp Gln Trp Phe His Phe145 150 155 160Asp Gly Thr Asp Trp Asp
Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys 165 170 175Phe Thr Gly Lys
Ala Trp Asp Trp Pro Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr Asp
Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro Asp Val 195 200
205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala Asn Glu Val Gly
210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys His Ile Lys Phe
Ser Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn Ala Arg Ala Ala
Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu Tyr Trp Gln Asn
Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala Lys Val Asn Tyr
Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His Tyr Asn Phe Tyr
Ala Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met 290 295 300Arg Asn Ile
Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys Ala305 310 315
320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser Val Phe Tyr Gly
Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr Arg Glu Ile Pro
Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu Lys Ala Arg Lys
Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp Tyr Ile Asp Asn
Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Thr Lys
Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro 420 425 430Gly
Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly Glu Ile 435 440
445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser
450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Asn Lys Gly Ser Val Ser
Val Trp465 470 475 480Val Gln Gln9483PRTArtificial
SequenceSynthetic amino acid sequence of the mature form of
CspAmy2-v6 9Ala Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr
Val Pro1 5 10 15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala
Pro Tyr Leu 20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro
Ala Tyr Lys Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr
Asp Leu Tyr Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg
Thr Lys Tyr Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn
Thr Leu His Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met
Asn His Lys Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala
Val Glu Val Asn Pro Ser Asn Arg Asn Gln Glu 115 120 125Thr Ser Gly
Glu Tyr Asn Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly
Arg Gly Thr Thr Tyr Ser Asn Phe Lys Trp Gln Trp Phe His Phe145 150
155 160Asp Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu Ser Arg Ile Phe
Lys 165 170 175Phe Thr Gly Lys Ala Trp Asp Trp Pro Val Ser Ser Glu
Asn Gly Asn 180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr
Asp His Pro Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val
Trp Tyr Ala Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp
Ala Val Lys His Ile Lys Phe Ser Phe225 230 235 240Leu Lys Asp Trp
Val Asp Asn Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255Phe Thr
Val Gly Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265
270Tyr Leu Ala Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu
275 280 285His Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Tyr Tyr
Asp Met 290 295 300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn
Pro Thr Lys Ala305 310 315 320Val Thr Leu Val Glu Asn His Asp Thr
Gln Pro Gly Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro Trp Phe
Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser Gly Gly
Tyr Pro Ser Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly
Thr Thr Thr Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380Glu
Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg385 390
395 400Asp Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly
Asp 405 410 415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr
Asp Gly Pro 420 425 430Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser
Asn Ala Gly Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly Asn Asn Ser
Thr Lys Ile Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala Thr Phe Pro
Val Asn Lys Gly Ser Val Ser Val Trp465 470 475 480Val Gln
Gln10483PRTArtificial SequenceSynthetic amino acid sequence of
mature CspAmy2-16A 10Ala Ala Thr Asn Gly Thr Met Met Gln Tyr Phe
Glu Trp Tyr Val Pro1 5 10 15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg
Thr Asp Ala Pro Tyr Leu 20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp
Thr Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr
Gly Pro Tyr Asp Leu Tyr Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly
Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser
Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp
Val Val Met Asn His Lys Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn
Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Tyr Gln Glu 115 120
125Thr Ser Gly Glu Tyr Asn Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro
130 135 140Gly Arg Gly Thr Thr Tyr Ser Asn Phe Lys Trp Gln Trp Phe
His Phe145 150 155 160Asp Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu
Ser Arg Ile Phe Lys 165 170 175Phe Thr Gly Lys Ala Trp Asp Trp Pro
Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr Asp Tyr Leu Met Tyr Ala
Asp Tyr Asp Tyr Asp His Pro Asp Val 195 200 205Val Asn Glu Met Lys
Lys Trp Gly Val Trp Tyr Ala Asn Glu Val Gly 210 215 220Leu Asp Gly
Tyr Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe225 230 235
240Leu Lys Asp Trp Val Asp Asn Ala Arg Ala Ala Thr Gly Lys Glu Met
245 250 255Phe Thr Val Gly Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu
Asn Asn 260 265 270Tyr Leu Ala Lys Val Asn Tyr Asn Gln Ser Leu Phe
Asp Ala Pro Leu 275 280 285His Tyr Asn Phe Tyr Ala Ala Ser Thr Gly
Gly Gly Tyr Tyr Asp Met 290 295 300Arg Asn Ile Leu Asn Asn Thr Leu
Val Ala Ser Asn Pro Thr Lys Ala305 310 315 320Val Thr Leu Val Glu
Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335Ser Thr Val
Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr
Arg Ser Gly Gly Tyr Pro Ser Val Phe Tyr Gly Asp Met Tyr Gly 355 360
365Thr Lys Gly Thr Thr Thr Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile
370 375 380Glu Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr
Gln Arg385 390 395 400Asp Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp
Thr Arg Glu Gly Asp 405 410 415Ser Thr Lys Ala Lys Ser Gly Leu Ala
Thr Val Ile Thr Asp Gly Pro 420 425 430Gly Gly Ser Lys Arg Met Tyr
Val Gly Thr Ser Asn Ala Gly Glu Ile 435 440 445Trp Tyr Asp Leu Thr
Gly Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser 450 455 460Asp Gly Tyr
Ala Thr Phe Pro Val Asn Gly Gly Ser Val Ser Val Trp465 470 475
480Val Gln Gln11483PRTArtificial SequenceSynthetic amino acid
sequence of mature CspAmy2-16B 11Ala Ala Thr Asn Gly Thr Met Met
Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10 15Asn Asp Gly Gln Gln Trp Asn
Arg Leu Arg Thr Asp Ala Pro Tyr Leu 20 25 30Ser Ser Val Gly Ile Thr
Ala Val Trp Thr Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser Gln Ala Asp
Val Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu 50 55 60Gly Glu Phe Asn
Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75 80Gly Glu
Leu Lys Ser Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln 85 90 95Val
Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp Tyr Thr 100 105
110Glu Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Tyr Gln Glu
115 120 125Thr Ser Gly Glu Tyr Asn Ile Gln Ala Trp Thr Gly Phe Asn
Phe Pro 130 135 140Gly Arg Gly Thr Thr Tyr Ser Asn Phe Lys Trp Gln
Trp Phe His Phe145 150 155 160Asp Gly Thr Asp Trp Asp Gln Ser Arg
Ser Leu Ser Arg Ile Phe Lys 165 170 175Phe Thr Gly Lys Ala Trp Asp
Trp Glu Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr
Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro Asp Val 195 200
205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala Asn Glu Val Gly
210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys His Ile Lys Phe
Gln Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn Ala Arg Ala Ala
Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu Tyr Trp Gln Asn
Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala Lys Val Asn Tyr
Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His Tyr Asn Phe Tyr
Ala Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met 290 295 300Arg Asn Ile
Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys Ala305 310 315
320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser Val Phe Tyr Gly
Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr Arg Glu Ile Pro
Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu Lys Ala Arg Lys
Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp Tyr Ile Asp Asn
Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Thr Lys
Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro 420 425 430Gly
Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly Glu Ile 435 440
445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser
450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Asn Gly Gly Ser Val Ser
Val Trp465 470 475 480Val Gln Gln12483PRTArtificial
SequenceSynthetic amino acid sequence of mature CspAmy2-16C 12Ala
Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10
15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu
20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys
Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr
Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His
Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys
Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val
Asn Pro Ser Asn Arg Tyr Gln Glu 115 120 125Thr Ser Gly Glu Tyr Asn
Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr
Thr Tyr Ser Asn Trp Lys Trp Gln Trp Phe His Phe145 150 155 160Asp
Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys 165 170
175Phe Thr Gly Lys Ala Trp Asp Trp Pro Val Ser Ser Glu Asn Gly Asn
180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro
Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala
Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys
His Ile Lys Phe Ser Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn
Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu
Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala
Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His
Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met 290 295
300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys
Ala305 310 315 320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser
Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr
Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu
Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp
Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410
415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro
420 425 430Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly
Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile
Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Asn Gly
Gly Ser Val Ser Val Trp465 470 475 480Val Gln Gln13483PRTArtificial
SequenceSynthetic amino acid sequence of mature CspAmy2-16D 13Ala
Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10
15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu
20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys
Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr
Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His
Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys
Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val
Asn Pro Ser Asn Arg Tyr Gln Glu 115 120 125Thr Ser Gly Glu Tyr Asn
Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr
Thr Tyr Ser Asn Trp Lys Trp Gln Trp Phe His Phe145 150 155 160Asp
Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys 165 170
175Phe Thr Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn
180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro
Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala
Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys
His Ile Lys Phe Gln Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn
Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu
Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala
Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His
Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met 290 295
300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys
Ala305 310 315 320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser
Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr
Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu
Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp
Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410
415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro
420 425 430Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly
Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile
Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Asn Gly
Gly Ser Val Ser Val Trp465 470 475 480Val Gln Gln14483PRTArtificial
SequenceSynthetic amino acid sequence of the mature CspAmy2-16E
14Ala Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1
5 10 15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr
Leu 20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr
Lys Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu
Tyr Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys
Tyr Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu
His Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His
Lys Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu
Val Asn Pro Ser Asn Arg Tyr Gln Glu 115 120 125Thr Ser Gly Glu Tyr
Asn Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly
Thr Thr Tyr Ser Asn Trp Lys Trp Gln Trp Phe His Phe145 150 155
160Asp Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys
165 170 175Phe His Gly Lys Ala Trp Asp Trp Pro Val Ser Ser Glu Asn
Gly Asn 180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp
His Pro Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val Trp
Tyr Ala Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala
Val Lys His Ile Lys Phe Ser Phe225 230 235 240Leu Lys Asp Trp Val
Asp Asn Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val
Gly Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr
Leu Ala Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280
285His Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met
290 295 300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr
Lys Ala305 310 315 320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro
Gly Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro
Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro
Ser Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr
Thr Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu
Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395
400Asp Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp
405 410 415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp
Gly Pro 420 425 430Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn
Ala Gly Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp
Lys Ile Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala Thr Phe Pro Val
Asn Gly Gly Ser Val Ser Val Trp465 470 475 480Val Gln
Gln15483PRTArtificial SequenceSynthetic amino acid sequence of
mature CspAmy2-16F 15Ala Ala Thr Asn Gly Thr Met Met Gln Tyr Phe
Glu Trp Tyr Val Pro1 5 10 15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg
Thr Asp Ala Pro Tyr Leu 20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp
Thr Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr
Gly Pro Tyr Asp Leu Tyr Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly
Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser
Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp
Val Val Met Asn His Lys Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn
Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Tyr Gln Glu 115 120
125Thr Ser Gly Glu Tyr Asn Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro
130 135 140Gly Arg Gly Thr Thr Tyr Ser Asn Trp Lys Trp Gln Trp Phe
His Phe145 150 155 160Asp Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu
Ser Arg Ile Phe Lys 165 170 175Phe His Gly Lys Ala Trp Asp Trp Glu
Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr Asp Tyr Leu Met Tyr Ala
Asp Tyr Asp Tyr Asp His Pro Asp Val 195 200 205Val Asn Glu Met Lys
Lys Trp Gly Val Trp Tyr Ala Asn Glu Val Gly 210 215 220Leu Asp Gly
Tyr Arg Leu Asp Ala Val Lys His Ile Lys Phe Gln Phe225 230 235
240Leu Lys Asp Trp Val Asp Asn Ala Arg Ala Ala Thr Gly Lys Glu Met
245 250 255Phe Thr Val Gly Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu
Asn Asn 260 265 270Tyr Leu Ala Lys Val Asn Tyr Asn Gln Ser Leu Phe
Asp Ala Pro Leu 275 280 285His Tyr Asn Phe Tyr Ala Ala Ser Thr Gly
Gly Gly Tyr Tyr Asp Met 290 295 300Arg Asn Ile Leu Asn Asn Thr Leu
Val Ala Ser Asn Pro Thr Lys Ala305 310 315 320Val Thr Leu Val Glu
Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335Ser Thr Val
Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr
Arg Ser Gly Gly Tyr Pro Ser Val Phe Tyr Gly Asp Met Tyr Gly 355 360
365Thr Lys Gly Thr Thr Thr Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile
370 375 380Glu Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr
Gln Arg385 390 395 400Asp Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp
Thr Arg Glu Gly Asp 405 410 415Ser Thr Lys Ala Lys Ser Gly Leu Ala
Thr Val Ile Thr Asp Gly Pro 420 425 430Gly Gly Ser Lys Arg Met Tyr
Val Gly Thr Ser Asn Ala Gly Glu Ile 435 440 445Trp Tyr Asp Leu Thr
Gly Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser 450 455 460Asp Gly Tyr
Ala Thr Phe Pro Val Asn Gly Gly Ser Val Ser Val Trp465 470 475
480Val Gln Gln16483PRTArtificial SequenceSynthetic amino acid
sequence of mature CspAmy2-16G 16Ala Ala Thr Asn Gly Thr Met Met
Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10 15Asn Asp Gly Gln Gln Trp Asn
Arg Leu Arg Thr Asp Ala Pro Tyr Leu 20 25 30Ser Ser Val Gly Ile Thr
Ala Val Trp Thr Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser Gln Ala Asp
Val Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu 50 55 60Gly Glu Phe Asn
Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75 80Gly Glu
Leu Lys Ser Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln 85 90 95Val
Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp Tyr Thr 100 105
110Glu Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Tyr Gln Glu
115 120 125Thr Ser Gly Glu Tyr Asn Ile Gln Ala Trp Thr Gly Phe Asn
Phe Pro 130 135 140Gly Arg Gly Thr Thr His Ser Asn Trp Lys Trp Gln
Trp Phe His Phe145 150 155 160Asp Gly Thr Asp Trp Asp Gln Ser Arg
Ser Asn Ser Arg Ile Phe Lys 165 170 175Phe Thr Gly Lys Ala Trp Asp
Trp Pro Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr Asp Tyr Leu Met
Tyr Ala Asp Tyr Asp Tyr Asp His Pro Asp Val 195
200 205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala Asn Glu Val
Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys His Ile Lys
Phe Ser Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn Ala Arg Ala
Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu Tyr Trp Gln
Asn Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala Lys Val Asn
Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His Tyr Asn Phe
Tyr Ala Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met 290 295 300Arg Asn
Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys Ala305 310 315
320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser Val Phe Tyr Gly
Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr Arg Glu Ile Pro
Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu Lys Ala Arg Lys
Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp Tyr Ile Asp Asn
Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Thr Lys
Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro 420 425 430Gly
Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly Glu Ile 435 440
445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser
450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Asn Gly Gly Ser Val Ser
Val Trp465 470 475 480Val Gln Gln17483PRTArtificial
SequenceSynthetic amino acid sequence of mature CspAmy2-16H 17Ala
Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10
15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu
20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys
Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr
Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His
Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys
Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val
Asn Pro Ser Asn Arg Tyr Gln Glu 115 120 125Thr Ser Gly Glu Tyr Asn
Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr
Thr His Ser Asn Trp Lys Trp Gln Trp Phe His Phe145 150 155 160Asp
Gly Thr Asp Trp Asp Gln Ser Arg Ser Asn Ser Arg Ile Phe Lys 165 170
175Phe Thr Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn
180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro
Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala
Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys
His Ile Lys Phe Gln Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn
Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu
Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala
Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His
Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met 290 295
300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys
Ala305 310 315 320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser
Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr
Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu
Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp
Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410
415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro
420 425 430Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly
Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile
Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Asn Gly
Gly Ser Val Ser Val Trp465 470 475 480Val Gln Gln18483PRTArtificial
SequenceSynthetic amino acid sequence of mature CspAmy2-16I 18Ala
Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10
15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu
20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys
Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr
Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His
Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys
Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val
Asn Pro Ser Asn Arg Tyr Gln Glu 115 120 125Thr Ser Gly Glu Tyr Asn
Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr
Thr His Ser Asn Trp Lys Trp Gln Trp Phe His Phe145 150 155 160Asp
Gly Thr Asp Trp Asp Gln Ser Arg Ser Asn Ser Arg Ile Phe Lys 165 170
175Phe His Gly Lys Ala Trp Asp Trp Pro Val Ser Ser Glu Asn Gly Asn
180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro
Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala
Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys
His Ile Lys Phe Ser Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn
Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu
Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala
Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His
Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met 290 295
300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys
Ala305 310 315 320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser
Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr
Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu
Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp
Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410
415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro
420 425 430Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly
Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile
Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Asn Gly
Gly Ser Val Ser Val Trp465 470 475 480Val Gln Gln19483PRTArtificial
SequenceSynthetic amino acid sequence of mature CspAmy2-16J 19Ala
Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10
15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu
20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys
Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr
Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His
Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys
Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val
Asn Pro Ser Asn Arg Tyr Gln Glu 115 120 125Thr Ser Gly Glu Tyr Asn
Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr
Thr His Ser Asn Trp Lys Trp Gln Trp Phe His Phe145 150 155 160Asp
Gly Thr Asp Trp Asp Gln Ser Arg Ser Asn Ser Arg Ile Phe Lys 165 170
175Phe His Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn
180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro
Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala
Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys
His Ile Lys Phe Gln Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn
Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu
Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala
Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His
Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met 290 295
300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys
Ala305 310 315 320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser
Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr
Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu
Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp
Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410
415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro
420 425 430Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly
Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile
Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Asn Gly
Gly Ser Val Ser Val Trp465 470 475 480Val Gln Gln20483PRTArtificial
SequenceSynthetic amino acid sequence of mature CspAmy2-v1-E187P
20Ala Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1
5 10 15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr
Leu 20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr
Lys Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu
Tyr Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys
Tyr Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu
His Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His
Lys Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu
Val Asn Pro Ser Asn Arg Asn Gln Glu 115 120 125Thr Ser Gly Glu Tyr
Asn Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly
Thr Thr Tyr Ser Asn Phe Lys Trp Gln Trp Phe His Phe145 150 155
160Asp Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys
165 170 175Phe Thr Gly Lys Ala Trp Asp Trp Pro Val Ser Ser Glu Asn
Gly Asn 180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp
His Pro Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val Trp
Tyr Ala Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala
Val Lys His Ile Lys Phe Ser Phe225 230 235 240Leu Lys Asp Trp Val
Asp Asn Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val
Gly Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr
Leu Ala Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280
285His Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met
290 295 300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr
Lys Ala305 310 315 320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro
Gly Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro
Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro
Ser Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr
Thr Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu
Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395
400Asp Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp
405 410 415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp
Gly Pro 420 425 430Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn
Ala Gly Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp
Lys Ile Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala Thr Phe Pro Val
Asn Gly Gly Ser Val Ser Val Trp465 470 475 480Val Gln
Gln21483PRTArtificial SequenceSynthetic amino acid sequence of
mature CspAmy2-v1-S241Q 21Ala Ala Thr Asn Gly Thr Met Met Gln Tyr
Phe Glu Trp Tyr Val Pro1 5 10 15Asn Asp Gly Gln Gln Trp Asn Arg Leu
Arg Thr Asp Ala Pro Tyr Leu 20 25 30Ser Ser Val Gly Ile Thr Ala Val
Trp Thr Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser Gln Ala Asp Val Gly
Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys
Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75 80Gly Glu Leu Lys
Ser Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly
Asp Val Val Met Asn His Lys Ala Gly Ala Asp Tyr Thr 100 105 110Glu
Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Asn Gln Glu 115 120
125Thr Ser Gly Glu Tyr Asn Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro
130 135 140Gly Arg Gly Thr Thr Tyr Ser Asn Phe Lys Trp Gln Trp Phe
His Phe145 150 155 160Asp Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu
Ser Arg Ile Phe Lys 165 170 175Phe Thr Gly Lys Ala Trp Asp Trp Glu
Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr Asp Tyr Leu Met Tyr Ala
Asp Ile Asp Tyr Asp His Pro Asp Val 195 200 205Val Asn Glu Met Lys
Lys Trp
Gly Val Trp Tyr Ala Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg
Leu Asp Ala Val Lys His Ile Lys Phe Gln Phe225 230 235 240Leu Lys
Asp Trp Val Asp Asn Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250
255Phe Thr Val Gly Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn
260 265 270Tyr Leu Ala Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala
Pro Leu 275 280 285His Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly
Tyr Tyr Asp Met 290 295 300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala
Ser Asn Pro Thr Lys Ala305 310 315 320Val Thr Leu Val Glu Asn His
Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro
Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser
Gly Gly Tyr Pro Ser Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr
Lys Gly Thr Thr Thr Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375
380Glu Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln
Arg385 390 395 400Asp Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr
Arg Glu Gly Asp 405 410 415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr
Val Ile Thr Asp Gly Pro 420 425 430Gly Gly Ser Lys Arg Met Tyr Val
Gly Thr Ser Asn Ala Gly Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly
Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala
Thr Phe Pro Val Asn Gly Gly Ser Val Ser Val Trp465 470 475 480Val
Gln Gln22483PRTArtificial SequenceSynthetic amino acid sequence of
mature CspAmy2-v171 22Ala Ala Thr Asn Gly Thr Met Met Gln Tyr Phe
Glu Trp Tyr Val Pro1 5 10 15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg
Thr Asp Ala Pro Tyr Leu 20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp
Thr Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr
Gly Pro Tyr Asp Leu Tyr Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly
Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser
Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp
Val Val Met Asn His Lys Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn
Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Asn Gln Glu 115 120
125Thr Ser Gly Glu Tyr Asn Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro
130 135 140Gly Arg Gly Thr Thr Tyr Ser Asn Phe Lys Trp Gln Trp Phe
His Phe145 150 155 160Asp Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu
Ser Arg Ile Phe Lys 165 170 175Phe Asp Gly Lys Ala Trp Asp Trp Pro
Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr Asp Tyr Leu Met Tyr Ala
Asp Tyr Asp Tyr Asp His Pro Asp Val 195 200 205Val Asn Glu Met Lys
Lys Trp Gly Val Trp Tyr Ala Asn Glu Val Gly 210 215 220Leu Asp Gly
Tyr Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe225 230 235
240Leu Lys Asp Trp Val Asp Asn Ala Arg Ala Ala Thr Gly Lys Glu Met
245 250 255Phe Thr Val Gly Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu
Asn Asn 260 265 270Tyr Leu Ala Lys Val Asn Tyr Asn Gln Ser Leu Phe
Asp Ala Pro Leu 275 280 285His Tyr Asn Phe Tyr Ala Ala Ser Thr Gly
Gly Gly Tyr Tyr Asp Met 290 295 300Arg Asn Ile Leu Asn Asn Thr Leu
Val Ala Ser Asn Pro Thr Lys Ala305 310 315 320Val Thr Leu Val Glu
Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335Ser Thr Val
Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr
Arg Ser Gly Gly Tyr Pro Ser Val Phe Tyr Gly Asp Met Tyr Gly 355 360
365Thr Lys Gly Thr Thr Thr Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile
370 375 380Glu Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr
Gln Arg385 390 395 400Asp Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp
Thr Arg Glu Gly Asp 405 410 415Ser Thr Lys Ala Lys Ser Gly Leu Ala
Thr Val Ile Thr Asp Gly Pro 420 425 430Gly Gly Ser Lys Arg Met Tyr
Val Gly Thr Ser Asn Ala Gly Glu Ile 435 440 445Trp Tyr Asp Leu Thr
Gly Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser 450 455 460Asp Gly Tyr
Ala Thr Phe Pro Val Asn Lys Gly Ser Val Ser Val Trp465 470 475
480Val Gln Gln23483PRTArtificial SequenceSynthetic amino acid
sequence of mature CspAmy2-v172 23Ala Ala Thr Asn Gly Thr Met Met
Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10 15Asn Asp Gly Gln Gln Trp Asn
Arg Leu Arg Thr Asp Ala Pro Tyr Leu 20 25 30Ser Ser Val Gly Ile Thr
Ala Val Trp Thr Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser Gln Ala Asp
Val Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu 50 55 60Gly Glu Phe Asn
Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75 80Gly Glu
Leu Lys Ser Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln 85 90 95Val
Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp Tyr Thr 100 105
110Glu Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Tyr Gln Glu
115 120 125Thr Ser Gly Glu Tyr Asn Ile Gln Ala Trp Thr Gly Phe Asn
Phe Pro 130 135 140Gly Arg Gly Thr Thr Tyr Ser Asn Phe Lys Trp Gln
Trp Phe His Phe145 150 155 160Asp Gly Thr Asp Trp Asp Gln Ser Arg
Ser Leu Ser Arg Ile Phe Lys 165 170 175Phe Asp Gly Lys Ala Trp Asp
Trp Pro Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr Asp Tyr Leu Met
Tyr Ala Asp Tyr Asp Tyr Asp His Pro Asp Val 195 200 205Val Asn Glu
Met Lys Lys Trp Gly Val Trp Tyr Ala Asn Glu Val Gly 210 215 220Leu
Asp Gly Tyr Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe225 230
235 240Leu Lys Asp Trp Val Asp Asn Ala Arg Ala Ala Thr Gly Lys Glu
Met 245 250 255Phe Thr Val Gly Glu Tyr Trp Gln Asn Asp Leu Gly Ala
Leu Asn Asn 260 265 270Tyr Leu Ala Lys Val Asn Tyr Asn Gln Ser Leu
Phe Asp Ala Pro Leu 275 280 285His Tyr Asn Phe Tyr Ala Ala Ser Thr
Gly Gly Gly Tyr Tyr Asp Met 290 295 300Arg Asn Ile Leu Asn Asn Thr
Leu Val Ala Ser Asn Pro Thr Lys Ala305 310 315 320Val Thr Leu Val
Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335Ser Thr
Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345
350Thr Arg Ser Gly Gly Tyr Pro Ser Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365Thr Lys Gly Thr Thr Thr Arg Glu Ile Pro Ala Leu Lys Ser
Lys Ile 370 375 380Glu Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr
Gly Thr Gln Arg385 390 395 400Asp Tyr Ile Asp Asn Pro Asp Val Ile
Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Thr Lys Ala Lys Ser Gly
Leu Ala Thr Val Ile Thr Asp Gly Pro 420 425 430Gly Gly Ser Lys Arg
Met Tyr Val Gly Thr Ser Asn Ala Gly Glu Ile 435 440 445Trp Tyr Asp
Leu Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser 450 455 460Asp
Gly Tyr Ala Thr Phe Pro Val Asn Lys Gly Ser Val Ser Val Trp465 470
475 480Val Gln Gln24483PRTArtificial SequenceSynthetic amino acid
sequence of the mature CspAmy2-C18P amylase polypeptide 24Ala Ala
Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10 15Asn
Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu 20 25
30Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys Gly
35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp
Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His Ser
Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys Ala
Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val Asn
Pro Ser Asn Arg Tyr Gln Glu 115 120 125Thr Ser Gly Glu Tyr Asn Ile
Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr Thr
Tyr Ser Asn Trp Lys Trp Gln Trp Phe His Phe145 150 155 160Asp Gly
Thr Asp Trp Asp Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys 165 170
175Phe Asp Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn
180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro
Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala
Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys
His Ile Lys Phe Gln Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn
Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu
Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala
Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His
Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met 290 295
300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys
Ala305 310 315 320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser
Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr
Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu
Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp
Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410
415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro
420 425 430Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly
Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile
Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Asn Gly
Gly Ser Val Ser Val Trp465 470 475 480Val Gln Gln25483PRTArtificial
SequenceSynthetic amino acid sequence of the mature CspAmy2-C25A
amylase polypeptide 25Ala Ala Thr Asn Gly Thr Met Met Gln Tyr Phe
Glu Trp Tyr Val Pro1 5 10 15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg
Thr Asp Ala Pro Tyr Leu 20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp
Thr Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr
Gly Pro Tyr Asp Leu Tyr Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly
Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser
Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp
Val Val Met Asn His Lys Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn
Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg Tyr Gln Glu 115 120
125Thr Ser Gly His Tyr Asn Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro
130 135 140Gly Arg Gly Thr Thr Tyr Ser Asn Trp Lys Trp Gln Trp Phe
His Phe145 150 155 160Asp Gly Thr Asp Trp Asp Glu Ser Arg Ser Leu
Ser Arg Ile Phe Lys 165 170 175Phe Asp Gly Lys Ala Trp Asp Trp Glu
Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr Asp Tyr Leu Met Tyr Ala
Asp Tyr Asp Tyr Asp His Pro Asp Val 195 200 205Val Asn Glu Met Lys
Lys Trp Gly Val Trp Tyr Ala Asn Glu Val Gly 210 215 220Leu Asp Gly
Tyr Arg Leu Asp Ala Val Lys His Ile Lys Phe Gln Phe225 230 235
240Leu Lys Asp Trp Val Asp Asn Ala Arg Ala Ala Thr Gly Lys Glu Met
245 250 255Phe Thr Val Gly Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu
Asn Asn 260 265 270Tyr Leu Phe Lys Val Asn Tyr Asn Gln Ser Leu Phe
Asp Ala Pro Leu 275 280 285His Tyr Asn Phe Tyr Ala Ala Ser Thr Gly
Gly Gly Tyr Tyr Asp Met 290 295 300Arg Asn Ile Leu Asn Asn Thr Leu
Val Ala Ser Asn Pro Thr Lys Ala305 310 315 320Val Thr Leu Val Glu
Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335Ser Thr Val
Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr
Arg Ser Gly Gly Tyr Pro Ser Val Phe Tyr Gly Asp Met Tyr Gly 355 360
365Thr Lys Gly Thr Thr Thr Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile
370 375 380Glu Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Lys
Gln Arg385 390 395 400Asp Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp
Thr Arg Glu Gly Asp 405 410 415Ser Thr Lys Ala Lys Ser Gly Leu Ala
Thr Val Ile Thr Asp Gly Pro 420 425 430Gly Gly Ser Lys Arg Met Tyr
Val Gly Thr Ser Asn Ala Gly Glu Ile 435 440 445Trp Tyr Asp Leu Thr
Gly Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser 450 455 460Asp Gly Tyr
Ala Thr Phe Pro Val Asn Gly Gly Ser Val Ser Val Trp465 470 475
480Val Gln Gln26483PRTArtificial SequenceSynthetic amino acid
sequence of the mature CspAmy2-C25B amylase polypeptide 26Ala Ala
Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10 15Asn
Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu 20 25
30Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys Gly
35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp
Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His Ser
Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys Ala
Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val Asn
Pro Ser Asn Arg Tyr Gln Glu 115 120 125Thr Ser Gly His Tyr Asn Ile
Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr Thr
Tyr Ser Asn Trp Lys Trp Gln Trp Phe His Phe145 150 155 160Asp Gly
Thr Asp Trp Asp Glu Ser Arg Ser Leu Ser Arg Ile Phe Lys 165 170
175Phe Asp Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn
180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro
Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val
Trp Tyr Ala Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp
Ala Val Lys His Ile Lys Phe Gln Phe225 230 235 240Leu Lys Asp Trp
Val Asp Asn Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255Phe Thr
Val Gly Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265
270Tyr Leu Phe Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu
275 280 285His Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Tyr Tyr
Asp Met 290 295 300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn
Pro Thr Lys Ala305 310 315 320Val Thr Leu Val Glu Asn His Asp Thr
Gln Pro Gly Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro Trp Phe
Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser Gly Gly
Tyr Pro Ser Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly
Thr Thr Thr Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380Glu
Pro Leu Leu Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg385 390
395 400Asp Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly
Asp 405 410 415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr
Asp Gly Pro 420 425 430Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser
Asn Ala Gly Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly Asn Arg Thr
Asp Lys Ile Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala Thr Phe Pro
Val Asn Gly Gly Ser Val Ser Val Trp465 470 475 480Val Gln
Gln27483PRTArtificial SequenceSynthetic amino acid sequence of the
mature CspAmy2-C25F amylase polypeptide 27Ala Ala Thr Asn Gly Thr
Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10 15Asn Asp Gly Gln Gln
Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu 20 25 30Ser Ser Val Gly
Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser Gln
Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu 50 55 60Gly Glu
Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75
80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln
85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp Tyr
Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg
Tyr Gln Glu 115 120 125Thr Ser Gly His Tyr Asn Ile Gln Ala Trp Thr
Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr Thr Tyr Ser Asn Trp
Lys Trp Gln Trp Phe His Phe145 150 155 160Asp Gly Thr Asp Trp Asp
Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys 165 170 175Phe Asp Gly Lys
Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr Asp
Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro Asp Val 195 200
205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala Asn Glu Val Gly
210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys His Ile Lys Phe
Gln Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn Ala Arg Ala Ala
Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu Tyr Trp Gln Asn
Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Phe Lys Val Asn Tyr
Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His Tyr Asn Phe Tyr
Ala Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met 290 295 300Arg Asn Ile
Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys Ala305 310 315
320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser Val Phe Tyr Gly
Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr Arg Glu Ile Pro
Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu Lys Ala Arg Lys
Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp Tyr Ile Asp Asn
Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Thr Lys
Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro 420 425 430Gly
Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly Glu Ile 435 440
445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser
450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Asn Gly Gly Ser Val Ser
Val Trp465 470 475 480Val Gln Gln28483PRTArtificial
SequenceSynthetic amino acid sequence of mature CspAmy2-v179 28Ala
Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10
15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu
20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys
Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr
Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His
Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys
Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val
Asn Pro Ser Asn Arg Tyr Gln Glu 115 120 125Thr Ser Gly Glu Tyr Asn
Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr
Thr Tyr Ser Asn Phe Lys Trp Gln Trp Phe His Phe145 150 155 160Asp
Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys 165 170
175Phe Asp Gly Lys Ala Trp Asp Trp Pro Val Ser Ser Glu Asn Gly Asn
180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro
Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala
Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys
His Ile Lys Phe Ser Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn
Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu
Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala
Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His
Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Asp Tyr Asp Met 290 295
300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys
Ala305 310 315 320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser
Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr
Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu
Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp
Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410
415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro
420 425 430Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly
Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile
Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Asn Thr
Glu Ser Val Ser Val Trp465 470 475 480Val Gln Gln29483PRTArtificial
SequenceSynthetic amino acid sequence of mature CspAmy2-v180 29Ala
Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10
15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu
20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys
Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr
Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His
Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys
Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val
Asn Pro Ser Asn Arg Tyr Gln Glu 115 120 125Thr Ser Gly Glu Tyr Asn
Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr
Thr Tyr Ser Asn Phe Lys Trp Gln Trp Phe His Phe145 150 155 160Asp
Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys 165 170
175Phe Asp Gly Lys Ala Trp Asp Trp Pro Val Ser Ser Glu Asn Gly Asn
180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro
Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala
Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys
His Ile Lys Phe Ser Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn
Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu
Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala
Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His
Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Asp Tyr Asp Met 290 295
300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys
Ala305 310 315 320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser
Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr
Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu
Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp
Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410
415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro
420 425 430Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly
Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile
Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Glu Gly
Gln Ser Val Ser Val Trp465 470 475 480Val Gln Gln30483PRTArtificial
SequenceSynthetic amino acid sequence of mature CspAmy2-v181 30Ala
Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10
15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu
20 25 30Ser Ser Val Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys
Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr
Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His
Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys
Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val
Asn Pro Ser Asn Arg Tyr Gln Glu 115 120 125Thr Ser Gly Glu Tyr Asn
Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr
Thr Tyr Ser Asn Phe Lys Trp Gln Trp Phe His Phe145 150 155 160Asp
Gly Thr Asp Trp Asp Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys 165 170
175Phe Asp Gly Lys Ala Trp Asp Trp Pro Val Ser Ser Glu Asn Gly Asn
180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro
Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala
Asn Glu Val Gly 210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys
His Ile Lys Phe Ser Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn
Ala Arg Ala Ala Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu
Tyr Trp Gln Asn Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala
Lys Val Asn Tyr Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His
Tyr Asn Phe Tyr Ala Ala Ser Thr Gly Gly Gly Asp Tyr Asp Met 290 295
300Arg Asn Ile Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys
Ala305 310 315 320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser
Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr
Arg Glu Ile Pro Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu
Lys Ala Arg Lys Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp
Tyr Ile Asp Asn Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410
415Ser Thr Lys Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro
420 425 430Gly Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly
Glu Ile 435 440 445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile
Thr Ile Gly Ser 450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Glu Thr
Arg Ser Val Ser Val Trp465 470 475 480Val Gln Gln31483PRTArtificial
SequenceSynthetic amino acid sequence of mature CspAmy2-v186 31Ala
Ala Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10
15Asn Asp Gly Gln Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu
20 25 30Ser Ser Val Gly Ile Asn Ala Val Trp Thr Pro Pro Ala Tyr Lys
Gly 35 40 45Thr Ser Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr
Asp Leu 50 55 60Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly Thr Lys65 70 75 80Gly Glu Leu Lys Ser Ala Val His Thr Leu His
Ser Asn Gly Ile Gln 85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys
Ala Gly Ala Asp Tyr Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val
Asn Pro Ser Asn Arg Tyr Gln Glu 115 120 125Ile Ser Gly Glu Tyr Met
Ile Gln Ala Trp Thr Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr
Thr Tyr Ser Asn Trp Lys Trp Gln Trp Phe His Phe145 150 155 160Asp
Gly Thr Asp Trp Asp Gln Ser Arg Ser Arg Ser Arg Ile Phe Lys 165 170
175Phe Asp Gly Lys Ala Trp Asp Trp Pro Val Ser Ser Glu Asn Gly Asn
180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro
Asp Val 195 200 205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala
Asn Glu Val Gly 210
215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser
Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn Ala Arg Ala Ala Thr
Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu Tyr Trp Gln Asn Asp
Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala Lys Val Asn Tyr Asn
Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His Tyr Asn Phe Tyr Ala
Ala Ser Thr Gly Gly Gly Tyr Tyr Asp Met 290 295 300Arg Asn Ile Leu
Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys Ala305 310 315 320Val
Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330
335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser Val Phe Tyr Gly Asp Met
Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr Arg Glu Ile Pro Ala Leu
Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu Lys Ala Arg Lys Asp Tyr
Ala Tyr Gly Thr Gln Arg385 390 395 400Asp Tyr Ile Asp Asn Pro Asp
Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Thr Lys Ala Lys
Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro 420 425 430Gly Gly Ser
Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly Glu Ile 435 440 445Trp
Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser 450 455
460Asp Gly Tyr Ala Thr Phe Pro Val Asn Lys Glu Ser Val Ser Val
Trp465 470 475 480Val Gln Gln32483PRTArtificial SequenceSynthetic
amino acid sequence of mature CspAmy2-v191 32Ala Ala Thr Asn Gly
Thr Met Met Gln Tyr Phe Glu Trp Tyr Val Pro1 5 10 15Asn Asp Gly Gln
Gln Trp Asn Arg Leu Arg Thr Asp Ala Pro Tyr Leu 20 25 30Ser Ser Val
Gly Ile Thr Ala Val Trp Thr Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser
Gln Ala Asp Val Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu 50 55 60Gly
Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75
80Gly Glu Leu Lys Ser Ala Val Asn Thr Leu His Ser Asn Gly Ile Gln
85 90 95Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp Tyr
Thr 100 105 110Glu Asn Val Thr Ala Val Glu Val Asn Pro Ser Asn Arg
Tyr Gln Glu 115 120 125Thr Ser Gly His Tyr Asn Ile Gln Ala Trp Thr
Gly Phe Asn Phe Pro 130 135 140Gly Arg Gly Thr Thr Tyr Ser Asn Phe
Lys Trp Gln Trp Phe His Phe145 150 155 160Asp Gly Thr Asp Trp Asp
Gln Ser Arg Ser Leu Ser Arg Ile Phe Lys 165 170 175Phe Asp Gly Lys
Ala Trp Asp Trp Pro Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr Asp
Tyr Leu Met Tyr Ala Asp Tyr Asp Tyr Asp His Pro Asp Val 195 200
205Val Asn Glu Met Lys Lys Trp Gly Val Trp Tyr Ala Asn Glu Val Gly
210 215 220Leu Asp Gly Tyr Arg Leu Asp Ala Val Lys His Ile Lys Phe
Ser Phe225 230 235 240Leu Lys Asp Trp Val Asp Asn Ala Arg Ala Ala
Thr Gly Lys Glu Met 245 250 255Phe Thr Val Gly Glu Tyr Trp Gln Asn
Asp Leu Gly Ala Leu Asn Asn 260 265 270Tyr Leu Ala Lys Val Asn Tyr
Asn Gln Ser Leu Phe Asp Ala Pro Leu 275 280 285His Tyr Asn Phe Tyr
Ala Ala Ser Thr Gly Gly Gly Asp Tyr Asp Met 290 295 300Arg Asn Ile
Leu Asn Asn Thr Leu Val Ala Ser Asn Pro Thr Lys Ala305 310 315
320Val Thr Leu Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335Ser Thr Val Gln Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe
Ile Leu 340 345 350Thr Arg Ser Gly Gly Tyr Pro Ser Val Phe Tyr Gly
Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Thr Thr Arg Glu Ile Pro
Ala Leu Lys Ser Lys Ile 370 375 380Glu Pro Leu Leu Lys Ala Arg Lys
Asp Tyr Ala Tyr Gly Thr Gln Arg385 390 395 400Asp Tyr Ile Asp Asn
Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Thr Lys
Ala Lys Ser Gly Leu Ala Thr Val Ile Thr Asp Gly Pro 420 425 430Gly
Gly Ser Lys Arg Met Tyr Val Gly Thr Ser Asn Ala Gly Glu Ile 435 440
445Trp Tyr Asp Leu Thr Gly Asn Arg Thr Asp Lys Ile Thr Ile Gly Ser
450 455 460Asp Gly Tyr Ala Thr Phe Pro Val Asn Thr Glu Ser Val Ser
Val Trp465 470 475 480Val Gln Gln331443DNAArtificial
SequenceSynthetic codon-optimized nucleotide sequence of the
PcuAmy1 gene 33gccgacaacg gcacaatcat gcagtatttc gagtggtacc
tgccgaacga cggagcgcac 60tggaacagac ttaataacga cgcacaaaac ctgaaaaatg
tgggcatcac ggcagtgtgg 120attcctccgg catacaaggg cggcagctca
gcagatgttg gctacggagt ttacgataca 180tacgacctgg gcgagttcaa
tcagaaaggc acggtcagaa caaagtacgg aacgaagagc 240gaactgattt
cagcggtcaa caatcttcac gcaaagggca ttgcggttta cggcgacgtg
300gtcctgaacc atagaatgaa tgcggatgca acggagcttg tggatgcggt
tgaggtggat 360ccgaacaaca gaaacgtcga gacgacaagc acgtatcaga
tccaggcatg gacgcaatac 420gatttcccgg gcagaggcaa cacgtacagc
agctttaaat ggagatggta tcacttcgac 480ggcgtcgact gggaccagag
cagaggcctg aacagaatct ataagctgag aggcgatggc 540aaggattggg
actgggaggt cgacagcgag tacggcaact acgattacct gatgggagcg
600gacctggact tcaaccaccc ggatgtggtt aacgaaacaa agacatgggg
caaatggttt 660gtgaacacgg tgaacctgga tggcgtcaga ctggacgcgg
ttaagcacat caagttcgac 720ttcatgagag actgggtgaa caacgtgaga
agcacgacgg gcaagaacct tttcgcagtt 780ggcgagtatt ggcactacga
cgtgaacaaa ctgaacagct acatcacgaa gacgaatggc 840acgatgagcc
tgttcgacgt gccgctgcac tttagatttt atgatgcaag caacggcgga
900ggcggctacg acatgagaaa cctgctgaat aacacgctga tgagcagcaa
cccgatgaag 960gcggttacat tcgttgagaa ccatgacaca caaccgacgc
aggccctgca atcaacggtc 1020caaagctggt ttaagccgct tgcgtatgct
acaatcctga cgagagagca aggctacccg 1080tgcgttttct acggcgacta
ttatggaaca agcgacggca aaattagcag ctacaagccg 1140atcatggata
agcttcttaa cgcgagaaag gtgtacgcct acggcacgca gagagattac
1200ttcgatcatc cggacatcgt tggctggaca agagaaggcg atgcagcaca
tgctggctca 1260ggactggcaa cgcttatcac agatggccct ggcggaagca
agtggatgta tgttggaacg 1320tcaaaggcag gccaggtctg gacggataaa
acaggaaaca gaagcggaac ggtgacgatt 1380gatgccaatg gctggggaaa
cttttgggtt aatggcggat cagttagcgt ttgggcaaaa 1440taa
144334478PRTArtificial SequenceSynthetic amino acid sequence of the
mature of PcuAmy1-v1A polypeptide 34Ala Asp Asn Gly Thr Ile Met Gln
Tyr Phe Glu Trp Tyr Leu Pro Asn1 5 10 15Asp Gly Ala His Trp Asn Arg
Leu Asn Asn Asp Ala Gln Asn Leu Lys 20 25 30Asn Val Gly Ile Thr Ala
Val Trp Ile Pro Pro Ala Tyr Lys Gly Gly 35 40 45Ser Ser Ala Asp Val
Gly Tyr Gly Val Tyr Asp Thr Tyr Asp Leu Gly 50 55 60Glu Phe Asn Gln
Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser65 70 75 80Glu Leu
Ile Ser Ala Val Asn Asn Leu His Ala Lys Gly Ile Ala Val 85 90 95Tyr
Gly Asp Val Val Leu Asn His Arg Met Asn Ala Asp Ala Thr Glu 100 105
110Leu Val Asp Ala Val Glu Val Asp Pro Asn Asn Arg Tyr Val Glu Thr
115 120 125Thr Ser Thr Tyr Gln Ile Gln Ala Trp Thr Gln Tyr Asp Phe
Pro Gly 130 135 140Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp
Tyr His Phe Asp145 150 155 160Gly Val Asp Trp Asp Gln Ser Arg Gly
Leu Asn Arg Ile Tyr Lys Leu 165 170 175Asp Gly Lys Asp Trp Asp Trp
Pro Val Asp Ser Glu Tyr Gly Asn Tyr 180 185 190Asp Tyr Leu Met Gly
Ala Asp Leu Asp Phe Asn His Pro Asp Val Val 195 200 205Asn Glu Thr
Lys Thr Trp Gly Lys Trp Phe Val Asn Thr Val Asn Leu 210 215 220Asp
Gly Val Arg Leu Asp Ala Val Lys His Ile Lys Phe Asp Phe Met225 230
235 240Arg Asp Trp Val Asn Asn Val Arg Ser Thr Thr Gly Lys Asn Leu
Phe 245 250 255Ala Val Gly Glu Tyr Trp His Tyr Asp Val Asn Lys Leu
Asn Ser Tyr 260 265 270Ile Thr Lys Thr Asn Gly Thr Met Ser Leu Phe
Asp Val Pro Leu His 275 280 285Phe Arg Phe Tyr Asp Ala Ser Asn Gly
Gly Gly Gly Tyr Asp Met Arg 290 295 300Asn Leu Leu Asn Asn Thr Leu
Met Ser Ser Asn Pro Met Lys Ala Val305 310 315 320Thr Phe Val Glu
Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser 325 330 335Thr Val
Gln Ser Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu Thr 340 345
350Arg Glu Gln Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr Tyr Gly Thr
355 360 365Ser Asp Gly Lys Ile Ser Ser Tyr Lys Pro Ile Met Asp Lys
Leu Leu 370 375 380Asn Ala Arg Lys Val Tyr Ala Tyr Gly Thr Gln Arg
Asp Tyr Phe Asp385 390 395 400His Pro Asp Ile Val Gly Trp Thr Arg
Glu Gly Asp Ala Ala His Ala 405 410 415Gly Ser Gly Leu Ala Thr Leu
Ile Thr Asp Gly Pro Gly Gly Ser Lys 420 425 430Trp Met Tyr Val Gly
Thr Ser Lys Ala Gly Gln Val Trp Thr Asp Lys 435 440 445Thr Gly Asn
Arg Ser Gly Thr Val Thr Ile Asp Ala Asn Gly Trp Gly 450 455 460Asn
Phe Trp Val Asn Lys Gly Ser Val Ser Val Trp Ala Lys465 470
47535478PRTArtificial SequenceSynthetic amino acid sequence of the
mature of PcuAmy1-v6 polypeptide 35Ala Asp Asn Gly Thr Ile Met Gln
Tyr Phe Glu Trp Tyr Leu Pro Asn1 5 10 15Asp Gly Ala His Trp Asn Arg
Leu Asn Asn Asp Ala Gln Asn Leu Lys 20 25 30Asn Val Gly Ile Thr Ala
Val Trp Ile Pro Pro Ala Tyr Lys Gly Gly 35 40 45Ser Ser Ala Asp Val
Gly Tyr Gly Val Tyr Asp Thr Tyr Asp Leu Gly 50 55 60Glu Phe Asn Gln
Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser65 70 75 80Glu Leu
Ile Ser Ala Val Asn Asn Leu His Ala Lys Gly Ile Ala Val 85 90 95Tyr
Gly Asp Val Val Leu Asn His Arg Met Asn Ala Asp Ala Thr Glu 100 105
110Leu Val Asp Ala Val Glu Val Asp Pro Asn Asn Arg Tyr Val Glu Thr
115 120 125Thr Ser Thr Tyr Gln Ile Gln Ala Trp Thr Gln Tyr Asp Phe
Pro Gly 130 135 140Arg Gly Asn Thr Tyr Ser Ser Trp Lys Trp Arg Trp
Tyr His Phe Asp145 150 155 160Gly Val Asp Trp Asp Gln Ser Arg Gly
Leu Asn Arg Ile Tyr Lys Leu 165 170 175Asp Gly Lys Asp Trp Asp Trp
Pro Val Asp Ser Glu Tyr Gly Asn Tyr 180 185 190Asp Tyr Leu Met Gly
Ala Asp Leu Asp Phe Asn His Pro Asp Val Val 195 200 205Asn Glu Thr
Lys Thr Trp Gly Lys Trp Phe Val Asn Thr Val Asn Leu 210 215 220Asp
Gly Val Arg Leu Asp Ala Val Lys His Ile Lys Phe Asp Phe Met225 230
235 240Arg Asp Trp Val Asn Asn Val Arg Ser Thr Thr Gly Lys Asn Leu
Phe 245 250 255Ala Val Gly Glu Tyr Trp His Tyr Asp Val Asn Lys Leu
Asn Ser Tyr 260 265 270Ile Thr Lys Thr Asn Gly Thr Met Ser Leu Phe
Asp Val Pro Leu His 275 280 285Phe Arg Phe Tyr Asp Ala Ser Asn Gly
Gly Gly Gly Tyr Asp Met Arg 290 295 300Asn Leu Leu Asn Asn Thr Leu
Met Ser Ser Asn Pro Met Lys Ala Val305 310 315 320Thr Phe Val Glu
Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser 325 330 335Thr Val
Gln Ser Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu Thr 340 345
350Arg Glu Gln Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr Tyr Gly Thr
355 360 365Ser Asp Gly Lys Ile Ser Ser Tyr Lys Pro Ile Met Asp Lys
Leu Leu 370 375 380Asn Ala Arg Lys Val Tyr Ala Tyr Gly Thr Gln Arg
Asp Tyr Phe Asp385 390 395 400His Pro Asp Ile Val Gly Trp Thr Arg
Glu Gly Asp Ala Ala His Ala 405 410 415Gly Ser Gly Leu Ala Thr Leu
Ile Thr Asp Gly Pro Gly Gly Ser Lys 420 425 430Trp Met Tyr Val Gly
Thr Ser Lys Ala Gly Gln Val Trp Thr Asp Lys 435 440 445Thr Gly Asn
Arg Ser Gly Thr Val Thr Ile Asp Ala Asn Gly Trp Gly 450 455 460Asn
Phe Trp Val Asn Lys Gly Ser Val Ser Val Trp Ala Lys465 470
47536478PRTArtificial SequenceSynthetic amino acid sequence of the
mature of PcuAmy1-v8 polypeptide 36Ala Asp Asn Gly Thr Ile Met Gln
Tyr Phe Glu Trp Tyr Leu Pro Asn1 5 10 15Asp Gly Ala His Trp Asn Arg
Leu Asn Asn Asp Ala Gln Asn Leu Lys 20 25 30Asn Val Gly Ile Thr Ala
Val Trp Ile Pro Pro Ala Tyr Lys Gly Gly 35 40 45Ser Ser Ala Asp Val
Gly Tyr Gly Val Tyr Asp Thr Tyr Asp Leu Gly 50 55 60Glu Phe Asn Gln
Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser65 70 75 80Glu Leu
Ile Ser Ala Val Asn Asn Leu His Ala Lys Gly Ile Ala Val 85 90 95Tyr
Gly Asp Val Val Leu Asn His Arg Met Asn Ala Asp Ala Thr Glu 100 105
110Leu Val Asp Ala Val Glu Val Asp Pro Asn Asn Arg Tyr Val Glu Thr
115 120 125Thr Ser Thr Tyr Gln Ile Gln Ala Trp Thr Gln Tyr Asp Phe
Pro Gly 130 135 140Arg Gly Asn Thr Tyr Ser Ser Trp Lys Trp Arg Trp
Tyr His Phe Asp145 150 155 160Gly Val Asp Trp Asp Gln Ser Arg Gly
Leu Asn Arg Ile Tyr Lys Leu 165 170 175Asp Gly Lys Asp Trp Asp Trp
Pro Val Asp Ser Glu Tyr Gly Asn Tyr 180 185 190Asp Tyr Leu Met Gly
Ala Asp Leu Asp Phe Asn His Pro Asp Val Val 195 200 205Asn Glu Thr
Lys Thr Trp Gly Lys Trp Phe Val Asn Thr Val Asn Leu 210 215 220Asp
Gly Val Arg Leu Asp Ala Val Lys His Ile Lys Phe Asp Phe Met225 230
235 240Arg Asp Trp Val Asn Asn Val Arg Ser Thr Thr Gly Lys Asn Leu
Phe 245 250 255Ala Val Gly Glu Tyr Trp His Tyr Asp Val Asn Lys Leu
Asn Ser Tyr 260 265 270Ile Thr Lys Thr Asn Gly Thr Met Ser Leu Phe
Asp Val Pro Leu His 275 280 285Phe Arg Phe Tyr Asp Ala Ser Asn Gly
Gly Gly Gly Tyr Asp Met Arg 290 295 300Asn Leu Leu Asn Asn Thr Leu
Met Ser Ser Asn Pro Met Lys Ala Val305 310 315 320Thr Phe Val Glu
Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser 325 330 335Thr Val
Gln Ser Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu Thr 340 345
350Arg Glu Gln Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr Tyr Gly Thr
355 360 365Ser Asp Gly Lys Ile Ser Ser Tyr Lys Pro Ile Met Asp Lys
Leu Leu 370 375 380Asn Ala Arg Lys Val Tyr Ala Tyr Gly Thr Gln Arg
Asp Tyr Phe Asp385 390 395 400His Pro Asp Ile Val Gly Trp Thr Arg
Glu Gly Asp Ala Ala His Ala 405 410 415Gly Ser Gly Leu Ala Thr Leu
Ile Thr Asp Gly Pro Gly Gly Ser Lys 420 425 430Trp Met Tyr Val Gly
Thr Ser Lys Ala Gly Gln Val Trp Thr Asp Lys 435 440 445Thr Gly Asn
Arg Ser Gly Thr Val Thr Ile Asp Ala Asn Gly Trp Gly 450 455 460Asn
Phe Trp
Val Asn Arg Arg Ser Val Ser Val Trp Ala Lys465 470
47537478PRTArtificial SequenceSynthetic amino acid sequence of the
mature of PcuAmy1-v16 polypeptide 37Ala Asp Asn Gly Thr Ile Met Gln
Tyr Phe Glu Trp Tyr Leu Pro Asn1 5 10 15Asp Gly Ala His Trp Asn Arg
Leu Asn Asn Asp Ala Gln Asn Leu Lys 20 25 30Asn Val Gly Ile Thr Ala
Val Trp Ile Pro Pro Ala Tyr Lys Gly Gly 35 40 45Ser Ser Ala Asp Val
Gly Tyr Gly Val Tyr Asp Thr Tyr Asp Leu Gly 50 55 60Glu Phe Asn Gln
Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser65 70 75 80Glu Leu
Ile Ser Ala Val Asn Asn Leu His Ala Lys Gly Ile Ala Val 85 90 95Tyr
Gly Asp Val Val Leu Asn His Arg Met Asn Ala Asp Ala Thr Glu 100 105
110Leu Val Asp Ala Val Glu Val Asp Pro Asn Asn Arg Tyr Val Glu Thr
115 120 125Thr Ser Thr Tyr Gln Ile Gln Ala Trp Thr Gln Tyr Asp Phe
Pro Gly 130 135 140Arg Gly Asn Thr Tyr Ser Ser Trp Lys Trp Arg Trp
Tyr His Phe Asp145 150 155 160Gly Val Asp Trp Asp Gln Ser Arg Gly
Leu Asn Arg Ile Tyr Lys Leu 165 170 175Asp Gly Lys Asp Trp Asp Trp
Pro Val Asp Ser Glu Tyr Gly Asn Tyr 180 185 190Asp Tyr Leu Met Gly
Ala Asp Leu Asp Phe Asp His Pro Asp Val Val 195 200 205Asn Glu Thr
Lys Thr Trp Gly Lys Trp Phe Val Asn Thr Val Asn Leu 210 215 220Asp
Gly Val Arg Leu Asp Ala Val Lys His Ile Lys Phe Asp Phe Met225 230
235 240Arg Asp Trp Val Asn Asn Val Arg Ser Thr Thr Gly Lys Asn Leu
Phe 245 250 255Ala Val Gly Glu Tyr Trp His Tyr Asp Val Asn Lys Leu
Asn Ser Tyr 260 265 270Ile Thr Lys Thr Asn Gly Thr Met Ser Leu Phe
Asp Val Pro Leu His 275 280 285Phe Arg Phe Tyr Asp Ala Ser Asn Gly
Gly Gly Gly Tyr Asp Met Arg 290 295 300Asn Leu Leu Asn Asn Thr Leu
Met Ser Ser Asn Pro Met Lys Ala Val305 310 315 320Thr Phe Val Glu
Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser 325 330 335Thr Val
Gln Ser Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu Thr 340 345
350Arg Glu Gln Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr Tyr Gly Thr
355 360 365Ser Asp Gly Lys Ile Ser Ser Tyr Lys Pro Ile Met Asp Lys
Leu Leu 370 375 380Asn Ala Arg Lys Val Tyr Ala Tyr Gly Thr Gln Arg
Asp Tyr Phe Asp385 390 395 400His Pro Asp Ile Val Gly Trp Thr Arg
Glu Gly Asp Ala Ala His Ala 405 410 415Gly Ser Gly Leu Ala Thr Leu
Ile Thr Asp Gly Pro Gly Gly Ser Lys 420 425 430Trp Met Tyr Val Gly
Thr Ser Lys Ala Gly Gln Val Trp Thr Asp Lys 435 440 445Thr Gly Asn
Arg Ser Gly Thr Val Thr Ile Asp Ala Asn Gly Trp Gly 450 455 460Asn
Phe Trp Val Asn Lys Gly Ser Val Ser Val Trp Ala Lys465 470
475381479DNAArtificial SequenceSynthetic codon-modified nucleic
acid sequence encoding the mature form of BASE 38tctgcagctt
cagcaaacac cgcgccgatt aacgaaacca tgatgcagta tttcgaatgg 60gatctgccga
acgatggcac cctgtggacc aaagtgaaaa acgaagcggc gaacctgagc
120agcctgggca ttaccgcgct gtggctgccg ccggcatata aaggcaccag
ccagagcgat 180gtgggctatg gcgtgtatga tctgtacgat ctgggcgaat
ttaaccagaa aggcaccatt 240cgtaccaaat atggcaccaa aacccagtat
attcaggcga tccaggcggc gaaagcggcg 300ggtatgcagg tgtatgcgga
tgtggtgttt aaccataaag cgggtgcgga tggcaccgaa 360tttgtggatg
cggtggaagt ggatccgagc aaccgtaacc aggaaaccag cggcacctat
420cagattcagg cgtggaccaa atttgatttt cccggccgtg gcaacaccta
tagcagcttt 480aaatggcgct ggtatcattt tgatggcacc gattgggatg
aaagccgtaa actgaaccgc 540atctataaat ttcgtagcac cggcaaagcg
tgggattggg aagtggatac cgaaaacggc 600aactatgatt acctgatgtt
cgcagacctg gatatggatc atccggaagt ggtgaccgaa 660ctgaaaaact
ggggcacctg gtatgtgaac accaccaaca ttgatggctt tcgtctggat
720gcggtgaaac acatcaaata cagctttttt ccggattggc tgacctatgt
gcgtaaccag 780accggcaaaa acctgtttgc ggtgggcgaa ttttggagct
atgatgtgaa caaactgcac 840aactacatca ccaaaaccaa cggcagcatg
agcctgtttg atgcgccgct gcataacaac 900ttttataccg cgagcaaaag
cagcggctat tttgatatgc gttatctgct gaacaacacc 960ctgatgaaag
atcagccgag cctggccgtg accctggtgg ataaccatga tacccagccg
1020ggccagagcc tgcaaagctg ggtggaaccg tggtttaaac cgctggccta
cgcgtttatt 1080ctgacccgtc aagagggcta tccgtgcgtt ttttatggcg
attattacgg catcccgaaa 1140tataacattc cgggcctgaa aagcaaaatt
gatccgctgc tgattgcgcg tcgtgattat 1200gcgtatggca cccagcgtga
ttatattgat caccaggata ttattggctg gacccgtgaa 1260ggcattgata
ccaaaccgaa cagcggcctg gccgcgctga ttaccgatgg cccgggtggc
1320agcaaatgga tgtatgtggg caaaaaacat gcgggcaaag tgttttatga
tctgaccggc 1380aaccgtagcg ataccgtgac cattaacgcg gatggctggg
gtgagtttaa agtgaacggc 1440ggcagcgtga gcatttgggt ggcgaaataa
gttaacaga 147939482PRTArtificial SequenceSynthetic amino acid
sequence of BASE-V28 39Asn Thr Ala Pro Ile Asn Glu Thr Met Met Gln
Tyr Phe Glu Trp Asp1 5 10 15Leu Pro Asn Asp Gly Thr Leu Trp Thr Lys
Val Lys Asn Glu Ala Ala 20 25 30Asn Leu Ser Ser Leu Gly Ile Thr Ala
Leu Trp Leu Pro Pro Ala Tyr 35 40 45Lys Gly Thr Ser Gln Ser Asp Val
Gly Tyr Gly Val Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln
Lys Gly Thr Ile Arg Thr Lys Tyr Gly65 70 75 80Thr Lys Thr Gln Tyr
Ile Gln Ala Ile Gln Ala Ala Lys Ala Ala Gly 85 90 95Met Gln Val Tyr
Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp 100 105 110Gly Thr
Glu Phe Val Asp Ala Val Glu Val Asp Pro Ser Asn Arg Tyr 115 120
125Gln Glu Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp
130 135 140Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg
Trp Tyr145 150 155 160His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg
Lys Leu Asn Arg Ile 165 170 175Tyr Lys Phe Thr Gly Lys Ala Trp Asp
Trp Pro Val Asp Thr Glu Asn 180 185 190Gly Asn Tyr Asp Tyr Leu Met
Phe Ala Asp Leu Asp Met Asp His Pro 195 200 205Glu Val Val Thr Glu
Leu Lys Asn Trp Gly Thr Trp Tyr Val Asn Thr 210 215 220Thr Asn Ile
Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Tyr225 230 235
240Ser Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Asn Gln Thr Gly Lys
245 250 255Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn
Lys Leu 260 265 270His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser
Leu Phe Asp Ala 275 280 285Pro Leu His Asn Asn Phe Tyr Thr Ala Ser
Lys Ser Ser Gly Tyr Phe 290 295 300Asp Met Arg Tyr Leu Leu Asn Asn
Thr Leu Met Lys Asp Gln Pro Ser305 310 315 320Leu Ala Val Thr Leu
Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser 325 330 335Leu Gln Ser
Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe 340 345 350Ile
Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr 355 360
365Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp
370 375 380Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln
Arg Asp385 390 395 400Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr
Arg Glu Gly Ile Asp 405 410 415Thr Lys Pro Asn Ser Gly Leu Ala Ala
Leu Ile Thr Asp Gly Pro Gly 420 425 430Gly Ser Lys Trp Met Tyr Val
Gly Lys Lys His Ala Gly Lys Val Phe 435 440 445Tyr Asp Leu Thr Gly
Asn Arg Ser Asp Thr Val Thr Ile Asn Ala Asp 450 455 460Gly Trp Gly
Glu Phe Lys Val Asn Arg Gly Ser Val Ser Ile Trp Val465 470 475
480Ala Lys40482PRTArtificial SequenceSynthetic amino acid sequence
of BASE-V29 40Asn Thr Ala Pro Ile Asn Glu Thr Met Met Gln Tyr Phe
Glu Trp Asp1 5 10 15Leu Pro Asn Asp Gly Thr Leu Trp Thr Lys Val Lys
Asn Glu Ala Ala 20 25 30Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp
Leu Pro Pro Ala Tyr 35 40 45Lys Gly Thr Ser Gln Ser Asp Val Gly Tyr
Gly Val Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly
Thr Ile Arg Thr Lys Tyr Gly65 70 75 80Thr Lys Thr Gln Tyr Ile Gln
Ala Ile Gln Ala Ala Lys Ala Ala Gly 85 90 95Met Gln Val Tyr Ala Asp
Val Val Phe Asn His Lys Ala Gly Ala Asp 100 105 110Gly Thr Glu Phe
Val Asp Ala Val Glu Val Asp Pro Ser Asn Arg Asn 115 120 125Gln Glu
Thr Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp 130 135
140Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Trp Lys Trp Arg Trp
Tyr145 150 155 160His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys
Leu Asn Arg Ile 165 170 175Tyr Lys Phe Thr Gly Lys Ala Trp Asp Trp
Pro Val Asp Thr Glu Asn 180 185 190Gly Asn Tyr Asp Tyr Leu Met Phe
Ala Asp Leu Asp Met Asp His Pro 195 200 205Glu Val Val Thr Glu Leu
Lys Asn Trp Gly Thr Trp Tyr Val Asn Thr 210 215 220Thr Asn Ile Asp
Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Tyr225 230 235 240Ser
Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Asn Gln Thr Gly Lys 245 250
255Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn Lys Leu
260 265 270His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe
Asp Ala 275 280 285Pro Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser
Ser Gly Tyr Phe 290 295 300Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu
Met Lys Asp Gln Pro Ser305 310 315 320Leu Ala Val Thr Leu Val Asp
Asn His Asp Thr Gln Pro Gly Gln Ser 325 330 335Leu Gln Ser Trp Val
Glu Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe 340 345 350Ile Leu Thr
Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr 355 360 365Tyr
Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp 370 375
380Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln Arg
Asp385 390 395 400Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg
Glu Gly Ile Asp 405 410 415Thr Lys Pro Asn Ser Gly Leu Ala Ala Leu
Ile Thr Asp Gly Pro Gly 420 425 430Gly Ser Lys Trp Met Tyr Val Gly
Lys Lys His Ala Gly Lys Val Phe 435 440 445Tyr Asp Leu Thr Gly Asn
Arg Ser Asp Thr Val Thr Ile Asn Ala Asp 450 455 460Gly Trp Gly Glu
Phe Lys Val Asn Arg Gly Ser Val Ser Ile Trp Val465 470 475 480Ala
Lys41482PRTArtificial SequenceSynthetic amino acid sequence of
BASE-V30 41Asn Thr Ala Pro Ile Asn Glu Thr Met Met Gln Tyr Phe Glu
Trp Asp1 5 10 15Leu Pro Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn
Glu Ala Ala 20 25 30Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu
Pro Pro Ala Tyr 35 40 45Lys Gly Thr Ser Gln Ser Asp Val Gly Tyr Gly
Val Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr
Ile Arg Thr Lys Tyr Gly65 70 75 80Thr Lys Thr Gln Tyr Ile Gln Ala
Ile Gln Ala Ala Lys Ala Ala Gly 85 90 95Met Gln Val Tyr Ala Asp Val
Val Phe Asn His Lys Ala Gly Ala Asp 100 105 110Gly Thr Glu Phe Val
Asp Ala Val Glu Val Asp Pro Ser Asn Arg Asn 115 120 125Gln Glu Thr
Ser Gly Glu Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp 130 135 140Phe
Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr145 150
155 160His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg
Ile 165 170 175Tyr Lys Phe His Gly Lys Ala Trp Asp Trp Pro Val Asp
Thr Glu Asn 180 185 190Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu
Asp Met Asp His Pro 195 200 205Glu Val Val Thr Glu Leu Lys Asn Trp
Gly Thr Trp Tyr Val Asn Thr 210 215 220Thr Asn Ile Asp Gly Phe Arg
Leu Asp Ala Val Lys His Ile Lys Tyr225 230 235 240Ser Phe Phe Pro
Asp Trp Leu Thr Tyr Val Arg Asn Gln Thr Gly Lys 245 250 255Asn Leu
Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn Lys Leu 260 265
270His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe Asp Ala
275 280 285Pro Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser Gly
Tyr Phe 290 295 300Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys
Asp Gln Pro Ser305 310 315 320Leu Ala Val Thr Leu Val Asp Asn His
Asp Thr Gln Pro Gly Gln Ser 325 330 335Leu Gln Ser Trp Val Glu Pro
Trp Phe Lys Pro Leu Ala Tyr Ala Phe 340 345 350Ile Leu Thr Arg Gln
Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr 355 360 365Tyr Gly Ile
Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp 370 375 380Pro
Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln Arg Asp385 390
395 400Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile
Asp 405 410 415Thr Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp
Gly Pro Gly 420 425 430Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His
Ala Gly Lys Val Phe 435 440 445Tyr Asp Leu Thr Gly Asn Arg Ser Asp
Thr Val Thr Ile Asn Ala Asp 450 455 460Gly Trp Gly Glu Phe Lys Val
Asn Arg Gly Ser Val Ser Ile Trp Val465 470 475 480Ala
Lys42482PRTArtificial SequenceSynthetic amino acid sequence of
BASE-V31 42Asn Thr Ala Pro Ile Asn Glu Thr Met Met Gln Tyr Phe Glu
Trp Asp1 5 10 15Leu Pro Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn
Glu Ala Ala 20 25 30Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu
Pro Pro Ala Tyr 35 40 45Lys Gly Thr Ser Gln Ser Asp Val Gly Tyr Gly
Val Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr
Ile Arg Thr Lys Tyr Gly65 70 75 80Thr Lys Thr Gln Tyr Ile Gln Ala
Ile Gln Ala Ala Lys Ala Ala Gly 85 90 95Met Gln Val Tyr Ala Asp Val
Val Phe Asn His Lys Ala Gly Ala Asp 100 105 110Gly Thr Glu Phe Val
Asp Ala Val Glu Val Asp Pro Ser Asn Arg Tyr 115 120 125Gln Glu Thr
Ser Gly Glu Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp 130 135 140Phe
Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr145 150
155 160His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg
Ile 165 170 175Tyr Lys Phe His Gly Lys Ala Trp Asp Trp Pro Val Asp
Thr Glu Asn 180 185 190Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu
Asp Met Asp His Pro 195 200 205Glu Val Val Thr Glu Leu Lys Asn Trp
Gly Thr Trp Tyr Val Asn Thr 210 215 220Thr Asn Ile Asp Gly Phe Arg
Leu Asp Ala
Val Lys His Ile Lys Tyr225 230 235 240Ser Phe Phe Pro Asp Trp Leu
Thr Tyr Val Arg Asn Gln Thr Gly Lys 245 250 255Asn Leu Phe Ala Val
Gly Glu Phe Trp Ser Tyr Asp Val Asn Lys Leu 260 265 270His Asn Tyr
Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe Asp Ala 275 280 285Pro
Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser Ser Gly Tyr Phe 290 295
300Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro
Ser305 310 315 320Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln
Pro Gly Gln Ser 325 330 335Leu Gln Ser Trp Val Glu Pro Trp Phe Lys
Pro Leu Ala Tyr Ala Phe 340 345 350Ile Leu Thr Arg Gln Glu Gly Tyr
Pro Cys Val Phe Tyr Gly Asp Tyr 355 360 365Tyr Gly Ile Pro Lys Tyr
Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp 370 375 380Pro Leu Leu Ile
Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln Arg Asp385 390 395 400Tyr
Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile Asp 405 410
415Thr Lys Pro Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly
420 425 430Gly Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys
Val Phe 435 440 445Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr
Ile Asn Ala Asp 450 455 460Gly Trp Gly Glu Phe Lys Val Asn Arg Gly
Ser Val Ser Ile Trp Val465 470 475 480Ala Lys43482PRTArtificial
SequenceSynthetic amino acid sequence of BASE-V32 43Asn Thr Ala Pro
Ile Asn Glu Thr Met Met Gln Tyr Phe Glu Trp Asp1 5 10 15Leu Pro Asn
Asp Gly Thr Leu Trp Thr Lys Val Lys Asn Glu Ala Ala 20 25 30Asn Leu
Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr 35 40 45Lys
Gly Thr Ser Gln Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr 50 55
60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly65
70 75 80Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala Lys Ala Ala
Gly 85 90 95Met Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly
Ala Asp 100 105 110Gly Thr Glu Phe Val Asp Ala Val Glu Val Asp Pro
Ser Asn Arg Tyr 115 120 125Gln Glu Thr Ser Gly Thr Tyr Gln Ile Gln
Ala Trp Thr Lys Phe Asp 130 135 140Phe Pro Gly Arg Gly Asn Thr Tyr
Ser Ser Trp Lys Trp Arg Trp Tyr145 150 155 160His Phe Asp Gly Thr
Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile 165 170 175Tyr Lys Phe
Thr Gly Lys Ala Trp Asp Trp Pro Val Asp Thr Glu Asn 180 185 190Gly
Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu Asp Met Asp His Pro 195 200
205Glu Val Val Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr Val Asn Thr
210 215 220Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile
Lys Tyr225 230 235 240Ser Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg
Asn Gln Thr Gly Lys 245 250 255Asn Leu Phe Ala Val Gly Glu Phe Trp
Ser Tyr Asp Val Asn Lys Leu 260 265 270His Asn Tyr Ile Thr Lys Thr
Asn Gly Ser Met Ser Leu Phe Asp Ala 275 280 285Pro Leu His Asn Asn
Phe Tyr Thr Ala Ser Lys Ser Ser Gly Tyr Phe 290 295 300Asp Met Arg
Tyr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro Ser305 310 315
320Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser
325 330 335Leu Gln Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr
Ala Phe 340 345 350Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe
Tyr Gly Asp Tyr 355 360 365Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly
Leu Lys Ser Lys Ile Asp 370 375 380Pro Leu Leu Ile Ala Arg Arg Asp
Tyr Ala Tyr Gly Thr Gln Arg Asp385 390 395 400Tyr Ile Asp His Gln
Asp Ile Ile Gly Trp Thr Arg Glu Gly Ile Asp 405 410 415Thr Lys Pro
Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly 420 425 430Gly
Ser Lys Trp Met Tyr Val Gly Lys Lys His Ala Gly Lys Val Phe 435 440
445Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ala Asp
450 455 460Gly Trp Gly Glu Phe Lys Val Asn Arg Gly Ser Val Ser Ile
Trp Val465 470 475 480Ala Lys44482PRTArtificial SequenceSynthetic
amino acid sequence of BASE-V33 44Asn Thr Ala Pro Ile Asn Glu Thr
Met Met Gln Tyr Phe Glu Trp Asp1 5 10 15Leu Pro Asn Asp Gly Thr Leu
Trp Thr Lys Val Lys Asn Glu Ala Ala 20 25 30Asn Leu Ser Ser Leu Gly
Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr 35 40 45Lys Gly Thr Ser Gln
Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu
Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly65 70 75 80Thr Lys
Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala Lys Ala Ala Gly 85 90 95Met
Gln Val Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp 100 105
110Gly Thr Glu Phe Val Asp Ala Val Glu Val Asp Pro Ser Asn Arg Asn
115 120 125Gln Glu Thr Ser Gly Glu Tyr Gln Ile Gln Ala Trp Thr Lys
Phe Asp 130 135 140Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Trp Lys
Trp Arg Trp Tyr145 150 155 160His Phe Asp Gly Thr Asp Trp Asp Glu
Ser Arg Lys Leu Asn Arg Ile 165 170 175Tyr Lys Phe His Gly Lys Ala
Trp Asp Trp Pro Val Asp Thr Glu Asn 180 185 190Gly Asn Tyr Asp Tyr
Leu Met Phe Ala Asp Leu Asp Met Asp His Pro 195 200 205Glu Val Val
Thr Glu Leu Lys Asn Trp Gly Thr Trp Tyr Val Asn Thr 210 215 220Thr
Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Tyr225 230
235 240Ser Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Asn Gln Thr Gly
Lys 245 250 255Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val
Asn Lys Leu 260 265 270His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met
Ser Leu Phe Asp Ala 275 280 285Pro Leu His Asn Asn Phe Tyr Thr Ala
Ser Lys Ser Ser Gly Tyr Phe 290 295 300Asp Met Arg Tyr Leu Leu Asn
Asn Thr Leu Met Lys Asp Gln Pro Ser305 310 315 320Leu Ala Val Thr
Leu Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser 325 330 335Leu Gln
Ser Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe 340 345
350Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr
355 360 365Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys
Ile Asp 370 375 380Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly
Thr Gln Arg Asp385 390 395 400Tyr Ile Asp His Gln Asp Ile Ile Gly
Trp Thr Arg Glu Gly Ile Asp 405 410 415Thr Lys Pro Asn Ser Gly Leu
Ala Ala Leu Ile Thr Asp Gly Pro Gly 420 425 430Gly Ser Lys Trp Met
Tyr Val Gly Lys Lys His Ala Gly Lys Val Phe 435 440 445Tyr Asp Leu
Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ala Asp 450 455 460Gly
Trp Gly Glu Phe Lys Val Asn Arg Gly Ser Val Ser Ile Trp Val465 470
475 480Ala Lys45482PRTArtificial SequenceSynthetic amino acid
sequence of BASE-V34 45Asn Thr Ala Pro Ile Asn Glu Thr Met Met Gln
Tyr Phe Glu Trp Asp1 5 10 15Leu Pro Asn Asp Gly Thr Leu Trp Thr Lys
Val Lys Asn Glu Ala Ala 20 25 30Asn Leu Ser Ser Leu Gly Ile Thr Ala
Leu Trp Leu Pro Pro Ala Tyr 35 40 45Lys Gly Thr Ser Gln Ser Asp Val
Gly Tyr Gly Val Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln
Lys Gly Thr Ile Arg Thr Lys Tyr Gly65 70 75 80Thr Lys Thr Gln Tyr
Ile Gln Ala Ile Gln Ala Ala Lys Ala Ala Gly 85 90 95Met Gln Val Tyr
Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp 100 105 110Gly Thr
Glu Phe Val Asp Ala Val Glu Val Asp Pro Ser Asn Arg Tyr 115 120
125Gln Glu Thr Ser Gly Glu Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp
130 135 140Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Trp Lys Trp Arg
Trp Tyr145 150 155 160His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg
Lys Leu Asn Arg Ile 165 170 175Tyr Lys Phe His Gly Lys Ala Trp Asp
Trp Pro Val Asp Thr Glu Asn 180 185 190Gly Asn Tyr Asp Tyr Leu Met
Phe Ala Asp Leu Asp Met Asp His Pro 195 200 205Glu Val Val Thr Glu
Leu Lys Asn Trp Gly Thr Trp Tyr Val Asn Thr 210 215 220Thr Asn Ile
Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Tyr225 230 235
240Ser Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Asn Gln Thr Gly Lys
245 250 255Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn
Lys Leu 260 265 270His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser
Leu Phe Asp Ala 275 280 285Pro Leu His Asn Asn Phe Tyr Thr Ala Ser
Lys Ser Ser Gly Tyr Phe 290 295 300Asp Met Arg Tyr Leu Leu Asn Asn
Thr Leu Met Lys Asp Gln Pro Ser305 310 315 320Leu Ala Val Thr Leu
Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser 325 330 335Leu Gln Ser
Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe 340 345 350Ile
Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr 355 360
365Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp
370 375 380Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln
Arg Asp385 390 395 400Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr
Arg Glu Gly Ile Asp 405 410 415Thr Lys Pro Asn Ser Gly Leu Ala Ala
Leu Ile Thr Asp Gly Pro Gly 420 425 430Gly Ser Lys Trp Met Tyr Val
Gly Lys Lys His Ala Gly Lys Val Phe 435 440 445Tyr Asp Leu Thr Gly
Asn Arg Ser Asp Thr Val Thr Ile Asn Ala Asp 450 455 460Gly Trp Gly
Glu Phe Lys Val Asn Arg Gly Ser Val Ser Ile Trp Val465 470 475
480Ala Lys46482PRTArtificial SequenceSynthetic amino acid sequence
of BASE-V35 46Asn Thr Ala Pro Ile Asn Glu Thr Met Met Gln Tyr Phe
Glu Trp Asp1 5 10 15Leu Pro Asn Asp Gly Thr Leu Trp Thr Lys Val Lys
Asn Glu Ala Ala 20 25 30Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp
Leu Pro Pro Ala Tyr 35 40 45Lys Gly Thr Ser Gln Ser Asp Val Gly Tyr
Gly Val Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly
Thr Ile Arg Thr Lys Tyr Gly65 70 75 80Thr Lys Thr Gln Tyr Ile Gln
Ala Ile Gln Ala Ala Lys Ala Ala Gly 85 90 95Met Gln Val Tyr Ala Asp
Val Val Phe Asn His Lys Ala Gly Ala Asp 100 105 110Gly Thr Glu Phe
Val Asp Ala Val Glu Val Asp Pro Ser Asn Arg Tyr 115 120 125Gln Glu
Thr Ser Gly His Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp 130 135
140Phe Pro Gly Arg Gly Asn Thr Tyr Ser Ser Trp Lys Trp Arg Trp
Tyr145 150 155 160His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys
Leu Asn Arg Ile 165 170 175Tyr Lys Phe Asp Gly Lys Ala Trp Asp Trp
Pro Val Asp Thr Glu Asn 180 185 190Gly Asn Tyr Asp Tyr Leu Met Phe
Ala Asp Leu Asp Met Asp His Pro 195 200 205Glu Val Val Thr Glu Leu
Lys Asn Trp Gly Thr Trp Tyr Val Asn Thr 210 215 220Thr Asn Ile Asp
Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Tyr225 230 235 240Ser
Phe Phe Pro Asp Trp Leu Thr Tyr Val Arg Asn Gln Thr Gly Lys 245 250
255Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn Lys Leu
260 265 270His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser Leu Phe
Asp Ala 275 280 285Pro Leu His Asn Asn Phe Tyr Thr Ala Ser Lys Ser
Ser Gly Tyr Phe 290 295 300Asp Met Arg Tyr Leu Leu Asn Asn Thr Leu
Met Lys Asp Gln Pro Ser305 310 315 320Leu Ala Val Thr Leu Val Asp
Asn His Asp Thr Gln Pro Gly Gln Ser 325 330 335Leu Gln Ser Trp Val
Glu Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe 340 345 350Ile Leu Thr
Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr 355 360 365Tyr
Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp 370 375
380Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln Arg
Asp385 390 395 400Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr Arg
Glu Gly Ile Asp 405 410 415Thr Lys Pro Asn Ser Gly Leu Ala Ala Leu
Ile Thr Asp Gly Pro Gly 420 425 430Gly Ser Lys Trp Met Tyr Val Gly
Lys Lys His Ala Gly Lys Val Phe 435 440 445Tyr Asp Leu Thr Gly Asn
Arg Ser Asp Thr Val Thr Ile Asn Ala Asp 450 455 460Gly Trp Gly Glu
Phe Lys Val Asn Arg Gly Ser Val Ser Ile Trp Val465 470 475 480Ala
Lys47482PRTArtificial SequenceSynthetic amino acid sequence of
BASE-V36 47Asn Thr Ala Pro Ile Asn Glu Thr Met Met Gln Tyr Phe Glu
Trp Asp1 5 10 15Leu Pro Asn Asp Gly Thr Leu Trp Thr Lys Val Lys Asn
Glu Ala Ala 20 25 30Asn Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu
Pro Pro Ala Tyr 35 40 45Lys Gly Thr Ser Gln Ser Asp Val Gly Tyr Gly
Val Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr
Ile Arg Thr Lys Tyr Gly65 70 75 80Thr Lys Thr Gln Tyr Ile Gln Ala
Ile Gln Ala Ala Lys Ala Ala Gly 85 90 95Met Gln Val Tyr Ala Asp Val
Val Phe Asn His Lys Ala Gly Ala Asp 100 105 110Gly Thr Glu Phe Val
Asp Ala Val Glu Val Asp Pro Ser Asn Arg Tyr 115 120 125Gln Glu Thr
Ser Gly Glu Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp 130 135 140Phe
Pro Gly Arg Gly Asn Thr Tyr Ser Ser Trp Lys Trp Arg Trp Tyr145 150
155 160His Phe Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg
Ile 165 170 175Tyr Lys Phe Gly Gly Lys Ala Trp Asp Trp Pro Val Asp
Thr Glu Asn 180 185 190Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asp Leu
Asp Met Asp His Pro 195 200 205Glu Val Val Thr Glu Leu Lys Asn Trp
Gly Thr Trp Tyr Val Asn Thr 210 215 220Thr Asn Ile Asp Gly Phe Arg
Leu Asp Ala Val Lys His Ile Lys Tyr225 230 235 240Ser Phe Phe Pro
Asp Trp Leu Thr Tyr Val Arg Asn Gln Thr Gly Lys
245 250 255Asn Leu Phe Ala Val Gly Glu Phe Trp Ser Tyr Asp Val Asn
Lys Leu 260 265 270His Asn Tyr Ile Thr Lys Thr Asn Gly Ser Met Ser
Leu Phe Asp Ala 275 280 285Pro Leu His Asn Asn Phe Tyr Thr Ala Ser
Lys Ser Ser Gly Tyr Phe 290 295 300Asp Met Arg Tyr Leu Leu Asn Asn
Thr Leu Met Lys Asp Gln Pro Ser305 310 315 320Leu Ala Val Thr Leu
Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser 325 330 335Leu Gln Ser
Trp Val Glu Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe 340 345 350Ile
Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr 355 360
365Tyr Gly Ile Pro Lys Tyr Asn Ile Pro Gly Leu Lys Ser Lys Ile Asp
370 375 380Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln
Arg Asp385 390 395 400Tyr Ile Asp His Gln Asp Ile Ile Gly Trp Thr
Arg Glu Gly Ile Asp 405 410 415Thr Lys Pro Asn Ser Gly Leu Ala Ala
Leu Ile Thr Asp Gly Pro Gly 420 425 430Gly Ser Lys Trp Met Tyr Val
Gly Lys Lys His Ala Gly Lys Val Phe 435 440 445Tyr Asp Leu Thr Gly
Asn Arg Ser Asp Thr Val Thr Ile Asn Ala Asp 450 455 460Gly Trp Gly
Glu Phe Lys Val Asn Arg Gly Ser Val Ser Ile Trp Val465 470 475
480Ala Lys484PRTArtificial SequenceSynthetic conserved amino acid
sequence motifmisc_feature(1)..(1)Xaa can be any naturally
occurring amino acidmisc_feature(2)..(2)Xaa is Gly or
Sermisc_feature(3)..(3)Xaa can be any naturally occurring amino
acid 48Xaa Xaa Xaa Gly1494PRTArtificial SequenceSynthetic peptide
49Arg Gly Thr Gly150478PRTArtificial SequenceSynthetic mature form
of PcuAmy1-v3 50Ala Asp Asn Gly Thr Ile Met Gln Tyr Phe Glu Trp Tyr
Leu Pro Asn1 5 10 15Asp Gly Ala His Trp Asn Arg Leu Asn Asn Asp Ala
Gln Asn Leu Lys 20 25 30Asn Val Gly Ile Thr Ala Val Trp Ile Pro Pro
Ala Tyr Lys Gly Gly 35 40 45Ser Ser Ala Asp Val Gly Tyr Gly Val Tyr
Asp Thr Tyr Asp Leu Gly 50 55 60Glu Phe Asn Gln Lys Gly Thr Val Arg
Thr Lys Tyr Gly Thr Lys Ser65 70 75 80Glu Leu Ile Ser Ala Val Asn
Asn Leu His Ala Lys Gly Ile Ala Val 85 90 95Tyr Gly Asp Val Val Leu
Asn His Arg Met Asn Ala Asp Ala Thr Glu 100 105 110Leu Val Asp Ala
Val Glu Val Asp Pro Asn Asn Arg Asn Val Glu Thr 115 120 125Thr Ser
Thr Tyr Gln Ile Gln Ala Trp Thr Gln Tyr Asp Phe Pro Gly 130 135
140Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His Phe
Asp145 150 155 160Gly Val Asp Trp Asp Gln Ser Arg Gly Leu Asn Arg
Ile Tyr Lys Leu 165 170 175Asp Gly Lys Asp Trp Asp Trp Pro Val Asp
Ser Glu Tyr Gly Asn Tyr 180 185 190Asp Tyr Leu Met Gly Ala Asp Leu
Asp Phe Asn His Pro Asp Val Val 195 200 205Asn Glu Thr Lys Thr Trp
Gly Lys Trp Phe Val Asn Thr Val Asn Leu 210 215 220Asp Gly Val Arg
Leu Asp Ala Val Lys His Ile Lys Phe Asp Phe Met225 230 235 240Arg
Asp Trp Val Asn Asn Val Arg Ser Thr Thr Gly Lys Asn Leu Phe 245 250
255Ala Val Gly Glu Tyr Trp His Tyr Asp Val Asn Lys Leu Asn Ser Tyr
260 265 270Ile Thr Lys Thr Asn Gly Thr Met Ser Leu Phe Asp Val Pro
Leu His 275 280 285Phe Arg Phe Tyr Asp Ala Ser Asn Gly Gly Gly Gly
Tyr Asp Met Arg 290 295 300Asn Leu Leu Asn Asn Thr Leu Met Ser Ser
Asn Pro Met Lys Ala Val305 310 315 320Thr Phe Val Glu Asn His Asp
Thr Gln Pro Thr Gln Ala Leu Gln Ser 325 330 335Thr Val Gln Ser Trp
Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu Thr 340 345 350Arg Glu Gln
Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr Tyr Gly Thr 355 360 365Ser
Asp Gly Lys Ile Ser Ser Tyr Lys Pro Ile Met Asp Lys Leu Leu 370 375
380Asn Ala Arg Lys Val Tyr Ala Tyr Gly Thr Gln Arg Asp Tyr Phe
Asp385 390 395 400His Pro Asp Ile Val Gly Trp Thr Arg Glu Gly Asp
Ala Ala His Ala 405 410 415Gly Ser Gly Leu Ala Thr Leu Ile Thr Asp
Gly Pro Gly Gly Ser Lys 420 425 430Trp Met Tyr Val Gly Thr Ser Lys
Ala Gly Gln Val Trp Thr Asp Lys 435 440 445Thr Gly Asn Arg Ser Gly
Thr Val Thr Ile Asp Ala Asn Gly Trp Gly 450 455 460Asn Phe Trp Val
Asn Lys Gly Ser Val Ser Val Trp Ala Lys465 470 475
* * * * *
References