U.S. patent application number 13/516891 was filed with the patent office on 2012-10-11 for alpha-amylases.
This patent application is currently assigned to NOVOZYMES NORTH AMERICA, INC.. Invention is credited to Carsten Andersen, Thomas Agersten Poulsen.
Application Number | 20120258497 13/516891 |
Document ID | / |
Family ID | 44246932 |
Filed Date | 2012-10-11 |
United States Patent
Application |
20120258497 |
Kind Code |
A1 |
Andersen; Carsten ; et
al. |
October 11, 2012 |
Alpha-Amylases
Abstract
The present invention relates to alpha-amylase variants,
polynucleotides encoding the variants and nucleic acid constructs,
vectors, and host cells comprising the polynucleotides, and methods
of using the variant enzymes.
Inventors: |
Andersen; Carsten;
(Vaerloese, DK) ; Poulsen; Thomas Agersten;
(Ballerup, DK) |
Assignee: |
NOVOZYMES NORTH AMERICA,
INC.
Franklinton
NC
NOVOZYMES A/S
Bagsvaerd
|
Family ID: |
44246932 |
Appl. No.: |
13/516891 |
Filed: |
January 4, 2011 |
PCT Filed: |
January 4, 2011 |
PCT NO: |
PCT/US11/20135 |
371 Date: |
June 18, 2012 |
Related U.S. Patent Documents
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Patent Number |
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61304092 |
Feb 12, 2010 |
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61333930 |
May 12, 2010 |
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61354775 |
Jun 15, 2010 |
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61354817 |
Jun 15, 2010 |
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61355230 |
Jun 16, 2010 |
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61362536 |
Jul 8, 2010 |
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Current U.S.
Class: |
435/69.6 ;
435/115; 435/138; 435/139; 435/145; 435/159; 435/160; 435/162;
435/196; 435/198; 435/201; 435/252.3; 435/252.31; 435/252.33;
435/252.34; 435/252.35; 435/254.11; 435/254.2; 435/254.21;
435/254.22; 435/254.23; 435/254.3; 435/254.4; 435/254.5; 435/254.6;
435/254.7; 435/254.8; 435/320.1; 435/325; 435/348; 435/412;
435/414; 435/415; 435/417; 435/419; 435/99; 510/392 |
Current CPC
Class: |
C12N 9/2414 20130101;
C12P 7/06 20130101; C12P 19/04 20130101; C12Y 302/01001 20130101;
C12P 7/14 20130101; C12P 19/02 20130101; C12P 19/14 20130101; Y02E
50/10 20130101; C12P 2203/00 20130101; C11D 3/38681 20130101; C12N
15/8257 20130101; C11D 3/386 20130101; Y02E 50/17 20130101; C12N
9/2417 20130101 |
Class at
Publication: |
435/69.6 ;
435/201; 435/198; 435/196; 435/99; 435/162; 435/160; 435/145;
435/139; 435/159; 435/138; 435/115; 435/252.3; 435/252.31;
435/252.34; 435/252.33; 435/252.35; 435/325; 435/348; 435/419;
435/254.11; 435/254.2; 435/254.21; 435/254.22; 435/254.23;
435/254.3; 435/254.7; 435/254.8; 435/254.5; 435/254.4; 435/254.6;
435/412; 435/417; 435/414; 435/415; 435/320.1; 510/392 |
International
Class: |
C12N 9/26 20060101
C12N009/26; C12N 9/16 20060101 C12N009/16; C12P 19/14 20060101
C12P019/14; C12P 7/14 20060101 C12P007/14; C12P 7/16 20060101
C12P007/16; C12P 7/46 20060101 C12P007/46; C12P 7/56 20060101
C12P007/56; C12P 7/20 20060101 C12P007/20; C12P 7/60 20060101
C12P007/60; C12P 13/08 20060101 C12P013/08; C12P 21/04 20060101
C12P021/04; C12N 1/21 20060101 C12N001/21; C12N 5/10 20060101
C12N005/10; C12N 1/15 20060101 C12N001/15; C12N 1/19 20060101
C12N001/19; C12N 15/63 20060101 C12N015/63; C11D 3/386 20060101
C11D003/386; C12N 9/20 20060101 C12N009/20 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 4, 2010 |
EP |
10150062.7 |
Jan 4, 2010 |
EP |
10150063.5 |
Claims
1-18. (canceled)
19. An isolated alpha-amylase, comprising an A-domain of a
calcium-sensitive alpha-amylase which has at least 70% and less
than 100% sequence identity with any of the A-domains of SEQ ID
NOS: 1-12, 29, and 30, a B-domain of a calcium-insensitive
alpha-amylase, and a C-domain of a calcium-sensitive alpha-amylase,
wherein the alpha-amylase has alpha-amylase activity.
20. An isolated alpha-amylase, comprising an A-domain of a
calcium-sensitive alpha-amylase, a B-domain of a
calcium-insensitive alpha-amylase, and a C-domain of a
calcium-sensitive alpha-amylase which has at least 70% and less
than 100% sequence identity with any of the C-domains of SEQ ID
NOS: 1-12, 29, and 30, wherein the alpha-amylase has alpha-amylase
activity.
21. The alpha-amylase of claim 19, wherein the amino acid at the
position corresponding to position 8 is Ala, Arg, Asn, Asp, Cys,
Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or
Val, e.g., Leu. the amino acid at the position corresponding to
position 27 is Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Gln. the
amino acid at the position corresponding to position 34 is Ala,
Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro,
Thr, Trp, Tyr, or Val, e.g., Lys. the amino acid at the position
corresponding to position 52 is any amino acid except Ala, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Gly. the amino acid at the position
corresponding to position 53 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val,
e.g., Tyr. the amino acid at the position corresponding to position
59 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys,
Met, Phe, Pro, Ser, Thr, Trp, or Tyr, e.g., Ala. the amino acid at
the position corresponding to position 80 is Ala, Arg, Asn, Asp,
Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp,
Tyr, or Val, e.g., Asp. the amino acid at the position
corresponding to position 86 is Ala, Arg, Asn, Asp, Cys, Glu, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Ser. the amino acid at the position corresponding to position
90 is Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,
Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ser. the amino acid at
the position corresponding to position 91 is Arg, Asn, Asp, Cys,
Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,
Tyr, or Val, e.g., Leu. the amino acid at the position
corresponding to position 100 is Arg, Asn, Asp, Cys, Gln, Glu, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Gly or Leu. the amino acid at the position corresponding to
position 204 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Leu. the
amino acid at the position corresponding to position 206 is Ala,
Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,
Pro, Ser, Thr, Trp, or Val, e.g., Met. the amino acid at the
position corresponding to position 220 is Ala, Arg, Asn, Asp, Cys,
Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or
Val, e.g., Pro. the amino acid at the position corresponding to
position 222 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile,
Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val, e.g., Val. the
amino acid at the position corresponding to position 224 is Ala,
Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Thr, Trp, Tyr, or Val, e.g., Leu. the amino acid at the
position corresponding to position 235 is Arg, Asn, Asp, Cys, Gln,
Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or
Val, e.g., Thr or Trp. the amino acid at the position corresponding
to position 242 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,
Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val, e.g., Gln or
Glu. the amino acid at the position corresponding to position 244
is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,
Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr. the amino acid at the
position corresponding to position 245 is Ala, Arg, Asn, Asp, Cys,
Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or
Val, e.g., Ala or Lys. the amino acid at the position corresponding
to position 270 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,
Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Leu. the
amino acid at the position corresponding to position 282 is Ala,
Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Thr, Trp, Tyr, or Val, e.g., Trp. the amino acid at the
position corresponding to position 284 is Ala, Arg, Asn, Asp, Cys,
Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or
Val, e.g., Gln, Thr, or Val. the amino acid at the position
corresponding to position 301 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val,
e.g., Lys. the amino acid at the position corresponding to position
303 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met,
Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Arg. the amino acid at
the position corresponding to position 304 is Arg, Asn, Asp, Cys,
Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,
Tyr, or Val, e.g., Asp. the amino acid at the position
corresponding to position 307 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Leu. the amino acid at the position corresponding to position
348 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys,
Met, Phe, Pro, Thr, Trp, Tyr, or Val, e.g., Thr. the amino acid at
the position corresponding to position 386 is Ala, Arg, Asn, Asp,
Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp,
Tyr, or Val, e.g., Gln, Glu, Thr, or Val. the amino acid at the
position corresponding to position 388 is Ala, Arg, Asn, Asp, Cys,
Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or
Val, e.g., Ile or Val.
22. The alpha-amylase of claim 19, wherein the amino acid at the
position corresponding to position 407 is Ala, Arg, Asp, Cys, Gln,
Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or
Val, e.g., Asp. the amino acid at the position corresponding to
position 430 is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp. the
amino acid at the position corresponding to position 432 is Ala,
Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Thr, Trp, Tyr, or Val, e.g., Pro. the amino acid at the
position corresponding to position 459 is Ala, Arg, Asn, Asp, Cys,
Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or
Val, e.g., Pro. the amino acid at the position corresponding to
position 475 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile,
Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val, e.g., Gln or
Lys.
23. A detergent composition comprising an alpha-amylase of claim 19
and a surfactant.
24. A composition comprising an alpha-amylase of claim 19 and one
or more enzymes selected from the group consisting of beta-amylase,
cellulase (beta-glucosidase, cellobiohydrolase, and endoglucanase)
glucoamylase, hemicellulase (e.g., xylanase), isoamylase,
isomerase, lipase, phytase, protease, and pullulanase.
25. A method of producing liquefied starch, comprising liquefying a
starch-containing material with an alpha-amylase of claim 19.
26. A process of producing a fermentation product, comprising (a)
liquefying a starch-containing material with an alpha-amylase of
claim 19 to produce a liquefied mash; (b) saccharifying the
liquefied mash to produce fermentable sugars; and (c) fermenting
the fermentable sugars in the presence of a fermenting
organism.
27. A process of producing a fermentation product, comprising
contacting a starch substrate with alpha-amylase of claim 19, a
glucoamylase, and a fermenting organism.
28. An isolated polynucleotide encoding the alpha-amylase of claim
19.
29. A nucleic acid construct comprising the polynucleotide of claim
28.
30. An expression vector comprising the nucleic acid construct of
claim 29.
31. A host cell comprising the nucleic acid construct of claim
15.
32. A method of producing an alpha-amylase, comprising: (a)
cultivating the host cell of claim 31 under conditions suitable for
the expression of the alpha-amylase; and (b) recovering the
alpha-amylase from the cultivation medium.
Description
REFERENCE TO SEQUENCE LISTING
[0001] This application contains a Sequence Listing in computer
readable form, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to alpha-amylase variants
having an improved property, e.g., improved stability,
polynucleotides encoding the variants, methods of producing the
variants, and methods of using the variants.
[0004] 2. Description of Related Art
[0005] Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C.
3.2.1.1) constitute a group of enzymes, which catalyze the
hydrolysis of starch and other linear and branched 1,4-glucosidic
oligo- and polysaccharides.
[0006] Alpha-amylases are used commercially for a variety of
purposes such as in the initial stages of starch processing (e.g.,
liquefaction); in wet milling processes; and in alcohol production
from carbohydrate sources. They are also used as cleaning agents or
adjuncts in detergent matrices; in the textile industry for starch
desizing; in baking applications; in the beverage industry; in oil
fields in drilling processes; in recycling processes, e.g., for
de-inking paper; and in animal feed.
[0007] One of the first bacterial alpha-amylases to be used was an
alpha-amylase from B. licheniformis, also known as Termamyl.TM.,
which has been extensively characterized and the crystal structure
has been determined for this enzyme. Alkaline amylases, such as the
alpha-amylase derived from Bacillus sp. strains NCIB 12289, NCIB
12512, NCIB 12513, and DSM 9375 (disclosed in WO 95/26397), form a
particular group of alpha-amylases that are useful in detergents.
Many of these known bacterial amylases have been modified in order
to improve their functionality in a particular application.
[0008] Termamyl.TM. and many highly efficient alpha-amylases
require calcium for activity. The crystal structure of Termamyl.TM.
shows that three calcium atoms are bound to the alpha-amylase
structure coordinated by negatively charged amino acid residues.
This requirement for calcium is a disadvantage in applications
where strong chelating compounds are present, such as in detergents
or during ethanol production from whole grains, where the plant
material comprises a large amount of natural chelators such as
phytate.
[0009] Calcium-insensitive amylases are known, e.g., the
alpha-amylases disclosed in EP 1022334 and WO 03/083054, and a
Bacillus circulans alpha-amylase having the sequence disclosed in
UNIPROT:Q03657.
[0010] It would therefore be beneficial to provide alpha-amylases
with reduced calcium sensitivity.
SUMMARY OF THE INVENTION
[0011] The present invention provides alpha-amylase variants
comprising an A-domain of a calcium-sensitive alpha-amylase which
has at least 70% and less than 100% sequence identity with any of
the A-domains of SEQ ID NOS: 1-12, 29, and 30, a B-domain of a
calcium-insensitive alpha-amylase, and a C-domain of a
calcium-sensitive alpha-amylase.
[0012] The present invention provides alpha-amylase variants
comprising an A-domain of a calcium-sensitive alpha-amylase, a
B-domain of a calcium-insensitive alpha-amylase, and a C-domain of
a calcium-sensitive alpha-amylase which has at least 70% and less
than 100% sequence identity with any of the C-domains of SEQ ID
NOS: 1-12, 29, and 30.
[0013] The present invention also relates to isolated
polynucleotides encoding an alpha-amylase variant, nucleic acid
constructs, vectors, and host cells comprising the polynucleotides,
and methods of producing a variant of a parent alpha-amylase.
[0014] The present invention also relates to the use of the
variants in starch processing (e.g., liquefaction); wet milling
processes; alcohol production from carbohydrate sources;
detergents; dishwashing compositions; starch desizing in the
textile industry; baking applications; the beverage industry; oil
fields in drilling processes; recycling processes, e.g., for
de-inking paper, and animal feed.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 shows an alignment of SEQ ID NOS: 1-16, 29, and
30.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention provides alpha-amylase variants
comprising an A-domain of a calcium-sensitive alpha-amylase which
has at least 70% and less than 100% sequence identity with any of
the A-domains of SEQ ID NOS: 1-12, 29 and 30, a B-domain of a
calcium-insensitive alpha-amylase, and a C-domain of a
calcium-sensitive alpha-amylase.
[0017] The present invention also provides alpha-amylase variants
comprising an A-domain of a calcium-sensitive alpha-amylase, a
B-domain of a calcium-insensitive alpha-amylase, and a C-domain of
a calcium-sensitive alpha-amylase which has at least 70% and less
than 100% sequence identity with any of the C-domains of SEQ ID
NOS: 1-12, 29, and 30.
DEFINITIONS
[0018] A, B and C-domains: The structure of alpha-amylases
comprises three distinct domains A, B and C, see, e.g., Machius et
al., 1995, J. Mol. Biol. 246: 545-559. The term "domain" means a
region of a polypeptide that in itself forms a distinct and
independent substructure of the whole molecule. Alpha-amylases
consist of a beta/alpha-8 barrel harboring the active site, which
is denoted the A-domain, a rather long loop between the beta-sheet
3 and alpha-helix 3, which is denoted the B-domain, and a C-domain
and in some cases also a carbohydrate binding domain (e.g., WO
2005/001064; Machius et al., supra).
[0019] The domains of an alpha-amylase can be determined by
structure analysis such as by using crystallographically
techniques. An alternative method for determining the domains of an
alpha-amylase is by sequence alignment of the amino acid sequence
of the alpha-amylase with another alpha-amylase for which the
domains have been determined. The sequence that aligns with, e.g.,
the B-domain sequence in the alpha-amylase for which the B-domain
has been determined can be considered the B-domain for the given
alpha-amylase.
[0020] Allelic variant: The term "allelic variant" means any of two
or more alternative forms of a gene occupying the same chromosomal
locus. Allelic variation arises naturally through mutation, and may
result in polymorphism within populations. Gene mutations can be
silent (no change in the encoded polypeptide) or may encode
polypeptides having altered amino acid sequences. An allelic
variant of a polypeptide is a polypeptide encoded by an allelic
variant of a gene.
[0021] Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C.
3.2.1.1) are a group of enzymes, which catalyze the hydrolysis of
starch and other linear and branched 1,4-glucosidic oligo- and
polysaccharides.
[0022] Calcium-insensitive amylase means an alpha-amylase that does
not require the presence of calcium for optimal activity and/or for
maintaining the active conformation/structure.
[0023] Calcium-sensitive amylase means an alpha-amylase that
requires the presence of calcium to retain its structure and/or to
have full enzymatic activity. For some calcium-sensitive amylases
it has been shown that they contains a calcium atom coordinated to
acidic amino acid residues in the active conformation. A large
number of calcium-sensitive alpha-amylases are known and have been
used industrially because of their beneficial properties.
Calcium-sensitive alpha-amylases are generally sensitive towards
conditions that leads to loss of the calcium atom coordinated in
their structure such as detergent compositions and fuel mass.
[0024] Calcium sensitivity is determined by incubating an
alpha-amylase in the presence of a strong chelator and analyzing
the impact of this incubation on the activity or stability of the
alpha-amylase. A calcium-sensitive alpha-amylase will be less
stable in the presence of a chelator or lose a major part or all of
its activity during incubation whereas a calcium-insensitive
alpha-amylase will not lose all of its activity or will lose only a
minor part of the activity during incubation. Chelator strength may
be evaluated using methods known in the art such as the methods
disclosed in Nielsen et al., 2003, Anal. Biochem. 314: 227-234; and
Nagarajan and Paine, 1984, J. Am. Oil Chem. Soc. 61(9): 1475-1478.
Examples of strong chelators that may be used for such an assay are
EGTA (ethylene glycol tetraacetic acid), EDTA (ethylene diamine
tetraacetic acid), DTPA (diethylene triamine pentaacetic acid),
DTMPA (diethylene triamine-penta-methylene phosphonic acid) and
HEDP (1-hydroxyethan-1,1-diylbis(phosphonic acid)). Other strong
chelators may be used to determine the calcium sensitivity of an
alpha-amylase.
[0025] Coding sequence: The term "coding sequence" means a
polynucleotide, which directly specifies the amino acid sequence of
its polypeptide product. The boundaries of the coding sequence are
generally determined by an open reading frame, which usually begins
with the ATG start codon or alternative start codons such as GTG
and TTG and ends with a stop codon such as TAA, TAG, and TGA. The
coding sequence may be a DNA, cDNA, synthetic, or recombinant
polynucleotide.
[0026] Control sequence: The term "control sequence" means all
components necessary for the expression of a polynucleotide
encoding a variant of the present invention. Each control sequence
may be native or foreign to the polynucleotide encoding the variant
or native or foreign to each other. Such control sequences include,
but are not limited to, a leader, polyadenylation sequence,
propeptide sequence, promoter, signal peptide sequence, and
transcription terminator. At a minimum, the control sequences
include a promoter, and transcriptional and translational stop
signals. The control sequences may be provided with linkers for the
purpose of introducing specific restriction sites facilitating
ligation of the control sequences with the coding region of the
polynucleotide encoding a variant.
[0027] Expression: The term "expression" includes any step involved
in the production of the polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0028] Expression vector: The term "expression vector" means a
linear or circular DNA molecule that comprises a polynucleotide
encoding a polypeptide of the present invention and is operably
linked to additional nucleotides that provide for its
expression.
[0029] Host cell: The term "host cell" means any cell type that is
susceptible to transformation, transfection, transduction, and the
like with a nucleic acid construct or expression vector comprising
a polynucleotide of the present invention. The term "host cell"
encompasses any progeny of a parent cell that is not identical to
the parent cell due to mutations that occur during replication.
[0030] Improved property: The term "improved property" means a
characteristic associated with a variant that is improved compared
to other alpha-amylases. Such improved properties include, but are
not limited to, altered temperature-dependent activity profile,
thermostability, pH activity, pH stability, substrate specificity,
product specificity, and chemical stability.
[0031] Isolated variant: The terms "isolated" and "purified" mean a
polypeptide or polynucleotide that is removed from at least one
component with which it is naturally associated. For example, a
variant may be at least 1% pure, e.g., at least 5% pure, at least
10% pure, at least 20% pure, at least 40% pure, at least 60% pure,
at least 80% pure, and at least 90% pure, as determined by SDS-PAGE
and a polynucleotide may be at least 1% pure, e.g., at least 5%
pure, at least 10% pure, at least 20% pure, at least 40% pure, at
least 60% pure, at least 80% pure, at least 90% pure, and at least
95% pure, as determined by agarose electrophoresis.
[0032] Mature polypeptide: The term "mature polypeptide" means a
polypeptide in its final form following translation and any
post-translational modifications, such as N-terminal processing,
C-terminal truncation, glycosylation, phosphorylation, etc. It is
known in the art that a host cell may produce a mixture of two of
more different mature polypeptides (i.e., with a different
C-terminal and/or N-terminal amino acid) expressed by the same
polynucleotide.
[0033] Mature polypeptide coding sequence: The term "mature
polypeptide coding sequence" means a nucleotide sequence that
encodes a mature polypeptide having alpha-amylase activity.
[0034] Nucleic acid construct: The term "nucleic acid construct"
means a nucleic acid molecule, either single- or double-stranded,
which is isolated from a naturally occurring gene or is modified to
contain segments of nucleic acids in a manner that would not
otherwise exist in nature or which is synthetic. The term nucleic
acid construct is synonymous with the term "expression cassette"
when the nucleic acid construct contains the control sequences
required for expression of a coding sequence.
[0035] Operably linked: The term "operably linked" means a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of the
polynucleotide sequence such that the control sequence directs the
expression of the coding sequence of a polypeptide.
[0036] Parent: The term "parent" alpha-amylase means an
alpha-amylase to which an alteration is made to produce a variant
of the present invention. The parent may be a naturally occurring
(wild-type) polypeptide, or a variant thereof, prepared by any
suitable means. For instance, the parent polypeptide may be a
variant of a naturally occurring polyptide which has a modified or
altered amino acid sequence. A parent may also be an allelic
variant.
[0037] Polypeptide fragment: The term "polypeptide fragment" means
a polypeptide having one or more (several) amino acids deleted from
the amino and/or carboxyl terminus of a mature polypeptide; wherein
the fragment has alpha-amylase activity. In one aspect, a fragment
contains at least 481 amino acid residues, e.g., at least 483, at
least 486, and at least 493 amino acid residues.
[0038] Sequence identity: The relatedness between two amino acid
sequences or between two nucleotide sequences is described by the
parameter "sequence identity".
[0039] For purposes of the present invention, the degree of
sequence identity between two amino acid sequences is determined
using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970,
J. Mol. Biol. 48: 443-453) as implemented in the Needle program of
the EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),
preferably version 3.0.0 or later. The optional parameters used are
gap open penalty of 10, gap extension penalty of 0.5, and the
EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The
output of Needle labeled "longest identity" (obtained using the
-nobrief option) is used as the percent identity and is calculated
as follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of
Gaps in Alignment)
[0040] For purposes of the present invention, the degree of
sequence identity between two deoxyribonucleotide sequences is
determined using the Needleman-Wunsch algorithm (Needleman and
Wunsch, 1970, supra) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, supra), preferably version 3.0.0
or later. The optional parameters used are gap open penalty of 10,
gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of
NCBI NUC4.4) substitution matrix. The output of Needle labeled
"longest identity" (obtained using the -nobrief option) is used as
the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides.times.100)/(Length of
Alignment-Total Number of Gaps in Alignment)
[0041] Subsequence: The term "subsequence" means a polynucleotide
sequence having one or more (several) nucleotides deleted from the
5' and/or 3' end of a mature polypeptide coding sequence; wherein
the subsequence encodes a polypeptide fragment having alpha-amylase
activity.
[0042] Variant: The term "variant" means a polypeptide having
alpha-amylase activity comprising an alteration, i.e., a
substitution, insertion, and/or deletion, of one or more (several)
amino acid residues at one or more (several) positions. A
substitution means a replacement of an amino acid occupying a
position with a different amino acid; a deletion means removal of
an amino acid occupying a position; and an insertion means adding
1-5 amino acids adjacent to and following an amino acid occupying a
position.
[0043] Wild-Type: The term "wild-type" means an alpha-amylase
expressed by a naturally occurring microorganism, such as a
bacterial, yeast, or filamentous fungus found in nature.
Conventions for Designation of Variants
[0044] For purposes of the present invention, unless otherwise
indicated, the hybrid polypeptide disclosed in SEQ ID NO: 27 (which
has the sequence of amino acids 1-104 of Bacillus
stearothermophilus alpha-amylase (SEQ ID NO: 4), followed by amino
acids 103-208 of Bacillus circulans alpha-amylase (SEQ ID NO: 13),
followed by amino acids 211-515 of Bacillus stearothermophilus
alpha-amylase (SEQ ID NO: 4)) is used to determine the
corresponding amino acid residue in another alpha-amylase. The
amino acid sequence of another alpha-amylase is aligned with the
mature polypeptide disclosed in SEQ ID NO: 27, and based on the
alignment, the amino acid position number corresponding to any
amino acid residue in the mature polypeptide disclosed in SEQ ID
NO: 27 can be determined using the Needleman-Wunsch algorithm
(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as
implemented in the Needle program of the EMBOSS package (EMBOSS:
The European Molecular Biology Open Software Suite, Rice et al.,
2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or
later.
[0045] Identification of the corresponding amino acid residue in
another alpha-amylase can be confirmed by an alignment of multiple
polypeptide sequences using "ClustalW" (Larkin et al., 2007,
Bioinformatics 23: 2947-2948).
[0046] When the other enzyme has diverged from the mature
polypeptide of SEQ ID NO: 27 such that a traditional sequence-based
comparison fails to detect their relationship (Lindahl and
Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pairwise
sequence comparison algorithms can be used. Greater sensitivity in
sequence-based searching can be attained using search programs that
utilize probabilistic representations of polypeptide families
(profiles) to search databases. For example, the PSI-BLAST program
generates profiles through an iterative database search process and
is capable of detecting remote homologs (Atschul et al., 1997,
Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can be
achieved if the family or superfamily for the polypeptide has one
or more (several) representatives in the protein structure
databases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol.
287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881)
utilize information from a variety of sources (PSI-BLAST, secondary
structure prediction, structural alignment profiles, and solvation
potentials) as input to a neural network that predicts the
structural fold for a query sequence. Similarly, the method of
Gough et al., 2000, J. Mol. Biol. 313: 903-919, can be used to
align a sequence of unknown structure with the superfamily models
present in the SCOP database. These alignments can in turn be used
to generate homology models for the polypeptide, and such models
can be assessed for accuracy using a variety of tools developed for
that purpose.
[0047] For proteins of known structure, several tools and resources
are available for retrieving and generating structural alignments.
For example the SCOP superfamilies of proteins have been
structurally aligned, and those alignments are accessible and
downloadable. Two or more protein structures can be aligned using a
variety of algorithms such as the distance alignment matrix (Holm
and Sander, 1998, Proteins 33: 88-96) or combinatorial extension
(Shindyalov and Bourne, 1998, Protein Eng. 11: 739-747), and
implementations of these algorithms can additionally be utilized to
query structure databases with a structure of interest in order to
discover possible structural homologs (e.g., Holm and Park, 2000,
Bioinformatics 16: 566-567). These structural alignments can be
used to predict the structurally and functionally corresponding
amino acid residues in proteins within the same structural
superfamily. This information, along with information derived from
homology modeling and profile searches, can be used to predict
which residues to mutate when moving mutations of interest from one
protein to a close or remote homolog.
[0048] In describing the alpha-amylase variants of the present
invention, the nomenclature described below is adapted for ease of
reference. In all cases, the accepted IUPAC single letter or triple
letter amino acid abbreviation is employed.
[0049] Substitutions.
[0050] For an amino acid substitution, the following nomenclature
is used: original amino acid, position, substituted amino acid.
Accordingly, the substitution of threonine with alanine at position
226 is designated as "Thr226Ala" or "T226A". Multiple mutations are
separated by addition marks ("+"), e.g., "Gly205Arg+Ser411Phe" or
"G205R+S411F", representing mutations at positions 205 and 411
substituting glycine (G) with arginine (R), and serine (S) with
phenylalanine (F), respectively.
[0051] Deletions.
[0052] For an amino acid deletion, the following nomenclature is
used: original amino acid, position, *. Accordingly, the deletion
of glycine at position 195 is designated as "Gly195*" or "G195*".
Multiple deletions are separated by addition marks ("+"), e.g.,
"Gly195*+Ser411*" or "G195*+S411*".
[0053] Insertions.
[0054] For an amino acid insertion, the following nomenclature is
used: original amino acid, position, original amino acid, new
inserted amino acid. Accordingly, the insertion of lysine after
glycine at position 195 is designated "Gly195GlyLys" or "G195GK".
Multiple insertions of amino acids are designated [Original amino
acid, position, original amino acid, new inserted amino acid #1,
new inserted amino acid #2; etc.]. For example, the insertion of
lysine and alanine after glycine at position 195 is indicated as
"Gly195GlyLysAla" or "G195GKA".
[0055] In such cases the inserted amino acid residue(s) are
numbered by the addition of lower case letters to the position
number of the amino acid residue preceding the inserted amino acid
residue(s). In the above example the sequence would thus be:
TABLE-US-00001 Parent: Variant: 195 195 195a 195b G G - K - A
[0056] Multiple Alterations.
[0057] Variants comprising multiple alterations are separated by
addition marks ("+"), e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E"
representing a substitution of tyrosine and glutamic acid for
arginine and glycine at positions 170 and 195, respectively.
[0058] Different Alterations.
[0059] Where different alterations can be introduced at a position,
the different alterations are separated by a comma, e.g.,
"Arg170Tyr,Glu" represents a substitution of arginge with tyrosine
or glutamic acid at position 170. Thus,
"Tyr167Gly,Ala+Arg170Gly,Ala" designates the following
variants:
Tyr167Gly+Arg170Gly, Tyr167Gly+Arg170Ala, Tyr167Ala+Arg170Gly, and
Tyr167Ala+Arg170Ala.
Domains
Calcium-Sensitive Alpha-Amylases
[0060] Examples of calcium-sensitive alpha-amylases include the
following alpha-amylases:
1. Bacillus amyloliquefaciens alpha-amylase having the amino acid
sequence of SEQ ID NO: 1; 2. Bacillus flavothermus amylase, AMY1048
described in WO 2005/001064, having the amino acid sequence of SEQ
ID NO: 2; 3. Bacillus licheniformis alpha-amylase having the amino
acid sequence of SEQ ID NO: 3, 4. Bacillus stearothermophilus
alpha-amylase having the amino acid sequence of SEQ ID NO: 4; 5.
Alpha-amylase AA560 derived from Bacillus sp. DSM 12649 described
in WO 00/60060, having the amino acid sequence of SEQ ID NO: 5; 6.
Alpha-amylase derived from Bacillus sp. strain NCIB 12512 described
in WO 95/26397, having the amino acid sequence of SEQ ID NO: 6; 7.
Alpha-amylase derived from Bacillus sp. strain NCIB 12513 described
in WO 95/26397, having the amino acid sequence of SEQ ID NO: 7; 8.
Alpha-amylase SP707 described by Tsukamoto et al., 1988, Biochem.
Biophys. Res. Comm. 151: 25-31, having the amino acid sequence of
SEQ ID NO: 8; 9. Alpha-amylase TS-22 having the amino acid sequence
of SEQ ID NO: 9; 10. Alpha-amylase TS-23 described in J. Appl.
Microbiology, 1997, 82: 325-334 (SWALL:q59222), having the amino
acid sequence of SEQ ID NO: 10; 11. Alpha-amylase derived from
Bacillus sp. KSM-AP1378 (FERM BP-3048) described in WO 97/00324,
having the amino acid sequence of SEQ ID NO: 11; 12. Alpha-amylase
derived from Bacillus sp. A 7-7 described in WO 02/10356, having
the amino acid sequence of SEQ ID NO: 12; 13. Alpha-amylase derived
from Bacillus stearothermophilus (Spezyme Xtra), having the amino
acid sequence of SEQ ID NO: 29. 14. Cytophaga alpha-amylase
described in Jeang et al., 2002, Appl. Environ. Microbiol.
68:3651-3654, having the amino acid sequence of SEQ ID NO: 30; as
well as hybrids and variants of any of these calcium-sensitive
alpha-amylases.
[0061] Other calcium-sensitive alpha-amylases include the
alpha-amylase produced by the B. licheniformis strain described in
EP 0252666 (ATCC 27811) and the alpha-amylases disclosed in WO
91/00353 and WO 94/18314.
[0062] The calcium-sensitive alpha-amylase may be a hybrid of two
or more calcium-sensitive alpha-amylases, such as a hybrid between
the alpha-amylase of Bacillus amyloliquefaciens and the
alpha-amylase of Bacillus licheniformis.
[0063] Commercially-available calcium-sensitive alpha-amylases are
the products sold under the following tradenames: Optitherm.TM. and
Takatherm.TM. (available from Danisco); Maxamyl.TM. (available from
Danisco), Spezym AA.TM., Spezyme Delta AA.TM., Spezyme Fred and
Spezyme Xtra (available from Danisco), and Keistase.TM. (available
from Daiwa), PURASTAR.TM. ST 5000E, and PURASTAR.TM. HPAM L (from
Genencor Int.).
[0064] The A-, B-, C-, and carbohydrate binding domains of these
calcium-sensitive alpha-amylases are provided in the following
table:
TABLE-US-00002 C-terminal A-Domain extension or (A1 and A2
Carbohydrate Alpha-Amylase Domains) B-Domain C-Domain Binding
Module Bacillus amyloliquefaciens 1-101 + 208-396 102-207 397-483
(SEQ ID NO: 1) Bacillus flavothermus 1-105 + 212-398 106-211
399-484 485-586 (SEQ ID NO: 2) Bacillus licheniformis 1-103 +
208-396 104-207 397-483 (SEQ ID NO: 3) Bacillus stearothermophilus
1-104 + 211-396 105-210 397-483 484-515 (SEQ ID NO: 4) Bacillus sp.
(SEQ ID NO: 5) 1-105 + 213-398 106-212 399-485 Bacillus sp. NCIB
12512 1-105 + 213-398 106-212 399-485 (SEQ ID NO: 6) Bacillus sp.
NCIB 12513 1-105 + 213-398 106-212 399-485 (SEQ ID NO: 7) SP707
(SEQ ID NO: 8) 1-105 + 213-398 106-212 399-485 TS-22 (SEQ ID NO: 9)
1-105 + 213-398 106-212 399-484 485-586 TS-23 (SEQ ID NO: 10) 1-105
+ 213-398 106-212 399-484 485-583 Bacillus sp. KSM-AP1378 1-105 +
213-398 106-212 399-485 (SEQ ID NO: 11) Bacillus sp. SP7-7 1-105 +
213-398 106-212 399-485 (SEQ ID NO: 12) Bacillus stearothermophilus
1-104 + 211-396 105-210 397-483 484-486 alpha-amylase (Spezyme
Xtra, SEQ ID NO: 29) Cytophaga alpha-amylase 1-102 + 209-397
103-208 398-484 (SEQ ID NO: 30)
Calcium-Insensitive Alpha-Amylases
[0065] Examples of calcium-insensitive alpha-amylases include the
following:
1. Bacillus circulans alpha-amylase having the sequence of SEQ ID
NO: 13; 2. KSM K-36 alpha-amylase having the sequence of SEQ ID NO:
14; 3. KSM K-38 alpha-amylase having the sequence of SEQ ID NO: 15;
4. Pyrococcus woesei alpha-amylase having the sequence of SEQ ID
NO: 16; 5. Pyrococcus hybrid alpha-amylase described in WO
03/083054 having the amino acid sequence of SEQ ID NO: 31; as well
as hybrids and variants of any of these alpha-amylases.
[0066] The A-, B-, C-, and carbohydrate binding domains of these
calcium-insensitive alpha-amylases are provided in the following
table:
TABLE-US-00003 C-terminal extension or Carbo- A-Domain hydrate (A1
and A2 B- C- Binding Alpha-Amylase Domains) Domain Domain Module
Bacillus circulans 1-102 + 209-395 103-208 396-482 483-492 (SEQ ID
NO: 13) KSM K-36 1-103 + 208-393 104-207 394-480 (SEQ ID NO: 14)
KSM K-38 1-103 + 208-393 104-207 394-480 (SEQ ID NO: 15) Pyrococcus
woesei 1-109 + 172-338 110-171 339-435 (SEQ ID NO: 16) Pyrococcus
hybrid 1-109 + 172-338 110-171 339-435 alpha-amylase (SEQ ID NO:
31)
[0067] The alpha-amylases may be produced by substituting the
B-domain or a portion thereof of a calcium-sensitive alpha-amylase
with the B-domain or a portion thereof of a calcium-insensitive
alpha-amylase. The alpha-amylases also may be produced by
substituting the A- and C-domains or a portion thereof of a
calcium-insensitive alpha-amylase with the A- and C-domains or a
portion thereof of a calcium-sensitive alpha-amylase. When
producing a hybrid alpha-amylase, no amino acids should be deleted
or inserted in the two splicing sites, i.e., the two sites where
the sequence of the calcium-sensitive alpha-amylase is combined
with the sequence of the calcium-insensitive alpha-amylase.
[0068] The boundaries of the A-, B- and C-domains of
calcium-sensitive and calcium-insensitive amylases provided in the
tables above are flexible, and some liberty regarding the sequences
is permitted. Thus, in general it is possible to deviate from the
exact boundaries for the domains by up to 20 amino acids, e.g.,
less than 20 amino acids, less than 10 amino acids, less than 6
amino acids, and less than 3 amino acids. In other words, the
sequence of the calcium-sensitive alpha-amylase to be replaced with
the sequence of a calcium-insensitive alpha-amylase may be within
20 amino acids of the boundaries of the B-domain, e.g., less than
10 amino acids, within 6 amino acids, and within 3 amino acids. For
example, the boundaries differ by one amino acid, two amino acids,
three amino acids, four amino acids, five amino acids, six amino
acids, seven amino acids, eight amino acids, nine amino acids, or
ten amino acids.
[0069] For example, for the B. amyloliquefaciens alpha-amylase (SEQ
ID NO: 1) where the B-domain has been determined as amino acid
residues 102-207, the sequence to be replaced by the corresponding
sequence of a calcium-insensitive alpha-amylase starts at a
position in the range of positions 92-112 and ending at a position
in the range of positions 197-217, e.g., starting at a position in
the range of positions 96-108 and ending at a position in the range
of positions 198-213 or starting at a position in the range of
positions 99-105 and ending at a position in the range of positions
204-210. The A and C-domains of the B. amyloliquefaciens
alpha-amylase were determined to be amino acid residues 1-101
(A1)+208-396 (A2) and 397-483, respectively. The alpha-amylases of
the present invention may comprise an A1-domain starting at a
position in the range of positions 1-5 and ending a position in the
range of positions 91-111, e.g., starting at a position in the
range of positions 1-3 and ending at a position in the range of
positions 96-101 or starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
101-106. The alpha-amylases of the present invention may comprise
A2 and C-domains starting at a position in the range of positions
198-218 and ending at a position in the range of positions 478-483,
e.g., starting at a position in the range of positions 203-208 and
ending at a position in the range of positions 480-483 or starting
at a position in the range of positions 208-213 and ending at a
position in the range of positions 480-483.
[0070] For the B. flavothermus alpha-amylase (SEQ ID NO: 2) where
the B-domain has been determined as amino acid residues 106-211,
the sequence to be replaced by the corresponding sequence of a
calcium-insensitive alpha-amylase starts at a position in the range
of positions 96-116 and ending at a position in the range of
positions 198-218, e.g., starting at a position in the range of
positions 100-112 and ending at a position in the range of
positions 202-214 or starting at a position in the range of
positions 103-109 and ending at a position in the range of
positions 205-212. The A and C-domains of the B. flavothermus
alpha-amylase were determined to be amino acid residues 1-105
(A1)+212-398 (A2) and 399-484, respectively. The alpha-amylases of
the present invention may comprise an A1-domain starting at a
position in the range of positions 1-5 and ending a position in the
range of positions 95-115, e.g., starting at a position in the
range of positions 1-3 and ending at a position in the range of
positions 100-105 or starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
105-110. The alpha-amylases of the present invention may comprise
A2 and C-domains starting at a position in the range of positions
202-222 and ending at a position in the range of positions 479-484,
e.g., starting at a position in the range of positions 207-212 and
ending at a position in the range of positions 481-484 or starting
at a position in the range of positions 212-217 and ending at a
position in the range of positions 481-484. The B. flavothermus
alpha-amylase further has a carbohydrate binding domain of amino
acid residues 485-586. The carbohydrate binding domain is not
required for the amylase activity and might be fully or partially
deleted.
[0071] For the B. licheniformis alpha-amylase (SEQ ID NO: 3) where
the B-domain has been determined as amino acid residues 104-207,
the sequence of B. licheniformis alpha-amylase to be replaced by
the corresponding sequence of a calcium-insensitive alpha-amylase
starts at a position in the range of positions 94-114 and ending at
a position in the range of positions 194-214, e.g., starting at a
position in the range of positions 98-110 and ending at a position
in the range of positions 198-210 or starting at a position in the
range of positions 101-107 and ending at a position in the range of
positions 201-207. The A and C-domains of the B. licheniformis
alpha-amylase were determined to be amino acid residues 1-103
(A1)+208-396 (A2) and 397-483, respectively. The alpha-amylases of
the present invention may comprise an A1-domain starting at a
position in the range of positions 1-5 and ending a position in the
range of positions 93-113, e.g., starting at a position in the
range of positions 1-3 and ending at a position in the range of
positions 98-103 or starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
103-108. The alpha-amylases of the present invention may comprise
A2 and C-domains starting at a position in the range of positions
198-218 and ending at a position in the range of positions 478-483,
e.g., starting at a position in the range of positions 203-208 and
ending at a position in the range of positions 480-483 or starting
at a position in the range of positions 208-213 and ending at a
position in the range of positions 480-483.
[0072] For the B. stearothermophilus alpha-amylase (SEQ ID NO: 4)
where the B-domain has been determined as amino acid residues
105-210, the sequence to be replaced by the corresponding sequence
of a calcium-insensitive alpha-amylase starts at a position in the
range of positions 95-115 and ending at a position in the range of
positions 197-213, e.g., starting at a position in the range of
positions 99-111 and ending at a position in the range of positions
201-213 or starting at a position in the range of positions 102-108
and ending at a position in the range of positions 204-210. The A
and C-domains of the B. stearothermophilus alpha-amylase were
determined to be amino acid residues 1-104 (A1)+211-396 (A2) and
397-483, respectively. The alpha-amylases of the present invention
may comprise an A1-domain starting at a position in the range of
positions 1-5 and ending a position in the range of positions
94-114, e.g., starting at a position in the range of positions 1-3
and ending at a position in the range of positions 99-104 or
starting at a position in the range of positions 1-3 and ending at
a position in the range of positions 104-109. The alpha-amylases of
the present invention may comprise A2 and C-domains starting at a
position in the range of positions 201-221 and ending at a position
in the range of positions 478-483, e.g., starting at a position in
the range of positions 206-211 and ending at a position in the
range of positions 480-483 or starting at a position in the range
of positions 211-216 and ending at a position in the range of
positions 480-483. The B. stearothermophilus alpha-amylase further
has a C-terminal extension of amino acid residues 484-586. The
C-terminal extension is not required for the amylase activity and
might be fully or partially deleted.
[0073] For the Bacillus alpha-amylase (SEQ ID NO: 5) where the
B-domain has been determined as amino acid residues 106-212, the
sequence to be replaced by the corresponding sequence of a
calcium-insensitive alpha-amylase starts at a position in the range
of positions 96-116 and ending at a position in the range of
positions 199-219, e.g., starting at a position in the range of
positions 100-112 and ending at a position in the range of
positions 203-215 or starting at a position in the range of
positions 103-109 and ending at a position in the range of
positions 206-212. The A and C-domains of the Bacillus
alpha-amylase were determined to be amino acid residues 1-105
(A1)+213-396 (A2) and 399-485, respectively. The alpha-amylases of
the present invention may comprise an A1-domain starting at a
position in the range of positions 1-5 and ending a position in the
range of positions 95-115, e.g., starting at a position in the
range of positions 1-3 and ending at a position in the range of
positions 100-105 or starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
105-110. The alpha-amylases of the present invention may comprise
A2 and C-domains starting at a position in the range of positions
203-223 and ending at a position in the range of positions 482-485,
e.g., starting at a position in the range of positions 208-213 and
ending at a position in the range of positions 482-485 or starting
at a position in the range of positions 213-218 and ending at a
position in the range of positions 482-485.
[0074] For the Bacillus alpha-amylase (SEQ ID NO: 6) where the
B-domain has been determined as amino acid residues 106-212, the
sequence to be replaced by the corresponding sequence of a
calcium-insensitive alpha-amylase starts at a position in the range
of positions 96-116 and ending at a position in the range of
positions 199-219, e.g., starting at a position in the range of
positions 100-112 and ending at a position in the range of
positions 203-215 or starting at a position in the range of
positions 103-109 and ending at a position in the range of
positions 206-212. The A and C-domains of the Bacillus
alpha-amylase were determined to be amino acid residues 1-105
(A1)+213-398 (A2) and 399-485, respectively. The alpha-amylases of
the present invention may comprise an A1-domain starting at a
position in the range of positions 1-5 and ending a position in the
range of positions 95-115, e.g., starting at a position in the
range of positions 1-3 and ending at a position in the range of
positions 100-105 or starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
105-110. The alpha-amylases of the present invention may comprise
A2 and C-domains starting at a position in the range of positions
203-223 and ending at a position in the range of positions 482-485,
e.g., starting at a position in the range of positions 208-213 and
ending at a position in the range of positions 482-485 or starting
at a position in the range of positions 213-218 and ending at a
position in the range of positions 482-485.
[0075] For the Bacillus alpha-amylase (SEQ ID NO: 7) where the
B-domain has been determined as amino acid residues 106-212, the
sequence to be replaced by the corresponding sequence of a
calcium-insensitive alpha-amylase starts at a position in the range
of positions 96-116 and ending at a position in the range of
positions 199-219, e.g., starting at a position in the range of
positions 100-112 and ending at a position in the range of
positions 203-215 or starting at a position in the range of
positions 103-109 and ending at a position in the range of
positions 206-212. The A and C-domains of the Bacillus
alpha-amylase were determined to be amino acid residues 1-105
(A1)+213-398 (A2) and 399-485, respectively. The alpha-amylases of
the present invention may comprise an A1-domain starting at a
position in the range of positions 1-5 and ending a position in the
range of positions 95-115, e.g., starting at a position in the
range of positions 1-3 and ending at a position in the range of
positions 100-105 or starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
105-110. The alpha-amylases of the present invention may comprise
A2 and C-domains starting at a position in the range of positions
203-223 and ending at a position in the range of positions 482-485,
e.g., starting at a position in the range of positions 208-213 and
ending at a position in the range of positions 482-485 or starting
at a position in the range of positions 213-218 and ending at a
position in the range of positions 482-485.
[0076] For the SP707 alpha-amylase (SEQ ID NO: 8) where the
B-domain has been determined as amino acid residues 106-212, the
sequence to be replaced by the corresponding sequence of a
calcium-insensitive alpha-amylase starts at a position in the range
of positions 96-116 and ending at a position in the range of
positions 199-219, e.g., starting at a position in the range of
positions 100-112 and ending at a position in the range of
positions 203-215 or starting at a position in the range of
positions 103-109 and ending at a position in the range of
positions 206-212. The A and C-domains of the SP707 alpha-amylase
were determined to be amino acid residues 1-105 (A1)+213-398 (A2)
and 399-485, respectively. The alpha-amylases of the present
invention may comprise an A1-domain starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 95-115, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
100-105 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 105-110. The
alpha-amylases of the present invention may comprise A2 and
C-domains starting at a position in the range of positions 203-223
and ending at a position in the range of positions 482-485, e.g.,
starting at a position in the range of positions 208-213 and ending
at a position in the range of positions 482-485 or starting at a
position in the range of positions 213-218 and ending at a position
in the range of positions 482-485.
[0077] For the TS-22 alpha-amylase (SEQ ID NO: 9) where the
B-domain has been determined as amino acid residues 106-212, the
sequence to be replaced by the corresponding sequence of a
calcium-insensitive alpha-amylase starts at a position in the range
of positions 96-116 and ending at a position in the range of
positions 199-219, e.g., starting at a position in the range of
positions 100-112 and ending at a position in the range of
positions 203-215 or starting at a position in the range of
positions 103-109 and ending at a position in the range of
positions 206-212. The A and C-domains of the TS-22 alpha-amylase
were determined to be amino acid residues 1-105 (A1)+213-398 (A2)
and 399-484, respectively. The alpha-amylases of the present
invention may comprise an A1-domain starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 95-115, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
100-105 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 105-110. The
alpha-amylases of the present invention may comprise A2 and
C-domains starting at a position in the range of positions 203-223
and ending at a position in the range of positions 481-484, e.g.,
starting at a position in the range of positions 208-213 and ending
at a position in the range of positions 482-484 or starting at a
position in the range of positions 213-218 and ending at a position
in the range of positions 482-484. The TS-22 alpha-amylase further
has a carbohydrate binding domain of amino acid residues 485-586.
The carbohydrate binding domain is not required for the amylase
activity and might be fully or partially deleted.
[0078] For the TS-23 alpha-amylase (SEQ ID NO: 10) where the
B-domain has been determined as amino acid residues 106-212, the
sequence to be replaced by the corresponding sequence of a
calcium-insensitive alpha-amylase starts at a position in the range
of positions 96-116 and ending at a position in the range of
positions 199-219, e.g., starting at a position in the range of
positions 100-112 and ending at a position in the range of
positions 203-215 or starting at a position in the range of
positions 103-109 and ending at a position in the range of
positions 206-212. The A and C-domains of the TS-23 alpha-amylase
were determined to be amino acid residues 1-105 (A1)+213-398 (A2)
and 399-484, respectively. The alpha-amylases of the present
invention may comprise an A1-domain starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 95-115, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
100-105 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 105-110. The
alpha-amylases of the present invention may comprise A2 and
C-domains starting at a position in the range of positions 203-223
and ending at a position in the range of positions 482-484, e.g.,
starting at a position in the range of positions 208-213 and ending
at a position in the range of positions 482-484 or starting at a
position in the range of positions 213-218 and ending at a position
in the range of positions 482-484. The TS-23 alpha-amylase further
has a carbohydrate binding domain of amino acid residues 485-583.
The carbohydrate binding domain is not required for the amylase
activity and might be fully or partially deleted.
[0079] For the KSM-AP1378 alpha-amylase (SEQ ID NO: 11) where the
B-domain has been determined as amino acid residues 106-212, the
sequence to be replaced by the corresponding sequence of a
calcium-insensitive alpha-amylase starts at a position in the range
of positions 96-116 and ending at a position in the range of
positions 199-219, e.g., starting at a position in the range of
positions 100-112 and ending at a position in the range of
positions 203-215 or starting at a position in the range of
positions 103-109 and ending at a position in the range of
positions 206-212. The A and C-domains of the KSM-AP1378
alpha-amylase were determined to be amino acid residues 1-105
(A1)+213-398 (A2) and 399-485, respectively. The alpha-amylases of
the present invention may comprise an A1-domain starting at a
position in the range of positions 1-5 and ending a position in the
range of positions 95-115, e.g., starting at a position in the
range of positions 1-3 and ending at a position in the range of
positions 100-105 or starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
105-110. The alpha-amylases of the present invention may comprise
A2 and C-domains starting at a position in the range of positions
203-223 and ending at a position in the range of positions 482-485,
e.g., starting at a position in the range of positions 208-213 and
ending at a position in the range of positions 482-485 or starting
at a position in the range of positions 213-218 and ending at a
position in the range of positions 482-485.
[0080] For the Bacillus SP7-7 alpha-amylase (SEQ ID NO: 12) where
the B-domain has been determined as amino acid residues 106-212,
the sequence to be replaced by the corresponding sequence of a
calcium-insensitive alpha-amylase starts at a position in the range
of positions 96-116 and ending at a position in the range of
positions 199-219, e.g., starting at a position in the range of
positions 100-112 and ending at a position in the range of
positions 203-215 or starting at a position in the range of
positions 103-109 and ending at a position in the range of
positions 206-212. The A and C-domains of the Bacillus SP7-7
alpha-amylase were determined to be amino acid residues 1-105
(A1)+213-398 (A2) and 399-485, respectively. The alpha-amylases of
the present invention may comprise an A1-domain starting at a
position in the range of positions 1-5 and ending a position in the
range of positions 95-115, e.g., starting at a position in the
range of positions 1-3 and ending at a position in the range of
positions 100-105 or starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
105-110. The alpha-amylases of the present invention may comprise
A2 and C-domains starting at a position in the range of positions
203-223 and ending at a position in the range of positions 482-485,
e.g., starting at a position in the range of positions 208-213 and
ending at a position in the range of positions 482-485 or starting
at a position in the range of positions 213-218 and ending at a
position in the range of positions 482-485.
[0081] For the B. stearothermophilus alpha-amylase (SEQ ID NO: 29)
where the B-domain has been determined as amino acid residues
105-210, the sequence to be replaced by the corresponding sequence
of a calcium-insensitive alpha-amylase starts at a position in the
range of positions 95-115 and ending at a position in the range of
positions 197-213, e.g., starting at a position in the range of
positions 99-111 and ending at a position in the range of positions
201-213 or starting at a position in the range of positions 102-108
and ending at a position in the range of positions 204-210. The A
and C-domains of the B. stearothermophilus alpha-amylase were
determined to be amino acid residues 1-104 (A1)+211-396 (A2) and
397-483, respectively. The alpha-amylases of the present invention
may comprise an A1-domain starting at a position in the range of
positions 1-5 and ending a position in the range of positions
94-114, e.g., starting at a position in the range of positions 1-3
and ending at a position in the range of positions 99-104 or
starting at a position in the range of positions 1-3 and ending at
a position in the range of positions 104-109. The alpha-amylases of
the present invention may comprise A2 and C-domains starting at a
position in the range of positions 201-221 and ending at a position
in the range of positions 478-483, e.g., starting at a position in
the range of positions 206-211 and ending at a position in the
range of positions 480-483 or starting at a position in the range
of positions 211-216 and ending at a position in the range of
positions 480-483. The B. stearothermophilus alpha-amylase further
has a C-terminal extension of amino acid residues 484-486. The
C-terminal extension is not required for the amylase activity and
might be fully or partially deleted.
[0082] For the Cytophagus alpha-amylase (SEQ ID NO: 30) where the
B-domain has been determined as amino acid residues 103-208, the
sequence to be replaced by the corresponding sequence of a
calcium-insensitive alpha-amylase starts at a position in the range
of positions 93-113 and ending at a position in the range of
positions 195-215, e.g., starting at a position in the range of
positions 97-109 and ending at a position in the range of positions
199-211 or starting at a position in the range of positions 100-106
and ending at a position in the range of positions 202-208. The A
and C-domains of the Cytophagus alpha-amylase were determined to be
amino acid residues 1-102 (A1)+209-397 (A2) and 398-484,
respectively. The alpha-amylases of the present invention may
comprise an A1-domain starting at a position in the range of
positions 1-5 and ending a position in the range of positions
92-112, e.g., starting at a position in the range of positions 1-3
and ending at a position in the range of positions 97-102 or
starting at a position in the range of positions 1-3 and ending at
a position in the range of positions 102-107. The alpha-amylases of
the present invention may comprise A2 and C-domains starting at a
position in the range of positions 199-219 and ending at a position
in the range of positions 479-484, e.g., starting at a position in
the range of positions 204-209 and ending at a position in the
range of positions 481-484 or starting at a position in the range
of positions 209-214 and ending at a position in the range of
positions 481-484.
[0083] For the Bacillus circulans alpha-amylase (SEQ ID NO: 13),
the B-domain has been determined as amino acid residues 103-208.
The alpha-amylases of the present invention may comprise a B-domain
starting at a position in the range of positions 93-113 and ending
at a position in the range of positions 195-215, e.g., starting at
a position in the range of positions 97-109 and ending at a
position in the range of positions 199-211 or starting at a
position in the range of positions 100-106 and ending at a position
in the range of positions 202-208. The A and C-domains of the
Bacillus circulans alpha-amylase were determined to be amino acid
residues 1-102 (A1)+209-395 (A2) and 396-482, respectively. The
A1-domain which can be replaced by the corresponding sequence of a
calcium-sensitive alpha-amylase starts at a position in the range
of positions 1-5 and ending at a position in the range of positions
92-112, e.g., starting at a position in the range of positions 1-3
and ending at a position in the range of positions 97-102 or
starting at a position in the range of positions 1-3 and ending at
a position in the range of positions 102-107. The A2-domain which
can be replaced by the corresponding sequence of a
calcium-sensitive alpha-amylase starts at a position in the range
of positions 199-219 and ending at a position in the range of
positions 385-405, e.g., starting at a position in the range of
positions 204-209 and ending at a position in the range of
positions 390-395 or starting at a position in the range of
positions 209-214 and ending at a position in the range of
positions 395-400. The A1 and A2 domains are preferably replaced
simultaneously by the corresponding sequence of a calcium-sensitive
alpha-amylase. The C-domain which can be replaced by the
corresponding sequence of a calcium-sensitive alpha-amylase starts
at a position in the range of positions 386-406 and ending at a
position in the range of positions 477-482, e.g., starting at a
position in the range of positions 391-396 and ending at a position
in the range of positions 479-482 or starting at a position in the
range of positions 396-401 and ending at a position in the range of
positions 479-482. The Bacillus circulans alpha-amylase further has
a C-terminal extension of amino acid residues 483-492. The
extension is not required for the amylase activity and might be
fully or partially deleted.
[0084] For the KSM-K36 alpha-amylase (SEQ ID NO: 14), the B-domain
has been determined as amino acid residues 104-207. The
alpha-amylases of the present invention may comprise a B-domain
starting at a position in the range of positions 93-113 and ending
at a position in the range of positions 195-215, e.g., starting at
a position in the range of positions 97-109 and ending at a
position in the range of positions 199-211 or starting at a
position in the range of positions 100-106 and ending at a position
in the range of positions 202-208. The A and C-domains of the
Bacillus circulans alpha-amylase were determined to be amino acid
residues 1-103 (A1)+208-393 (A2) and 394-480, respectively. The
A1-domain which can be replaced by the corresponding sequence of a
calcium-sensitive alpha-amylase starts at a position in the range
of positions 1-5 and ending at a position in the range of positions
93-113, e.g., starting at a position in the range of positions 1-3
and ending at a position in the range of positions 98-103 or
starting at a position in the range of positions 1-3 and ending at
a position in the range of positions 103-108. The A2-domain which
can be replaced by the corresponding sequence of a
calcium-sensitive alpha-amylase starts at a position in the range
of positions 198-218 and ending at a position in the range of
positions 383-403, e.g., starting at a position in the range of
positions 203-208 and ending at a position in the range of
positions 388-393 or starting at a position in the range of
positions 208-213 and ending at a position in the range of
positions 393-398. The A1 and A2 domains are preferably replaced
simultaneously by the corresponding sequence of a calcium-sensitive
alpha-amylase. The C-domain which can be replaced by the
corresponding sequence of a calcium-sensitive alpha-amylase starts
at a position in the range of positions 384-404 and ending at a
position in the range of positions 475-480, e.g., starting at a
position in the range of positions 389-394 and ending at a position
in the range of positions 477-480 or starting at a position in the
range of positions 394-399 and ending at a position in the range of
positions 477-480.
[0085] For the KSM-K38 alpha-amylase (SEQ ID NO: 15), the B-domain
has been determined as amino acid residues 104-207. The
alpha-amylases of the present invention may comprise a B-domain
starting at a position in the range of positions 93-113 and ending
at a position in the range of positions 195-215, e.g., starting at
a position in the range of positions 97-109 and ending at a
position in the range of positions 199-211 or starting at a
position in the range of positions 100-106 and ending at a position
in the range of positions 202-208. The A and C-domains of the
Bacillus circulans alpha-amylase were determined to be amino acid
residues 1-103 (A1)+208-393 (A2) and 394-480, respectively. The
A1-domain which can be replaced by the corresponding sequence of a
calcium-sensitive alpha-amylase starts at a position in the range
of positions 1-5 and ending at a position in the range of positions
93-113, e.g., starting at a position in the range of positions 1-3
and ending at a position in the range of positions 98-103 or
starting at a position in the range of positions 1-3 and ending at
a position in the range of positions 103-108. The A2-domain which
can be replaced by the corresponding sequence of a
calcium-sensitive alpha-amylase starts at a position in the range
of positions 198-218 and ending at a position in the range of
positions 383-403, e.g., starting at a position in the range of
positions 203-208 and ending at a position in the range of
positions 388-393 or starting at a position in the range of
positions 208-213 and ending at a position in the range of
positions 393-398. The A1 and A2 domains are preferably replaced
simultaneously by the corresponding sequence of a calcium-sensitive
alpha-amylase. The C-domain which can be replaced by the
corresponding sequence of a calcium-sensitive alpha-amylase starts
at a position in the range of positions 384-404 and ending at a
position in the range of positions 475-480, e.g., starting at a
position in the range of positions 389-394 and ending at a position
in the range of positions 477-480 or starting at a position in the
range of positions 394-399 and ending at a position in the range of
positions 477-480.
[0086] For the Pyrococcus woesei alpha-amylase (SEQ ID NO: 16), the
B-domain has been determined as amino acid residues 110-171. The
alpha-amylases of the present invention may comprise a B-domain
starting at a position in the range of positions 100-120 and ending
at a position in the range of positions 161-181, e.g., starting at
a position in the range of positions 105-115 and ending at a
position in the range of positions 166-171 or starting at a
position in the range of positions 107-113 and ending at a position
in the range of positions 171-176. The A and C-domains of the
Bacillus circulans alpha-amylase were determined to be amino acid
residues 1-109 (A1)+172-338 (A2) and 339-435, respectively. The
A1-domain which can be replaced by the corresponding sequence of a
calcium-sensitive alpha-amylase starts at a position in the range
of positions 1-5 and ending at a position in the range of positions
99-119, e.g., starting at a position in the range of positions 1-3
and ending at a position in the range of positions 104-109 or
starting at a position in the range of positions 1-3 and ending at
a position in the range of positions 109-114. The A2-domain which
can be replaced by the corresponding sequence of a
calcium-sensitive alpha-amylase starts at a position in the range
of positions 161-181 and ending at a position in the range of
positions 328-348, e.g., starting at a position in the range of
positions 167-172 and ending at a position in the range of
positions 333-338 or starting at a position in the range of
positions 172-177 and ending at a position in the range of
positions 338-343. The A1 and A2 domains are preferably replaced
simultaneously by the corresponding sequence of a calcium-sensitive
alpha-amylase. The C-domain which can be replaced by the
corresponding sequence of a calcium-sensitive alpha-amylase starts
at a position in the range of positions 329-349 and ending at a
position in the range of positions 430-435, e.g., starting at a
position in the range of positions 324-329 and ending at a position
in the range of positions 432-435 or starting at a position in the
range of positions 329-344 and ending at a position in the range of
positions 432-435.
[0087] For the Pyrococcus hybrid alpha-amylase (SEQ ID NO: 31), the
B-domain has been determined as amino acid residues 110-171. The
alpha-amylases of the present invention may comprise a B-domain
starting at a position in the range of positions 100-120 and ending
at a position in the range of positions 161-181, e.g., starting at
a position in the range of positions 105-115 and ending at a
position in the range of positions 166-171 or starting at a
position in the range of positions 107-113 and ending at a position
in the range of positions 171-176. The A and C-domains of the
Bacillus circulans alpha-amylase were determined to be amino acid
residues 1-109 (A1)+172-338 (A2) and 339-435, respectively. The
A1-domain which can be replaced by the corresponding sequence of a
calcium-sensitive alpha-amylase starts at a position in the range
of positions 1-5 and ending at a position in the range of positions
99-119, e.g., starting at a position in the range of positions 1-3
and ending at a position in the range of positions 104-109 or
starting at a position in the range of positions 1-3 and ending at
a position in the range of positions 109-114. The A2-domain which
can be replaced by the corresponding sequence of a
calcium-sensitive alpha-amylase starts at a position in the range
of positions 161-181 and ending at a position in the range of
positions 328-348, e.g., starting at a position in the range of
positions 167-172 and ending at a position in the range of
positions 333-338 or starting at a position in the range of
positions 172-177 and ending at a position in the range of
positions 338-343. The A1 and A2 domains are preferably replaced
simultaneously by the corresponding sequence of a calcium-sensitive
alpha-amylase. The C-domain which can be replaced by the
corresponding sequence of a calcium-sensitive alpha-amylase starts
at a position in the range of positions 329-349 and ending at a
position in the range of positions 430-435, e.g., starting at a
position in the range of positions 334-339 and ending at a position
in the range of positions 432-435 or starting at a position in the
range of positions 339-344 and ending at a position in the range of
positions 432-435.
Variants
[0088] The variants of the present invention have alpha-amylase
activity and comprise A-, B-, and C-domains and, optionally, a
carbohydrate-binding module.
[0089] A-Domain of the Variants
[0090] In an embodiment, the variants of the present invention
comprise an A-domain which has at least 70% and less than 100%
sequence identity with any of the A-domains of SEQ ID NOS:
1-12.
[0091] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 1.
[0092] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 2.
[0093] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 3.
[0094] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 4.
[0095] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 5.
[0096] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 6.
[0097] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 7.
[0098] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 8.
[0099] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 9.
[0100] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 10.
[0101] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 11.
[0102] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 12.
[0103] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 29.
[0104] In an embodiment, the A-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 30.
[0105] The amino acid sequence of the A-domain has at least one
difference, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, at a position
corresponding to position 8, 52, 53, 59, 91, 220, 224, 242, 244,
245, 282, 284, 301, 303, 304, 307, 348, 386, or 388.
[0106] In an embodiment, the A-domain has 1-15 differences compared
with any of the A-domains of SEQ ID NOS: 1-12, i.e., 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 differences.
[0107] In an embodiment, a difference may be at the position
corresponding to position 8, i.e., the amino acid is Ala, Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr,
Trp, Tyr, or Val, e.g., Leu.
[0108] In an embodiment, a difference may be at the position
corresponding to position 27, i.e., the amino acid is Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Gln.
[0109] In an embodiment, a difference may be at the position
corresponding to position 34, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Thr, Trp, Tyr, or Val, e.g., Lys.
[0110] In an embodiment, a difference may be at the position
corresponding to position 52, i.e., the amino acid is Ala, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Gly.
[0111] In an embodiment, a difference may be at the position
corresponding to position 53, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Thr, Trp, Tyr, or Val, e.g., Tyr.
[0112] In an embodiment, a difference may be at the position
corresponding to position 59, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Thr, Trp, or Tyr, e.g., Ala.
[0113] In an embodiment, a difference may be at the position
corresponding to position 80, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Trp, Tyr, or Val, e.g., Asp.
[0114] In an embodiment, a difference may be at the position
corresponding to position 86, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Ser.
[0115] In an embodiment, a difference may be at the position
corresponding to position 90, i.e., the amino acid is Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Ser.
[0116] In an embodiment, a difference may be at the position
corresponding to position 91, i.e., the amino acid is Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Leu.
[0117] In an embodiment, a difference may be at the position
corresponding to position 100, i.e., the amino acid is Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Glu or Leu.
[0118] In an embodiment, a difference may be at the position
corresponding to position 204, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Leu.
[0119] In an embodiment, a difference may be at the position
corresponding to position 206, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Thr, Trp, or Val, e.g., Met.
[0120] In an embodiment, a difference may be at the position
corresponding to position 220, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Pro.
[0121] In an embodiment, a difference may be at the position
corresponding to position 224, i.e., the amino acid is Ala, Arg,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Leu.
[0122] In an embodiment, a difference may be at the position
corresponding to position 235, i.e., the amino acid is Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Thr or Trp.
[0123] In an embodiment, a difference may be at the position
corresponding to position 242, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Thr, Trp, Tyr, or Val, e.g., Gln or Glu.
[0124] In an embodiment, a difference may be at the position
corresponding to position 244, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Tyr.
[0125] In an embodiment, a difference may be at the position
corresponding to position 245, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser,
Thr, Trp, Tyr, or Val, e.g., Ala or Lys.
[0126] In an embodiment, a difference may be at the position
corresponding to position 270, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Leu.
[0127] In an embodiment, a difference may be at the position
corresponding to position 282, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Trp.
[0128] In an embodiment, a difference may be at the position
corresponding to position 284, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Gln, Thr, or Val.
[0129] In an embodiment, a difference may be at the position
corresponding to position 301, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Thr, Trp, Tyr, or Val, e.g., Lys.
[0130] In an embodiment, a difference may be at the position
corresponding to position 303, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Arg.
[0131] In an embodiment, a difference may be at the position
corresponding to position 304, i.e., the amino acid is Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Asp.
[0132] In an embodiment, a difference may be at the position
corresponding to position 307, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Leu.
[0133] In an embodiment, a difference may be at the position
corresponding to position 348, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Thr, Trp, Tyr, or Val, e.g., Thr.
[0134] In an embodiment, a difference may be at the position
corresponding to position 386, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser,
Thr, Trp, Tyr, or Val, e.g., Gln, Glu, Thr, or Val.
[0135] In an embodiment, a difference may be at the position
corresponding to position 388, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Ile or Val.
[0136] In an embodiment, the A-domain of the variant is different
from the A-domain of SEQ ID NOS: 1-12, 29, or 30 at the positions
corresponding to positions 52 and 53. For example, the amino acids
at the positions corresponding to positions 52 and 53 are Gly and
Tyr, respectively.
[0137] In an embodiment, the A-domain of the variant is different
from the A-domain of SEQ ID NOS: 1-12, 29, or 30 at the positions
corresponding to positions 59, 242, and 307. For example, the amino
acids at the positions corresponding to positions 59, 242, and 307
are Ala; Gln or Glu; and Leu, respectively.
[0138] In an embodiment, the A-domain of the variant is different
from the A-domain of SEQ ID NOS: 1-12, 29, or 30 at the positions
corresponding to positions 220 and 224. For example, the amino
acids at the positions corresponding to positions 220 and 224 are
Pro and Leu, respectively.
[0139] In an embodiment, the A-domain of the variant is different
from the A-domain of SEQ ID NOS: 1-12, 29, or 30 at the positions
corresponding to positions 242 and 282. For example, the amino
acids at the positions corresponding to positions 242 and 282 are
Gln or Glu; and Trp, respectively.
[0140] In an embodiment, the A-domain of the variant is different
from the A-domain of SEQ ID NOS: 1-12, 29, or 30 at the positions
corresponding to positions 242 and 284. For example, the amino
acids at the positions corresponding to positions 242 and 284 are
Gln or Glu; and Gln, Thr, or Val, respectively.
[0141] In an embodiment, the A-domain of the variant is different
from the A-domain of SEQ ID NOS: 1-12, 29, or 30 at the positions
corresponding to positions 242, 284, and 307. For example, the
amino acids at the positions corresponding to positions 242, 284,
and 307 are Gln or Glu; Gln, Thr, or Val; and Leu,
respectively.
[0142] In an embodiment, the A-domain of the variant is different
from the A-domain of SEQ ID NOS: 1-12, 29, or 30 at the positions
corresponding to positions 242 and 307. For example, the amino
acids at the positions corresponding to positions 242 and 307 are
Gln or Glu; and Leu, respectively.
[0143] In an embodiment, the A-domain of the variant is different
from the A-domain of SEQ ID NOS: 1-12, 29, or 30 at the positions
corresponding to positions 244 and 284. For example, the amino
acids at the positions corresponding to positions 244 and 284 are
Tyr; and Gln, Thr, or Val, respectively.
[0144] In an embodiment, the A-domain of the variant is different
from the A-domain of SEQ ID NOS: 1-12, 29, or 30 at the positions
corresponding to positions 244, 284, and 307. For example, the
amino acids at the positions corresponding to positions 244, 284,
and 307 are Tyr; Gln, Thr, or Val; and Leu, respectively.
[0145] In an embodiment, the A-domain of the variant is different
from the A-domain of SEQ ID NOS: 1-12, 29, or 30 at the positions
corresponding to positions 244 and 307. For example, the amino
acids at the positions corresponding to positions 244 and 307 are
Tyr and Leu, respectively.
[0146] In an embodiment, the A-domain of the variant is different
from the A-domain of SEQ ID NOS: 1-12, 29, or 30 at the positions
corresponding to positions 284 and 307. For example, the amino
acids at the positions corresponding to positions 284 and 307 are
Gln, Thr, or Val; and Leu, respectively.
[0147] In an embodiment, the A-domain of the variant is different
from the A-domain of SEQ ID NOS: 1-12, 29, or 30 at the positions
corresponding to positions 301, 303, and 304. For example, the
amino acids at the positions corresponding to positions 301, 303,
and 304 are Lys, Arg, and Asp, respectively.
[0148] B-Domain of the Variants
[0149] The variants of the present invention comprise a B-domain of
a calcium-insenstive alpha-amylase.
[0150] In an embodiment, the B-domain has at least 60% sequence
identity, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, 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%, at least 99%, or 100% sequence identity with the
B-domain of SEQ ID NO: 13.
[0151] In an embodiment, the B-domain has at least 60% sequence
identity, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, 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%, at least 99%, or 100% sequence identity with the
B-domain of SEQ ID NO: 14.
[0152] In an embodiment, the B-domain has at least 60% sequence
identity, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, 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%, at least 99%, or 100% sequence identity with the
B-domain of SEQ ID NO: 15.
[0153] In an embodiment, the B-domain has at least 60% sequence
identity, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, 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%, at least 99%, or 100% sequence identity with the
B-domain of SEQ ID NO: 16.
[0154] In an embodiment, the B-domain has at least 55% and less
than 100% sequence identity with the B-domain of SEQ ID NO: 13. For
example, the B-domain may have at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, 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 at least 99% sequence
identity with the B-domain of SEQ ID NO: 13.
[0155] In an embodiment, the B-domain has at least 55% and less
than 100% sequence identity with the B-domain of SEQ ID NO: 13. For
example, the B-domain may have at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, 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 at least 99% sequence
identity with the B-domain of SEQ ID NO: 31.
[0156] The amino acid sequence of the B-domain has at least one
difference, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, at a position
corresponding to position 105, 115, 117, 129, 132, 134, 135, 150,
157, 159, 160, 164, 166, 168, 169, 170, 171, 172, 174, 176, 177,
179, 180, 181, 182, 184, 187, 188, 191, 206, 208, or 210.
[0157] In an embodiment, the B-domain has 1-15 differences compared
with the B-domain of SEQ ID NO: 13, i.e., 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14 or 15 differences.
[0158] In an embodiment, the amino acid sequence of the B-domain
has at least one difference, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10, at a position corresponding to position 105, 115, 117, 129,
132, 134, 135, 150, 157, 159, 160, 164, 166, 168, 169, 170, 171,
172, 174, 176, 177, 184, 187, 188, 191, 206, 208, or 210, and the
amino acids at at least two positions corresponding to positions
179-182 are absent.
[0159] In an embodiment, a difference may be at the position
corresponding to position 105, i.e., the amino acid is Ala, Arg,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Asp.
[0160] In an embodiment, a difference may be at the position
corresponding to position 115, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Thr, Trp, or Tyr, e.g., Trp.
[0161] In an embodiment, a difference may be at the position
corresponding to position 117, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Asp.
[0162] In an embodiment, a difference may be at the position
corresponding to position 129, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Val.
[0163] In an embodiment, a difference may be at the position
corresponding to position 132, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Asp.
[0164] In an embodiment, a difference may be at the position
corresponding to position 134, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Glu or Tyr.
[0165] In an embodiment, a difference may be at the position
corresponding to position 135, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Asn or Gln.
[0166] In an embodiment, a difference may be at the position
corresponding to position 150, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Thr.
[0167] In an embodiment, a difference may be at the position
corresponding to position 157, i.e., the amino acid is Ala, Arg,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Tyr.
[0168] In an embodiment, a difference may be at the position
corresponding to position 159, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Tyr.
[0169] In an embodiment, a difference may be at the position
corresponding to position 160, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Tyr.
[0170] In an embodiment, a difference may be at the position
corresponding to position 164, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Trp, Tyr, or Val, e.g., Val.
[0171] In an embodiment, a difference may be at the position
corresponding to position 166, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Trp.
[0172] In an embodiment, a difference may be at the position
corresponding to position 168, i.e., the amino acid is Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Glu.
[0173] In an embodiment, a difference may be at the position
corresponding to position 169, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Ser.
[0174] In an embodiment, a difference may be at the position
corresponding to position 170, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Arg.
[0175] In an embodiment, a difference may be at the position
corresponding to position 171, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Lys.
[0176] In an embodiment, a difference may be at the position
corresponding to position 172, i.e., the amino acid is Ala, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Leu.
[0177] In an embodiment, a difference may be at the position
corresponding to position 174, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Arg.
[0178] In an embodiment, a difference may be at the position
corresponding to position 176, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Tyr.
[0179] In an embodiment, a difference may be at the position
corresponding to position 177, i.e., the amino acid is Ala, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Leu.
[0180] In an embodiment, a difference may be at the position
corresponding to position 179, i.e., the amino acid is Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Asn, Asp, Gln, or Glu, or is
absent.
[0181] In an embodiment, a difference may be at the position
corresponding to position 180, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Ala, Pro, or Ser, or is absent.
[0182] In an embodiment, a difference may be at the position
corresponding to position 181, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Ala, Asp, Cys, Leu, or Pro, or is
absent.
[0183] In an embodiment, a difference may be at the position
corresponding to position 182, i.e., the amino acid is Ala, Arg,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Ala, Pro, or Ser, or is absent.
[0184] In an embodiment, a difference may be at the position
corresponding to position 184, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Ala.
[0185] In an embodiment, a difference may be at the position
corresponding to position 187, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Trp.
[0186] In an embodiment, a difference may be at the position
corresponding to position 188, i.e., the amino acid is Ala, Arg,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Glu.
[0187] In an embodiment, a difference may be at the position
corresponding to position 191, i.e., the amino acid is Ala, Arg,
Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Thr.
[0188] In an embodiment, a difference may be at the position
corresponding to position 202, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Leu.
[0189] In an embodiment, a difference may be at the position
corresponding to position 204, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Thr, Trp, or Val, e.g., Lys or Met.
[0190] In an embodiment, a difference may be at the position
corresponding to position 206, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Thr, Trp, or Val, e.g., Lys or Met.
[0191] In an embodiment, a difference may be at the position
corresponding to position 208, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Tyr.
[0192] In an embodiment, a difference may be at the position
corresponding to position 210, i.e., the amino acid is Ala, Arg,
Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Tyr or Val.
[0193] In an embodiment, a difference may be at the position
corresponding to position 222, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Thr, Trp, or Val, e.g., Val.
[0194] In an embodiment, the amino acids at the positions
corresponding to positions 179 and 180 are absent.
[0195] In an embodiment, the amino acids at the positions
corresponding to positions 179 and 181 are absent.
[0196] In an embodiment, the amino acids at the positions
corresponding to positions 179 and 182 are absent.
[0197] In an embodiment, the amino acids at the positions
corresponding to positions 180 and 181 are absent.
[0198] In an embodiment, the amino acids at the positions
corresponding to positions 180 and 182 are absent.
[0199] In an embodiment, the amino acids at the positions
corresponding to positions 181 and 182 are absent.
[0200] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 117, 150, and 184. For example, the amino acids
at the positions corresponding to positions 105, 117, 150, and 184
are Asp, Asp, Thr, and Ala, respectively.
[0201] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 129, 177, and 179. For example, the amino acids
at the positions corresponding to positions 105, 129, 177, and 179
are Asp; Val; Leu; and Asn, Asp, Gln, or Glu, or is absent; and
Ala, respectively.
[0202] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 132, and 184. For example, the amino acids at the
positions corresponding to positions 105, 132, and 184 are Asp,
Asp, and Ala, respectively.
[0203] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 134, and 184. For example, the amino acids at the
positions corresponding to positions 105, 134, and 184 are Asp,
Glu, and Ala, respectively.
[0204] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 135, 179, and 184. For example, the amino acids
at the positions corresponding to positions 105, 135, 179, and 184
are Asp; Asn; Asn, Asp, Gln, or Glu, or is absent; and Ala,
respectively.
[0205] In an embodiment, the amino acid sequence of the B-domain of
the variant is different from the amino acid sequence of SEQ ID NO:
13 at the positions corresponding to positions 105 and 150. For
example, the amino acids at the positions corresponding to
positions 105 and 150 are Asp and Thr, respectively.
[0206] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 150, 164, 166, 168, 171, and 184. For example,
the amino acids at the positions corresponding to positions 105,
150, 164, 166, 168, 171, and 184 are Asp, Thr, Val, Trp, Glu, Lys,
and Ala, respectively.
[0207] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 150, 164, and 184. For example, the amino acids
at the positions corresponding to positions 105, 150, 164, and 184
are Asp, Thr, Val, and Ala, respectively.
[0208] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 150, 166, 168, and 171. For example, the amino
acids at the positions corresponding to positions 105, 150, 166,
168, and 171 are Asp, Thr, Trp, Glu, and Lys, respectively.
[0209] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 150, 166, 168, 171, and 184. For example, the
amino acids at the positions corresponding to positions 105, 150,
166, 168, 171, and 184 are Asp, Thr, Trp, Glu, Lys, and Ala,
respectively.
[0210] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 150, and 184. For example, the amino acids at the
positions corresponding to positions 105, 150, and 184 are Asp,
Thr, and Ala, respectively.
[0211] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 150, 184, and 206. For example, the amino acids
at the positions corresponding to positions 105, 150, 184, and 206
are Asp, Thr, Ala, and Met, respectively.
[0212] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 157, 159, 160, 184, 208, and 210. For example,
the amino acids at the positions corresponding to positions 105,
157, 159, 160, 184, 208, and 210 are Asp; Tyr; Tyr; Tyr; Ala; Tyr;
and Tyr or Val, respectively.
[0213] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 160, and 184. For example, the amino acids at the
positions corresponding to positions 105, 160, and 184 are Asp,
Tyr, and Ala, respectively.
[0214] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 164, and 184. For example, the amino acids at the
positions corresponding to positions 105, 164, and 184 are Asp,
Val, and Ala, respectively.
[0215] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 166, 168, and 171. For example, the amino acids
at the positions corresponding to positions 105, 166, 168, and 171
are Asp, Trp, Glu, and Lys, respectively.
[0216] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 179, and 184. For example, the amino acids at the
positions corresponding to positions 105, 179, and 184 are Asp;
Asn, Asp, Gln, or Glu, or is absent; and Ala, respectively.
[0217] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105 and 184. For example, the amino acids at the
positions corresponding to positions 105 and 184 are Asp and Ala,
respectively.
[0218] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105, 184, and 210. For example, the amino acids at the
positions corresponding to positions 105, 184, and 210 are Asp;
Ala; and Tyr or Val, respectively.
[0219] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 105 and 206. For example, the amino acids at the
positions corresponding to positions 105 and 206 are Asp and Met,
respectively.
[0220] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 166, 168, and 171. For example, the amino acids at the
positions corresponding to positions 166, 168, and 171 are Trp,
Glu, and Lys, respectively.
[0221] In an embodiment, the B-domain of the variant is different
from the B-domain of SEQ ID NO: 13 at the positions corresponding
to positions 187, 188, and 191. For example, the amino acids at the
positions corresponding to positions 187, 188, and 191 are Trp,
Glu, and Thr, respectively.
[0222] The alpha-amylases of the invention may further comprise
additional substitutions, insertions or deletions in the B-domain
derived from the calcium-insensitive alpha-amylase. Examples of
suitable substitutions, insertions or deletions in the B-domain of
a calcium-insensitive alpha-amylase are the alterations
corresponding to the following alterations in B. circulans
alpha-amylase: E179*, N180*, E185W, N186E and D189T (SEQ ID NO: 13
numbering), which correspond to E181*, N182*, E187W, N188E and
D191T in SEQ ID NO: 27 numbering.
[0223] In another embodiment, the alpha-amylases of the present
invention comprise the substitution Q150T.
[0224] In another embodiment, the alpha-amylases of the present
invention comprise the substitution T164V.
[0225] In another embodiment, the alpha-amylases of the present
invention comprise the substitution K184A.
[0226] C-Domain of the Variants
[0227] In an embodiment, the variants of the present invention
comprise a C-domain which has at least 70% and less than 100%
sequence identity with any of the C-domains of SEQ ID NOS:
1-12.
[0228] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 1.
[0229] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 2.
[0230] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 3.
[0231] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 4.
[0232] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 5.
[0233] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 6.
[0234] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 7.
[0235] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 8.
[0236] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 9.
[0237] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 10.
[0238] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 11.
[0239] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 12.
[0240] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 29.
[0241] In an embodiment, the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 30.
[0242] In an embodiment, the C-domain has 1-15 differences compared
with any of the C-domains of SEQ ID NOS: 1-12, 29, and 30, i.e., 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 differences.
[0243] In an embodiment, a difference may be at the position
corresponding to position 407, i.e., the amino acid is Ala, Arg,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Asp.
[0244] In an embodiment, a difference may be at the position
corresponding to position 430, i.e., the amino acid is Ala, Arg,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Asp.
[0245] In an embodiment, a difference may be at the position
corresponding to position 432, i.e., the amino acid is Ala, Arg,
Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Pro.
[0246] In an embodiment, a difference may be at the position
corresponding to position 459, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Trp, Tyr, or Val, e.g., Pro.
[0247] In an embodiment, a difference may be at the position
corresponding to position 475, i.e., the amino acid is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,
Ser, Trp, Tyr, or Val, e.g., Gln or Lys.
[0248] In an embodiment, the C-domain of the variant is different
from any of the C-domains of SEQ ID NOS: 1-12 at the positions
corresponding to positions 407 and 430. For example, the amino
acids at the positions corresponding to positions 407 and 430 are
Asp.
[0249] In an embodiment, the C-domain of the variant is different
from any of the C-domains of SEQ ID NOS: 1-12 at the positions
corresponding to positions 407, 430, and 432. For example, the
amino acids at the positions corresponding to positions 407, 430,
and 432 are Asp, Asp, and Pro, respectively.
[0250] In an embodiment, the C-domain of the variant is different
from any of the C-domains of SEQ ID NOS: 1-12 at the positions
corresponding to positions 407 and 432. For example, the amino
acids at the positions corresponding to positions 407 and 432 are
Asp and Pro, respectively.
[0251] In an embodiment, the C-domain of the variant is different
from any of the C-domains of SEQ ID NOS: 1-12 at the positions
corresponding to positions 430 and 432. For example, the amino
acids at the positions corresponding to positions 430 and 432 are
Asp and Pro, respectively.
[0252] In an embodiment, the alpha-amylase variants have a ratio of
activity measured by the Phadebas activity to the activity measured
by the G7-pNG assay greater than 0.1, preferably of more than 0.25,
even more preferred more than 0.5 and most preferred more than
1.
[0253] The Phadebas assay is an assay for determining alpha-amylase
activity using a cross-linked insoluble blue-colored starch
polymer. (Phadebas.RTM. Amylase Test, supplied by Magle Life
Sciences, Lund, Sweden).
[0254] The G7-pNG assay is an assay for determining alpha-amylase
activity using a soluble chromogen compound,
p-nitrophenyl-alpha-D-maltoheptaoside. Kits containing PNP-G7
substrate and alpha-glucosidase is manufactured by
Boehringer-Mannheim (cat. no. 1054635).
[0255] In order to determine the amylase activity using the
Phadebas and the G7-pNG assays, a reference amylase with known
activity must be included in the assay and the activity is
determined relative to the known reference. For purposes of the
present invention, the reference alpha-amylase which is considered
to have the same activity when measured by the Phadebas and the
G7-pNG assays, is the Bacillus licheniformis alpha-amylase sold by
Novozymes NS under the tradename Termamyl.RTM., which has the
sequence of SEQ ID NO: 3. Thus, the reference alpha-amylase has a
ratio of 1 when measuring the activity by the Phadebas assay
relative to the activity measured by the G7-pNP assay.
[0256] The ratio of activity on insoluble substrate to activity on
soluble substrate is determined by measuring the activities on the
two particular selected substrates and calculating the ratio.
Preferably the ratio is at least 1.5 fold higher than for the
calcium-insensitive alpha-amylase, e.g., at least 2 fold higher, at
least 2.5 fold higher and at least 3 fold higher.
[0257] Using the methods disclosed below for determining the ratio
of activity by the Phadebas assay to the activity of the G7pNG
assay, the B. circulans alpha-amylase having the amino acid
sequence of SEQ ID NO: 13 was found to have a ratio of
approximately 0.014.
[0258] Additional Mutation(s)
[0259] The alpha-amylases of the invention may comprise one or more
substitutions, insertions and/or deletions known in the art to
improve the properties of alpha-amylases.
[0260] For example, oxidizable amino acid residues may be
substituted with a non-oxidizable amino acid residue in order to
improve the stability of the enzyme under oxidizing conditions,
e.g., in the presence of bleach, in accordance with the teachings
of WO 94/02597 and WO 94/18314, which are incorporated herein by
reference.
[0261] Further beneficial substitutions that may be introduced are
disclosed in WO 99/23211, WO 01/66712 and WO 2006/002643, which are
incorporated herein by reference.
[0262] The variants of the present invention preferably consist of
481 to 515, 481 to 493, or 481 to 486 amino acids.
Preparation of Variants
[0263] The present invention also relates to methods for obtaining
a variant having alpha-amylase activity, comprising: (a)
introducing into a parent alpha-amylase a substitution at one or
more (several) positions corresponding to positions 8, 52, 53, 59,
91, 220, 224, 242, 244, 245, 282, 284, 301, 303, 304, 307, 348,
386, 388, 407, 430, 432, and 459, wherein the variant has
alpha-amylase activity; and (b) recovering the variant.
[0264] The variants can be prepared according to any mutagenesis
procedure known in the art, such as site-directed mtagenesis,
synthetic gene construction, semi-synthetic gene construction,
random mutagenesis, shuffling, etc.
[0265] Site-directed mutagenesis is a technique in which one or
more (several) mutations are created at a defined site in a
polynucleotide molecule encoding the parent alpha-amylase. The
technique can be performed in vitro or in vivo.
[0266] Synthetic gene construction entails in vitro synthesis of a
designed polynucleotide molecule to encode a polypeptide molecule
of interest. Gene synthesis can be performed utilizing a number of
techniques, such as the multiplex microchip-based technology
described by Tian et al., 2004, Nature 432: 1050-1054, and similar
technologies wherein olgionucleotides are synthesized and assembled
upon photo-programable microfluidic chips.
[0267] Site-directed mutagenesis can be accomplished in vitro by
PCR involving the use of oligonucleotide primers containing the
desired mutation. Site-directed mutagenesis can also be performed
in vitro by cassette mutagenesis involving the cleavage by a
restriction enzyme at a site in the plasmid comprising a
polynucleotide encoding the parent alpha-amylase and subsequent
ligation of an oligonucleotide containing the mutation in the
polynucleotide. Usually the restriction enzyme that digests at the
plasmid and the oligonucleotide is the same, permitting sticky ends
of the plasmid and insert to ligate to one another. See, for
example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:
4949-4955; and Barton et al., 1990, Nucleic Acids Research 18:
7349-4966.
[0268] Site-directed mutagenesis can be accomplished in vivo by
methods known in the art. See, for example, U.S. Patent Application
Publication No. 2004/0171154; Storici et al., 2001, Nature
Biotechnology 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290;
and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:
15-16.
[0269] Any site-directed mutagenesis procedure can be used in the
present invention. There are many commercial kits available that
can be used to prepare variants of a parent alpha-amylase.
[0270] Single or multiple amino acid substitutions, deletions,
and/or insertions can be made and tested using known methods of
mutagenesis, recombination, and/or shuffling, followed by a
relevant screening procedure, such as those disclosed by
Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and
Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413;
or WO 95/22625. Other methods that can be used include error-prone
PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:
10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) and
region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145;
Ner et al., 1988, DNA 7: 127).
[0271] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells.
Mutagenized DNA molecules that encode active polypeptides can be
recovered from the host cells and rapidly sequenced using standard
methods in the art. These methods allow the rapid determination of
the importance of individual amino acid residues in a polypeptide
of interest.
[0272] Semi-synthetic gene construction is accomplished by
combining aspects of synthetic gene construction, and/or
site-directed mutagenesis, and/or random mutagenesis, and/or
shuffling. Semi-synthetic construction is typified by a process
utilizing polynucleotide fragments that are synthesized, in
combination with PCR techniques. Defined regions of genes may thus
be synthesized de novo, while other regions may be amplified using
site-specific mutagenic primers, while yet other regions may be
subjected to error-prone PCR or non-error prone PCR amplification.
Polynucleotide fragments may then be shuffled.
Polynucleotides
[0273] The present invention also relates to isolated
polynucleotides that encode any of the variants of the present
invention.
Nucleic Acid Constructs
[0274] The present invention also relates to nucleic acid
constructs comprising a polynucleotide encoding a variant of the
present invention operably linked to one or more (several) control
sequences that direct the expression of the coding sequence in a
suitable host cell under conditions compatible with the control
sequences.
[0275] An isolated polynucleotide encoding a variant may be
manipulated in a variety of ways to provide for expression of the
variant. Manipulation of the polynucleotide prior to its insertion
into a vector may be desirable or necessary depending on the
expression vector. The techniques for modifying polynucleotides
utilizing recombinant DNA methods are well known in the art.
[0276] The control sequence may be a promoter sequence, which is
recognized by a host cell for expression of the polynucleotide. The
promoter sequence contains transcriptional control sequences that
mediate the expression of the variant. The promoter may be any
nucleic acid sequence that shows transcriptional activity in the
host cell including mutant, truncated, and hybrid promoters, and
may be obtained from genes encoding extracellular or intracellular
polypeptides either homologous or heterologous to the host
cell.
[0277] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention, especially in a bacterial host cell, are the promoters
obtained from the Bacillus amyloliquefaciens alpha-amylase gene
(amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus
licheniformis penicillinase gene (penP), Bacillus
stearothermophilus maltogenic amylase gene (amyM), Bacillus
subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB
genes, E. coli lac operon, Streptomyces coelicolor agarase gene
(dagA), and prokaryotic beta-lactamase gene (VIIIa-Kamaroff et al.,
1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac
promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:
21-25). Further promoters are described in "Useful proteins from
recombinant bacteria" in Scientific American, 1980, 242: 74-94; and
in Sambrook et al., 1989, supra.
[0278] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a filamentous fungal host cell are promoters obtained
from the genes for Aspergillus nidulans acetamidase, Aspergillus
niger neutral alpha-amylase, Aspergillus niger acid stable
alpha-amylase, Aspergillus niger or Aspergillus awamori
glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus
oryzae alkaline protease, Aspergillus oryzae triose phosphate
isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787),
Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium
venenatum Dana (WO 00/56900), Fusarium venenatum Quinn (WO
00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic
proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,
Trichoderma reesei endoglucanase I, Trichoderma reesei
endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma
reesei endoglucanase IV, Trichoderma reesei endoglucanase V,
Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,
Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter
(a modified promoter including a gene encoding a neutral
alpha-amylase in Aspergilli in which the untranslated leader has
been replaced by an untranslated leader from a gene encoding triose
phosphate isomerase in Aspergilli; non-limiting examples include
modified promoters including the gene encoding neutral
alpha-amylase in Aspergillus niger in which the untranslated leader
has been replaced by an untranslated leader from the gene encoding
triose phosphate isomerase in Aspergillus nidulans or Aspergillus
oryzae); and mutant, truncated, and hybrid promoters thereof.
[0279] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,
ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase
(TPI), Saccharomyces cerevisiae metallothionein (CUP1), and
Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful
promoters for yeast host cells are described by Romanos et al.,
1992, Yeast 8: 423-488.
[0280] The control sequence may also be a suitable transcription
terminator sequence, which is recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3'-terminus of the polynucleotide encoding the variant. Any
terminator that is functional in the host cell may be used in the
present invention.
[0281] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, Aspergillus niger
glucoamylase, Aspergillus oryzae TAKA amylase, and Fusarium
oxysporum trypsin-like protease.
[0282] Preferred terminators for yeast host cells are obtained from
the genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are described by Romanos et al., 1992,
supra.
[0283] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA that is important for translation
by the host cell. The leader sequence is operably linked to the
5'-terminus of the polynucleotide encoding the variant. Any leader
sequence that is functional in the host cell may be used in the
present invention.
[0284] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0285] Suitable leaders for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae
alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
[0286] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3'-terminus of the
polypeptide-encoding sequence and, when transcribed, is recognized
by the host cell as a signal to add polyadenosine residues to
transcribed mRNA. Any polyadenylation sequence that is functional
in the host cell may be used in the present invention.
[0287] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus nidulans
anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus
niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and
Fusarium oxysporum trypsin-like protease.
[0288] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:
5983-5990.
[0289] The control sequence may also be a signal peptide coding
region that codes for an amino acid sequence linked to the amino
terminus of a variant and directs the encoded polypeptide into the
cell's secretory pathway. The 5'-end of the coding sequence of the
polynucleotide may inherently contain a signal peptide coding
region naturally linked in translation reading frame with the
segment of the coding region that encodes the secreted variant.
Alternatively, the 5'-end of the coding sequence may contain a
signal peptide coding region that is foreign to the coding
sequence. The foreign signal peptide coding region may be required
where the coding sequence does not naturally contain a signal
peptide coding region. Alternatively, the foreign signal peptide
coding region may simply replace the natural signal peptide coding
region in order to enhance secretion of the variant. However, any
signal peptide coding region that directs the expressed polypeptide
into the secretory pathway of a host cell may be used in the
present invention.
[0290] Effective signal peptide coding sequences for bacterial host
cells are the signal peptide coding sequences obtained from the
genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus
licheniformis subtilisin, Bacillus licheniformis beta-lactamase,
Bacillus stearothermophilus alpha-amylase, Bacillus
stearothermophilus neutral proteases (nprT, nprS, nprM), and
Bacillus subtilis prsA. Further signal peptides are described by
Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[0291] Effective signal peptide coding sequences for filamentous
fungal host cells are the signal peptide coding sequences obtained
from the genes for Aspergillus niger neutral amylase, Aspergillus
niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola
insolens cellulase, Humicola insolens endoglucanase V, Humicola
lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.
[0292] Useful signal peptides for yeast host cells are obtained
from the genes for Saccharomyces cerevisiae alpha-factor and
Saccharomyces cerevisiae invertase. Other useful signal peptide
coding sequences are described by Romanos et al., 1992, supra.
[0293] The control sequence may also be a propeptide coding region
that codes for an amino acid sequence positioned at the amino
terminus of a variant. The resultant polypeptide is known as a
proenzyme or propolypeptide (or a zymogen in some cases). A
propolypeptide is generally inactive and can be converted to a
mature active polypeptide by catalytic or autocatalytic cleavage of
the propeptide from the propolypeptide. The propeptide coding
region may be obtained from the genes for Myceliophthora
thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic
proteinase, and Saccharomyces cerevisiae alpha-factor.
[0294] Where both signal peptide and propeptide regions are present
at the amino terminus of a polypeptide, the propeptide region is
positioned next to the amino terminus of a polypeptide and the
signal peptide region is positioned next to the amino terminus of
the propeptide region.
[0295] It may also be desirable to add regulatory sequences that
allow the regulation of the expression of the variant relative to
the growth of the host cell. Examples of regulatory systems are
those that cause the expression of the gene to be turned on or off
in response to a chemical or physical stimulus, including the
presence of a regulatory compound. Regulatory systems in
prokaryotic systems include the lac, tac, and trp operator systems.
In yeast, the ADH2 system or GAL1 system may be used. In
filamentous fungi, the Aspergillus niger glucoamylase promoter,
Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus
oryzae glucoamylase promoter may be used as regulatory sequences.
Other examples of regulatory sequences are those that allow for
gene amplification. In eukaryotic systems, these regulatory
sequences include the dihydrofolate reductase gene that is
amplified in the presence of methotrexate, and the metallothionein
genes that are amplified with heavy metals. In these cases, the
polynucleotide encoding the variant would be operably linked with
the regulatory sequence.
Expression Vectors
[0296] The present invention also relates to recombinant expression
vectors comprising a polynucleotide encoding a variant of the
present invention, a promoter, and transcriptional and
translational stop signals. The various nucleotide and control
sequences described above may be joined together to produce a
recombinant expression vector that may include one or more
(several) convenient restriction sites to allow for insertion or
substitution of the polynucleotide encoding the variant at such
sites. Alternatively, the polynucleotide may be expressed by
inserting the polynucleotide or a nucleic acid construct comprising
the polynucleotide into an appropriate vector for expression. In
creating the expression vector, the coding sequence is located in
the vector so that the coding sequence is operably linked with the
appropriate control sequences for expression.
[0297] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) that can be conveniently subjected to recombinant
DNA procedures and can bring about the expression of the
polynucleotide. The choice of the vector will typically depend on
the compatibility of the vector with the host cell into which the
vector is to be introduced. The vectors may be linear or closed
circular plasmids.
[0298] The vector may be an autonomously replicating vector, i.e.,
a vector that exists as an extrachromosomal entity, the replication
of which is independent of chromosomal replication, e.g., a
plasmid, an extrachromosomal element, a minichromosome, or an
artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
that, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids that together contain the total DNA to
be introduced into the genome of the host cell, or a transposon,
may be used.
[0299] The vectors of the present invention preferably contain one
or more (several) selectable markers that permit easy selection of
transformed, transfected, transduced, or the like cells. A
selectable marker is a gene the product of which provides for
biocide or viral resistance, resistance to heavy metals,
prototrophy to auxotrophs, and the like.
[0300] Examples of bacterial selectable markers are the dal genes
from Bacillus subtilis or Bacillus licheniformis, or markers that
confer antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol, or tetracycline resistance. Suitable markers for
yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
Selectable markers for use in a filamentous fungal host cell
include, but are not limited to, amdS (acetamidase), argB
(ornithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hph (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), and trpC (anthranilate synthase),
as well as equivalents thereof. Preferred for use in an Aspergillus
cell are the amdS and pyrG genes of Aspergillus nidulans or
Aspergillus oryzae and the bar gene of Streptomyces
hygroscopicus.
[0301] The vectors of the present invention preferably contain an
element(s) that permits integration of the vector into the host
cell's genome or autonomous replication of the vector in the cell
independent of the genome.
[0302] For integration into the host cell genome, the vector may
rely on the polynucleotide's sequence encoding the polypeptide or
any other element of the vector for integration into the genome by
homologous or non-homologous recombination. Alternatively, the
vector may contain additional nucleotide sequences for directing
integration by homologous recombination into the genome of the host
cell at a precise location(s) in the chromosome(s). To increase the
likelihood of integration at a precise location, the integrational
elements should preferably contain a sufficient number of nucleic
acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000
base pairs, and most preferably 800 to 10,000 base pairs, which
have a high degree of identity to the corresponding target sequence
to enhance the probability of homologous recombination. The
integrational elements may be any sequence that is homologous with
the target sequence in the genome of the host cell. Furthermore,
the integrational elements may be non-encoding or encoding
nucleotide sequences. On the other hand, the vector may be
integrated into the genome of the host cell by non-homologous
recombination.
[0303] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. The origin of
replication may be any plasmid replicator mediating autonomous
replication that functions in a cell. The term "origin of
replication" or "plasmid replicator" is defined herein as a
nucleotide sequence that enables a plasmid or vector to replicate
in vivo.
[0304] Examples of bacterial origins of replication are the origins
of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184
permitting replication in E. coli, and pUB110, pE194, pTA1060, and
pAMR1 permitting replication in Bacillus.
[0305] Examples of origins of replication for use in a yeast host
cell are the 2 micron origin of replication, ARS1, ARS4, the
combination of ARS1 and CEN3, and the combination of ARS4 and
CEN6.
[0306] Examples of origins of replication useful in a filamentous
fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67;
Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO
00/24883). Isolation of the AMA1 gene and construction of plasmids
or vectors comprising the gene can be accomplished according to the
methods disclosed in WO 00/24883.
[0307] More than one copy of a polynucleotide of the present
invention may be inserted into the host cell to increase production
of an alpha-amylase variant. An increase in the copy number of the
polynucleotide can be obtained by integrating at least one
additional copy of the sequence into the host cell genome or by
including an amplifiable selectable marker gene with the
polynucleotide where cells containing amplified copies of the
selectable marker gene, and thereby additional copies of the
polynucleotide, can be selected for by cultivating the cells in the
presence of the appropriate selectable agent.
[0308] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra) to obtain substantially pure
alpha-amylase variants.
Host Cells
[0309] The present invention also relates to recombinant host
cells, comprising a polynucleotide encoding a variant, which are
advantageously used in the recombinant production of the variant. A
vector comprising a polynucleotide of the present invention is
introduced into a host cell so that the vector is maintained as a
chromosomal integrant or as a self-replicating extra-chromosomal
vector as described earlier. The choice of a host cell will to a
large extent depend upon the gene encoding the polypeptide and its
source.
[0310] The host cell may be any cell useful in the recombinant
production of a variant, e.g., a prokaryote or a eukaryote.
[0311] The prokaryotic host cell may be any gram-positive bacterium
or gram-negative bacterium. Gram-positive bacteria include, but are
not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus,
Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus,
Streptococcus, and Streptomyces. Gram-negative bacteria include,
but are not limited to, Campylobacter, E. coli, Flavobacterium,
Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas,
Salmonella, and Ureaplasma.
[0312] The bacterial host cell may be any Bacillus cell, including,
but not limited to, Bacillus alkalophilus, Bacillus
amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus
clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,
Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,
Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis,
and Bacillus thuringiensis cells.
[0313] The bacterial host cell may also be any Streptococcus cell,
including, but not limited to, Streptococcus equisimilis,
Streptococcus pyogenes, Streptococcus uberis, and Streptococcus
equi subsp. Zooepidemicus cells.
The bacterial host cell may also be any Streptomyces cell,
including, but not limited to, Streptomyces achromogenes,
Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces
griseus, and Streptomyces lividans cells.
[0314] The introduction of DNA into a Bacillus cell may, for
instance, be effected by protoplast transformation (see, e.g.,
Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), by using
competent cells (see, e.g., Young and Spizizen, 1961, J. Bacteriol.
81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol.
56: 209-221), by electroporation (see, e.g., Shigekawa and Dower,
1988, Biotechniques 6: 742-751), or by conjugation (see, e.g.,
Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The
introduction of DNA into an E coli cell may, for instance, be
effected by protoplast transformation (see, e.g., Hanahan, 1983, J.
Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et
al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of
DNA into a Streptomyces cell may, for instance, be effected by
protoplast transformation and electroporation (see, e.g., Gong et
al., 2004, Folia Microbiol. (Praha) 49: 399-405), by conjugation
(see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585),
or by transduction (see, e.g., Burke et al., 2001, Proc. Natl.
Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a
Pseudomonas cell may, for instance, be effected by electroporation
(see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397)
or by conjugation (see, e.g., Pinedo and Smets, 2005, Appl.
Environ. Microbiol. 71: 51-57). The introduction of DNA into a
Streptococcus cell may, for instance, be effected by natural
competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun.
32: 1295-1297), by protoplast transformation (see, e.g., Catt and
Jollick, 1991, Microbios 68: 189-2070, by electroporation (see,
e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65:
3800-3804) or by conjugation (see, e.g., Clewell, 1981, Microbiol.
Rev. 45: 409-436). However, any method known in the art for
introducing DNA into a host cell can be used.
[0315] The host cell may also be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0316] In one aspect, the host cell is a fungal cell. "Fungi" as
used herein includes the phyla Ascomycota, Basidiomycota,
Chytridiomycota, and Zygomycota as well as the Oomycota and all
mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth
and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB
International, University Press, Cambridge, UK).
[0317] In another aspect, the fungal host cell is a yeast cell.
"Yeast" as used herein includes ascosporogenous yeast
(Endomycetales), basidiosporogenous yeast, and yeast belonging to
the Fungi Imperfecti (Blastomycetes). Since the classification of
yeast may change in the future, for the purposes of this invention,
yeast shall be defined as described in Biology and Activities of
Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds,
Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[0318] In another aspect, the yeast host cell is a Candida,
Hansenula, Kluyveromyces, Pichia, Saccharomyces,
Schizosaccharomyces, or Yarrowia cell.
[0319] In another aspect, the yeast host cell is a Kluyveromyces
lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae,
Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces
kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or
Yarrowia lipolytica cell.
[0320] In another aspect, the fungal host cell is a filamentous
fungal cell. "Filamentous fungi" include all filamentous forms of
the subdivision Eumycota and Oomycota (as defined by Hawksworth et
al., 1995, supra). The filamentous fungi are generally
characterized by a mycelial wall composed of chitin, cellulose,
glucan, chitosan, mannan, and other complex polysaccharides.
Vegetative growth is by hyphal elongation and carbon catabolism is
obligatory aerobic. In contrast, vegetative growth by yeasts such
as Saccharomyces cerevisiae is by budding of a unicellular thallus
and carbon catabolism may be fermentative.
[0321] In another aspect, the filamentous fungal host cell is an
Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,
Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium,
Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,
Neocallimastix, Neurospora, Paecilomyces, Penicillium,
Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum,
Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or
Trichoderma cell.
[0322] In another aspect, the filamentous fungal host cell is an
Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus,
Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger,
Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina,
Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis
pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,
Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium
keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium,
Chrysosporium pannicola, Chrysosporium queenslandicum,
Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus,
Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis,
Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,
Fusarium graminum, Fusarium heterosporum, Fusarium negundi,
Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium
sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides,
Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,
Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor
miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Phanerochaete chrysosporium, Phlebia radiata,
Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes
versicolor, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride cell.
[0323] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus and Trichoderma host
cells are described in EP 238023 and Yelton et al., 1984, Proc.
Natl. Acad. Sci. USA 81: 1470-1474. Suitable methods for
transforming Fusarium species are described by Malardier et al.,
1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed
using the procedures described by Becker and Guarente, In Abelson,
J. N. and Simon, M. I., editors, Guide to Yeast Genetics and
Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187,
Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol.
153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:
1920.
Methods of Production
[0324] The present invention also relates to methods of producing a
variant, comprising: (a) cultivating a host cell of the present
invention under conditions suitable for the expression of the
variant; and (b) recovering the variant from the cultivation
medium.
[0325] The host cells are cultivated in a nutrient medium suitable
for production of the variant using methods known in the art. For
example, the cell may be cultivated by shake flask cultivation, or
small-scale or large-scale fermentation (including continuous,
batch, fed-batch, or solid state fermentations) in laboratory or
industrial fermentors performed in a suitable medium and under
conditions allowing the polypeptide to be expressed and/or
isolated. The cultivation takes place in a suitable nutrient medium
comprising carbon and nitrogen sources and inorganic salts, using
procedures known in the art. Suitable media are available from
commercial suppliers or may be prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection). If the polypeptide is secreted into the nutrient
medium, the polypeptide can be recovered directly from the medium.
If the polypeptide is not secreted, it can be recovered from cell
lysates.
[0326] In an alternative aspect, the variant is not recovered, but
rather a host cell of the present invention expressing a variant is
used as a source of the variant.
[0327] The variant may be detected using methods known in the art
that are specific for the variants. These detection methods may
include use of specific antibodies, formation of an enzyme product,
or disappearance of an enzyme substrate. For example, an enzyme
assay may be used to determine the activity of the polypeptide as
described herein in the Examples.
[0328] The variant may be recovered by methods known in the art.
For example, the polypeptide may be recovered from the nutrient
medium by conventional procedures including, but not limited to,
collection, centrifugation, filtration, extraction, spray-drying,
evaporation, or precipitation.
[0329] A variant of the present invention may be purified by a
variety of procedures known in the art including, but not limited
to, chromatography (e.g., ion exchange, affinity, hydrophobic,
chromatofocusing, and size exclusion), electrophoretic procedures
(e.g., preparative isoelectric focusing), differential solubility
(e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction
(see, e.g., Protein Purification, J.-C. Janson and Lars Ryden,
editors, VCH Publishers, New York, 1989) to obtain substantially
pure variants.
Plants
[0330] The present invention also relates to plants, e.g., a
transgenic plant, plant part, or plant cell, comprising an isolated
polynucleotide encoding a variant of the present invention so as to
express and produce the variant in recoverable quantities. The
variant may be recovered from the plant or plant part.
Alternatively, the plant or plant part containing the recombinant
variant may be used as such for improving the quality of a food or
feed, e.g., improving nutritional value, palatability, and
rheological properties, or to destroy an antinutritive factor.
[0331] The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot). Examples of monocot plants are
grasses, such as meadow grass (blue grass, Poa), forage grass such
as Festuca, Lolium, temperate grass, such as Agrostis, and cereals,
e.g., wheat, oats, rye, barley, rice, sorghum, and maize
(corn).
[0332] Examples of dicot plants are tobacco, legumes, such as
lupins, potato, sugar beet, pea, bean and soybean, and cruciferous
plants (family Brassicaceae), such as cauliflower, rape seed, and
the closely related model organism Arabidopsis thaliana.
[0333] Examples of plant parts are stem, callus, leaves, root,
fruits, seeds, and tubers as well as the individual tissues
comprising these parts, e.g., epidermis, mesophyll, parenchyme,
vascular tissues, meristems. Specific plant cell compartments, such
as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and
cytoplasm are also considered to be a plant part. Furthermore, any
plant cell, whatever the tissue origin, is considered to be a plant
part. Likewise, plant parts such as specific tissues and cells
isolated to facilitate the utilization of the invention are also
considered plant parts, e.g., embryos, endosperms, aleurone and
seed coats.
[0334] Also included within the scope of the present invention are
the progeny of such plants, plant parts, and plant cells.
[0335] The transgenic plant or plant cell expressing a variant of
the present invention may be constructed in accordance with methods
known in the art. In short, the plant or plant cell is constructed
by incorporating one or more (several) expression constructs
encoding the variant into the plant host genome or chloroplast
genome and propagating the resulting modified plant or plant cell
into a transgenic plant or plant cell.
[0336] The expression construct is conveniently a nucleic acid
construct that comprises a polynucleotide encoding the variant
operably linked with appropriate regulatory sequences required for
expression of the nucleotide sequence in the plant or plant part of
choice. Furthermore, the expression construct may comprise a
selectable marker useful for identifying host cells into which the
expression construct has been integrated and DNA sequences
necessary for introduction of the construct into the plant in
question (the latter depends on the DNA introduction method to be
used).
[0337] The choice of regulatory sequences, such as promoter and
terminator sequences and optionally signal or transit sequences, is
determined, for example, on the basis of when, where, and how the
variant is desired to be expressed. For instance, the expression of
the gene encoding a variant may be constitutive or inducible, or
may be developmental, stage or tissue specific, and the gene
product may be targeted to a specific tissue or plant part such as
seeds or leaves. Regulatory sequences are, for example, described
by Tague et al., 1988, Plant Physiol. 86: 506.
[0338] For constitutive expression, the 35S-CaMV, the maize
ubiquitin 1, and the rice actin 1 promoter may be used (Franck et
al., 1980, Cell 21: 285-294; Christensen et al., 1992, Plant Mol.
Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165).
Organ-specific promoters may be, for example, a promoter from
storage sink tissues such as seeds, potato tubers, and fruits
(Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from
metabolic sink tissues such as meristems (Ito et al., 1994, Plant
Mol. Biol. 24: 863-878), a seed specific promoter such as the
glutelin, prolamin, globulin, or albumin promoter from rice (Wu et
al., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter
from the legumin B4 and the unknown seed protein gene from Vicia
faba (Conrad et al., 1998, J. Plant Physiol. 152: 708-711), a
promoter from a seed oil body protein (Chen et al., 1998, Plant
Cell Physiol. 39: 935-941), the storage protein napA promoter from
Brassica napus, or any other seed specific promoter known in the
art, e.g., as described in WO 91/14772. Furthermore, the promoter
may be a leaf specific promoter such as the rbcs promoter from rice
or tomato (Kyozuka et al., 1993, Plant Physiol. 102: 991-1000, the
chlorella virus adenine methyltransferase gene promoter (Mitra and
Higgins, 1994, Plant Mol. Biol. 26: 85-93), or the aldP gene
promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:
668-674), or a wound inducible promoter such as the potato pin2
promoter (Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise,
the promoter may inducible by abiotic treatments such as
temperature, drought, or alterations in salinity or induced by
exogenously applied substances that activate the promoter, e.g.,
ethanol, oestrogens, plant hormones such as ethylene, abscisic
acid, and gibberellic acid, and heavy metals.
[0339] A promoter enhancer element may also be used to achieve
higher expression of a polypeptide of the present invention in the
plant. For instance, the promoter enhancer element may be an intron
that is placed between the promoter and the nucleotide sequence
encoding a polypeptide of the present invention. For instance, Xu
et al., 1993, supra, disclose the use of the first intron of the
rice actin 1 gene to enhance expression.
[0340] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0341] The nucleic acid construct is incorporated into the plant
genome according to conventional techniques known in the art,
including Agrobacterium-mediated transformation, virus-mediated
transformation, microinjection, particle bombardment, biolistic
transformation, and electroporation (Gasser et al., 1990, Science
244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al.,
1989, Nature 338: 274).
[0342] Presently, Agrobacterium tumefaciens-mediated gene transfer
is the method of choice for generating transgenic dicots (for a
review, see Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19:
15-38) and can also be used for transforming monocots, although
other transformation methods are often used for these plants.
Presently, the method of choice for generating transgenic monocots
is particle bombardment (microscopic gold or tungsten particles
coated with the transforming DNA) of embryonic calli or developing
embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto, 1994,
Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,
Bio/Technology 10: 667-674). An alternative method for
transformation of monocots is based on protoplast transformation as
described by Omirulleh et al., 1993, Plant Molecular Biology 21:
415-428. Additional transformation methods for use in accordance
with the present disclosure include those described in U.S. Pat.
Nos. 6,395,966 and 7,151,204 (both of which are herein incorporated
by reference in their entirety).
[0343] Following transformation, the transformants comprising the
expression construct are selected and regenerated into whole plants
according to methods well known in the art. Often the
transformation procedure is designed for the selective elimination
of selection genes either during regeneration or in the following
generations by using, for example, co-transformation with two
separate T-DNA constructs or site specific excision of the
selection gene by a specific recombinase.
[0344] In addition to direct transformation of a particular plant
genotype with a construct prepared according to the present
invention, transgenic plants may be made by crossing a plant having
a construct of the present invention to a second plant lacking the
construct. For example, a construct encoding a variant or a portion
thereof can be introduced into a particular plant variety by
crossing, without the need for ever directly transforming a plant
of that given variety. Therefore, the present invention not only
encompasses a plant directly regenerated from cells which have been
transformed in accordance with the present invention, but also the
progeny of such plants. As used herein, progeny may refer to the
offspring of any generation of a parent plant prepared in
accordance with the present invention. Such progeny may include a
DNA construct prepared in accordance with the present invention, or
a portion of a DNA construct prepared in accordance with the
present invention. Crossing results in a transgene of the present
invention being introduced into a plant line by cross pollinating a
starting line with a donor plant line that includes a transgene of
the present invention. Non-limiting examples of such steps are
further articulated in U.S. Pat. No. 7,151,204.
[0345] Plants may be generated through a process of backcross
conversion. For example, plants include plants referred to as a
backcross converted genotype, line, inbred, or hybrid.
[0346] Genetic markers may be used to assist in the introgression
of one or more transgenes of the invention from one genetic
background into another. Marker assisted selection offers
advantages relative to conventional breeding in that it can be used
to avoid errors caused by phenotypic variations. Further, genetic
markers may provide data regarding the relative degree of elite
germplasm in the individual progeny of a particular cross. For
example, when a plant with a desired trait which otherwise has a
non-agronomically desirable genetic background is crossed to an
elite parent, genetic markers may be used to select progeny which
not only possess the trait of interest, but also have a relatively
large proportion of the desired germplasm. In this way, the number
of generations required to introgress one or more traits into a
particular genetic background is minimized.
[0347] The present invention also relates to methods of producing a
variant of the present invention comprising: (a) cultivating a
transgenic plant or a plant cell comprising a polynucleotide
encoding the variant under conditions conducive for production of
the variant; and (b) recovering the variant.
Compositions
[0348] The present invention also relates to compositions
comprising an alpha-amylase variant and at least one additional
enzyme. The additional enzyme(s) may be selected from the group
consisting of beta-amylase, cellulase (beta-glucosidase,
cellobiohydrolase and endoglucanase), glucoamylase, hemicellulsae
(e.g., xylanase), isoamylase, isomerase, lipase, phytase, protease,
pullulanase, and/or other enzymes useful in a commercial process in
conjunction with an alpha-amylase. The additional enzyme may also
be a second alpha-amylase. Such enzymes are known in the art in
starch processing, sugar conversion, fermentations for alcohol and
other useful end-products, commercial detergents and cleaning aids,
stain removal, fabric treatment or desizing, and the like.
Methods of Using the Alpha-Amylase Variants--Industrial
Applications
[0349] The variants of the present invention possess valuable
properties allowing for a variety of industrial applications. In
particular, the variants may be used in detergents, in particular
laundry detergent compositions and dishwashing detergent
compositions, hard surface cleaning compositions, and for desizing
textiles, fabrics or garments, production of pulp and paper, beer
making, ethanol production, and starch conversion processes.
[0350] The alpha-amylase variants may be used for starch processes,
in particular starch conversion, especially liquefaction of starch
(see, e.g., U.S. Pat. No. 3,912,590, EP 252730 and EP 063909, WO
99/19467, and WO 96/28567, which are all hereby incorporated by
reference). Also contemplated are compositions for starch
conversion purposes, which may beside the variant of the invention
also comprise an AMG, pullulanase, and other alpha-amylases.
[0351] Further, the variants are particularly useful in the
production of sweeteners and ethanol (see, e.g., U.S. Pat. No.
5,231,017, which is hereby incorporated by reference), such as
fuel, drinking and industrial ethanol, from starch or whole
grains.
[0352] The variants may also be used for desizing of textiles,
fabrics, and garments (see, e.g., WO 95/21247, U.S. Pat. No.
4,643,736, and EP 119920, which are incorporated herein by
reference), beer making or brewing, and in pulp and paper
production or related processes.
Starch Processing
[0353] Native starch consists of microscopic granules, which are
insoluble in water at room temperature. When an aqueous starch
slurry is heated, the granules swell and eventually burst,
dispersing the starch molecules into the solution. During this
"gelatinization" process there is a dramatic increase in viscosity.
As the solids level is 30-40% in a typical industrial process, the
starch has to be thinned or "liquefied" so that it can be suitably
processed. This reduction in viscosity is primarily attained by
enzymatic degradation in current commercial practice.
[0354] Conventional starch-conversion processes, such as
liquefaction and saccharification processes are described, e.g., in
U.S. Pat. No. 3,912,590, EP 252730 and EP 063909, which are
incorporated herein by reference.
[0355] In an embodiment, the conversion process degrading starch to
lower molecular weight carbohydrate components such as sugars or
fat replacers includes a debranching step.
[0356] In the case of converting starch into a sugar, the starch is
depolymerized. Such a depolymerization process consists of, e.g., a
pre-treatment step and two or three consecutive process steps,
i.e., a liquefaction process, a saccharification process, and
depending on the desired end-product, an optional isomerization
process.
[0357] When the desired final sugar product is, e.g., high fructose
syrup the dextrose syrup may be converted into fructose. After the
saccharification process, the pH is increased to a value in the
range of 6-8, preferably pH 7.5, and the calcium is removed by ion
exchange. The dextrose syrup is then converted into high fructose
syrup using, e.g., an immobilized glucose isomerase.
Production of Fermentation Products
[0358] In general, alcohol production (ethanol) from whole grain
can be separated into 4 main steps: milling, liquefaction,
saccharification, and fermentation.
[0359] The grain is milled in order to open up the structure and
allow for further processing. Two processes used are wet or dry
milling. In dry milling, the whole kernel is milled and used in the
remaining part of the process. Wet milling gives a very good
separation of germ and meal (starch granules and protein) and is
with a few exceptions applied at locations where there is a
parallel production of syrups.
[0360] In the liquefaction process the starch granules are
solubilized by hydrolysis to maltodextrins mostly of a DP higher
than 4. The hydrolysis may be carried out by acid treatment or
enzymatically by an alpha-amylase. Acid hydrolysis is used on a
limited basis. The raw material can be milled whole grain or a side
stream from starch processing.
[0361] During a typical enzymatic liquefaction, the long-chained
starch is degraded into branched and linear shorter units
(maltodextrins) by an alpha-amylase. Enzymatic liquefaction is
generally carried out as a three-step hot slurry process. The
slurry is heated to between 60-95.degree. C. (e.g., 77-86.degree.
C., 80-85.degree. C., or 83-85.degree. C.) and the enzyme(s) is
(are) added. The liquefaction process is carried out at 85.degree.
C. for 1-2 hours. The pH is generally between 5.5 and 6.2. In order
to ensure optimal enzyme stability under these conditions, 1 mM of
calcium is added (to provide about 40 ppm free calcium ions). After
such treatment, the liquefied starch will have a "dextrose
equivalent" (DE) of 10-15.
[0362] The slurry is subsequently jet-cooked at between
95-140.degree. C., e.g., 105-125.degree. C., cooled to
60-95.degree. C. and more enzyme(s) is (are) added to obtain the
final hydrolysis. The liquefaction process is carried out at pH
4.5-6.5, typically at a pH between 5 and 6. Milled and liquefied
grain is also known as mash.
[0363] Liquefied starch-containing material is saccharified in the
presence of saccharifying enzymes such as glucoamylases. The
saccharification process may last for 12 hours to 120 hours (e.g.,
12 to 90 hours, 12 to 60 hours and 12 to 48 hours).
[0364] However, it is common to perform a pre-saccharification step
for about 30 minutes to 2 hours (e.g., 30 to 90 minutes) at a
temperature of 30 to 65.degree. C., typically around 60.degree. C.,
which is followed by a complete saccharification during
fermentation referred to as simultaneous saccharification and
fermentation (SSF). The pH is usually between 4.2-4.8, e.g., 4.5.
In a simultaneous saccharification and fermentation (SSF) process,
there is no holding stage for saccharification, rather, the yeast
and enzymes are added together.
[0365] In a typical saccharification process, maltodextrins
produced during liquefaction are converted into dextrose by adding
a glucoamylase and a debranching enzyme, such as an isoamylase
(U.S. Pat. No. 4,335,208) or a pullulanase. The temperature is
lowered to 60.degree. C., prior to the addition of a glucoamylase
and debranching enzyme. The saccharification process proceeds for
24-72 hours.
[0366] Prior to addition of the saccharifying enzymes, the pH is
reduced to below 4.5, while maintaining a high temperature (above
95.degree. C.), to inactivate the liquefying alpha-amylase. This
process reduces the formation of short oligosaccharide called
"panose precursors," which cannot be hydrolyzed properly by the
debranching enzyme. Normally, about 0.2-0.5% of the
saccharification product is the branched trisaccharide panose (Glc
p.alpha.1-6Glc p.alpha.1-4Glc), which cannot be degraded by a
pullulanase. If active amylase from the liquefaction remains
present during saccharification (i.e., no denaturing), the amount
of panose can be as high as 1-2%, which is highly undesirable since
it lowers the saccharification yield significantly.
[0367] Fermentable sugars (e.g., dextrins, monosaccharides,
particularly glucose) are produced from enzymatic saccharification.
These fermentable sugars may be further purified and/or converted
to useful sugar products. In addition, the sugars may be used as a
fermentation feedstock in a microbial fermentation process for
producing end-products, such as alcohol (e.g., ethanol and
butanol), organic acids (e.g., succinic acid and lactic acid),
sugar alcohols (e.g., glycerol), ascorbic acid intermediates (e.g.,
gluconate, 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and
2-keto-L-gulonic acid), amino acids (e.g., lysine), proteins (e.g.,
antibodies and fragment thereof).
[0368] In an embodiment, the fermentable sugars obtained during the
liquefaction process steps are used to produce alcohol and
particularly ethanol. In ethanol production, an SSF process is
commonly used wherein the saccharifying enzymes and fermenting
organisms (e.g., yeast) are added together and then carried out at
a temperature of 30-40.degree. C.
[0369] The organism used in fermentation will depend on the desired
end-product. Typically, if ethanol is the desired end product yeast
will be used as the fermenting organism. In some preferred
embodiments, the ethanol-producing microorganism is a yeast and
specifically Saccharomyces such as strains of S. cerevisiae (U.S.
Pat. No. 4,316,956). A variety of S. cerevisiae are commercially
available and these include but are not limited to FALI
(Fleischmann's Yeast), SUPERSTART (Alltech), FERMIOL (DSM
Specialties), RED STAR (Lesaffre) and Angel alcohol yeast (Angel
Yeast Company, China). The amount of starter yeast employed in the
methods is an amount effective to produce a commercially
significant amount of ethanol in a suitable amount of time, (e.g.,
to produce at least 10% ethanol from a substrate having between
25-40% DS in less than 72 hours). Yeast cells are generally
supplied in amounts of about 10.sup.4 to about 10.sup.12, and
preferably from about 10.sup.7 to about 10.sup.10 viable yeast
count per mL of fermentation broth. After yeast is added to the
mash, it is typically subjected to fermentation for about 24-96
hours, e.g., 35-60 hours. The temperature is between about
26-34.degree. C., typically at about 32.degree. C., and the pH is
from pH 3-6, e.g., around pH 4-5.
[0370] The fermentation may include, in addition to a fermenting
microorganisms (e.g., yeast), nutrients, and additional enzymes,
including phytases. The use of yeast in fermentation is well known
in the art.
[0371] In further embodiments, use of appropriate fermenting
microorganisms, as is known in the art, can result in fermentation
end product including, e.g., glycerol, 1,3-propanediol, gluconate,
2-keto-D-gluconate, 2,5-diketo-D-gluconate, 2-keto-L-gulonic acid,
succinic acid, lactic acid, amino acids, and derivatives thereof.
More specifically when lactic acid is the desired end product, a
Lactobacillus sp. (L. casei) may be used; when glycerol or
1,3-propanediol are the desired end-products E. coli may be used;
and when 2-keto-D-gluconate, 2,5-diketo-D-gluconate, and
2-keto-L-gulonic acid are the desired end products, Pantoea citrea
may be used as the fermenting microorganism. The above enumerated
list are only examples and one skilled in the art will be aware of
a number of fermenting microorganisms that may be used to obtain a
desired end product.
Processes for Producing Fermentation Products from Ungelatinized
Starch-Containing Material
[0372] The invention relates to processes for producing
fermentation products from starch-containing material without
gelatinization (i.e., without cooking) of the starch-containing
material. The fermentation product, such as ethanol, can be
produced without liquefying the aqueous slurry containing the
starch-containing material and water. In one embodiment a process
of the invention includes saccharifying (e.g., milled)
starch-containing material, e.g., granular starch, below the
initial gelatinization temperature, preferably in the presence of
alpha-amylase and/or carbohydrate-source generating enzyme(s) to
produce sugars that can be fermented into the fermentation product
by a suitable fermenting organism. In this embodiment the desired
fermentation product, e.g., ethanol, is produced from ungelatinized
(i.e., uncooked), preferably milled, cereal grains, such as corn.
Accordingly, in the first aspect the invention relates to processes
for producing fermentation products from starch-containing material
comprising simultaneously saccharifying and fermenting
starch-containing material using a carbohydrate-source generating
enzyme and a fermenting organism at a temperature below the initial
gelatinization temperature of said starch-containing material. In
an embodiment a protease is also present. The protease may be any
acid fungal protease or metalloprotease. The fermentation product,
e.g., ethanol, may optionally be recovered after fermentation,
e.g., by distillation. Typically amylase(s), such as
glucoamylase(s) and/or other carbohydrate-source generating
enzymes, and/or alpha-amylase(s), is(are) present during
fermentation. Examples of glucoamylases and other
carbohydrate-source generating enzymes include raw starch
hydrolyzing glucoamylases. Examples of alpha-amylase(s) include
acid alpha-amylases such as acid fungal alpha-amylases. Examples of
fermenting organisms include yeast, e.g., a strain of Saccharomyces
cerevisiae. The term "initial gelatinization temperature" means the
lowest temperature at which starch gelatinization commences. In
general, starch heated in water begins to gelatinize between about
50.degree. C. and 75.degree. C.; the exact temperature of
gelatinization depends on the specific starch and can readily be
determined by the skilled artisan. Thus, the initial gelatinization
temperature may vary according to the plant species, to the
particular variety of the plant species as well as with the growth
conditions. In the context of this invention the initial
gelatinization temperature of a given starch-containing material
may be determined as the temperature at which birefringence is lost
in 5% of the starch granules using the method described by
Gorinstein and Lii, 1992, Starch/Starke 44(12): 461-466. Before
initiating the process a slurry of starch-containing material, such
as granular starch, having 10-55, e.g., 25-45 and 30-40, w/w % dry
solids (DS) of starch-containing material may be prepared. The
slurry may include water and/or process waters, such as stillage
(backset), scrubber water, evaporator condensate or distillate,
side-stripper water from distillation, or process water from other
fermentation product plants. Because the process of the invention
is carried out below the initial gelatinization temperature, and
thus no significant viscosity increase takes place, high levels of
stillage may be used if desired. In an embodiment the aqueous
slurry contains from about 1 to about 70 vol. %, preferably 15-60
vol. %, especially from about 30 to 50 vol. % water and/or process
waters, such as stillage (backset), scrubber water, evaporator
condensate or distillate, side-stripper water from distillation, or
process water from other fermentation product plants, or
combinations thereof, or the like. The starch-containing material
may be prepared by reducing the particle size, preferably by dry or
wet milling, to 0.05 to 3.0 mm, preferably 0.1-0.5 mm. After being
subjected to a process of the invention 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 preferably at least 99%
of the dry solids in the starch-containing material are converted
into a soluble starch hydrolyzate. A process in this aspect of the
invention is conducted at a temperature below the initial
gelatinization temperature, which means that the temperature
typically lies in the range between 30-75.degree. C., preferably
between 45-60.degree. C. In a preferred embodiment the process
carried at a temperature from 25.degree. C. to 40.degree. C., such
as from 28.degree. C. to 35.degree. C., such as from 30.degree. C.
to 34.degree. C., preferably around 32.degree. C. In an embodiment
the process is carried out so that the sugar level, such as glucose
level, is kept at a low level, such as below 6 w/w %, such as below
about 3 w/w %, such as below about 2 w/w %, such as below about 1
w/w %, such as below about 0.5 w/w %, or below 0.25 w/w %, such as
below about 0.1 w/w %. Such low levels of sugar can be accomplished
by simply employing adjusted quantities of enzyme and fermenting
organism. A skilled person in the art can easily determine which
doses/quantities of enzyme and fermenting organism to use. The
employed quantities of enzyme and fermenting organism may also be
selected to maintain low concentrations of maltose in the
fermentation broth. For instance, the maltose level may be kept
below about 0.5 w/w %, such as below about 0.2 w/w %. The process
of the invention may be carried out at a pH from about 3 and 7,
preferably from pH 3.5 to 6, or more preferably from pH 4 to 5. In
an embodiment fermentation is ongoing for 6 to 120 hours, in
particular 24 to 96 hours.
Processes for Producing Fermentation Products from Gelatinized
Starch-Containing Material
[0373] In this aspect the invention relates to processes for
producing fermentation products, especially ethanol, from
starch-containing material, which process includes a liquefaction
step and sequentially or simultaneously performed saccharification
and fermentation steps. Consequently, the invention relates to
processes for producing fermentation products from
starch-containing material comprising the steps of:
[0374] (a) liquefying starch-containing material in the presence of
an alpha-amylase variant, or;
[0375] (b) saccharifying the liquefied material obtained in step
(a) using a carbohydrate-source generating enzyme;
[0376] (c) fermenting using a fermenting organism.
[0377] In an aspect, a pullulanase such as a family GH57
pullulanase is also used in the liquefaction step. In an embodiment
a protease, such as an acid fungal protease or a metallo protease
is added before, during and/or after liquefaction. In an embodiment
the metalloprotease is derived from a strain of Thermoascus, e.g.,
a strain of Thermoascus aurantiacus, especially Thermoascus
aurantiacus CGMCC No. 0670. In an embodiment the
carbohydrate-source generating enzyme is a glucoamylase derived
from a strain of Aspergillus, e.g., Aspergillus niger or
Aspergillus awamori, a strain of Talaromyces, especially
Talaromyces emersonii; or a strain of Athelia, especially Athelia
rolfsii; a strain of Trametes, e.g., Trametes cingulata; a strain
of the genus Pachykytospora, e.g., a strain of Pachykytospora
papyracea; or a strain of the genus Leucopaxillus, e.g.,
Leucopaxillus giganteus; or a strain of the genus Peniophora, e.g.,
a strain of the species Peniophora rufomarginata; or a mixture
thereof. Saccharification step (b) and fermentation step (c) may be
carried out either sequentially or simultaneously. A pullulanase
and/or metalloprotease may be added during saccharification and/or
fermentation when the process is carried out as a sequential
saccharification and fermentation process and before or during
fermentation when steps (b) and (c) are carried out simultaneously
(SSF process). The pullulanase and/or metalloprotease may also
advantageously be added before liquefaction (pre-liquefaction
treatment), i.e., before or during step (a), and/or after
liquefaction (post liquefaction treatment), i.e., after step (a).
The pullulanase is most advantageously added before or during
liquefaction, i.e., before or during step (a). The fermentation
product, such as especially ethanol, may optionally be recovered
after fermentation, e.g., by distillation. The fermenting organism
is preferably yeast, preferably a strain of Saccharomyces
cerevisiae. In a particular embodiment, the process of the
invention further comprises, prior to step (a), the steps of:
[0378] x) reducing the particle size of the starch-containing
material, preferably by milling (e.g., using a hammer mill);
[0379] y) forming a slurry comprising the starch-containing
material and water.
[0380] In an embodiment the particle size is smaller than a #7
screen, e.g., a #6 screen. A #7 screen is usually used in
conventional prior art processes. The aqueous slurry may contain
from 10-55 w/w % dry solids (DS), e.g., 25-45 and 30-40 w/w % dry
solids (DS) of starch-containing material. The slurry is heated to
above the gelatinization temperature and an alpha-amylase variant
may be added to initiate liquefaction (thinning). The slurry may in
an embodiment be jet-cooked to further gelatinize the slurry before
being subjected to alpha-amylase in step (a). Liquefaction may in
an embodiment be carried out as a three-step hot slurry process.
The slurry is heated to between 60-95.degree. C., preferably
between 70-90.degree. C., such as preferably between 80-85.degree.
C. at pH 4-6, preferably 4.5-5.5, and alpha-amylase variant,
optionally together with a pullulanase and/or protease, preferably
metalloprotease, are added to initiate liquefaction (thinning). In
an embodiment the slurry may then be jet-cooked at a temperature
between 95-140.degree. C., preferably 100-135.degree. C., such as
105-125.degree. C., for about 1-15 minutes, preferably for about
3-10 minutes, especially around about 5 minutes. The slurry is
cooled to 60-95.degree. C. and more alpha-amylase variant and
optionally pullulanase variant and/or protease, preferably
metalloprotease, is(are) added to finalize hydrolysis (secondary
liquefaction). The liquefaction process is usually carried out at
pH 4.0-6, in particular at a pH from 4.5 to 5.5. Saccharification
step (b) may be carried out using conditions well known in the art.
For instance, a full saccharification process may last up to from
about 24 to about 72 hours, however, it is common only to do a
pre-saccharification of typically 40-90 minutes at a temperature
between 30-65.degree. C., typically about 60.degree. C., followed
by complete saccharification during fermentation in a simultaneous
saccharification and fermentation process (SSF process).
Saccharification is typically carried out at temperatures from
20-75.degree. C., preferably from 40-70.degree. C., typically
around 60.degree. C., and at a pH between 4 and 5, normally at
about pH 4.5. The most widely used process to produce a
fermentation product, especially ethanol, is a simultaneous
saccharification and fermentation (SSF) process, in which there is
no holding stage for the saccharification, meaning that a
fermenting organism, such as yeast, and enzyme(s), may be added
together. SSF may typically be carried out at a temperature from
25.degree. C. to 40.degree. C., such as from 28.degree. C. to
35.degree. C., such as from 30.degree. C. to 34.degree. C.,
preferably around about 32.degree. C. In an embodiment fermentation
is ongoing for 6 to 120 hours, in particular 24 to 96 hours.
Beer Making
[0381] The alpha-amylase variants may also be used in a beer-making
process and similar fermentations; the alpha-amylases will
typically be added during the mashing process. The process is
substantially similar to the milling, liquefaction,
saccharification, and fermentation processes described above.
Starch Slurry Processing with Stillage
[0382] Milled starch-containing material is combined with water and
recycled thin-stillage resulting in an aqueous slurry. The slurry
can comprise between 15 to 55% ds w/w (e.g., 20 to 50%, 25 to 50%,
25 to 45%, 25 to 40%, 20 to 35% and 30-36% ds). In some
embodiments, the recycled thin-stillage (backset) is in the range
of about 10 to 70% v/v (e.g., 10 to 60%, 10 to 50%, 10 to 40%, 10
to 30%, 10 to 20%, 20 to 60%, 20 to 50%, 20 to 40% and also 20 to
30%).
[0383] Once the milled starch-containing material is combined with
water and backset, the pH is not adjusted in the slurry. Further
the pH is not adjusted after the addition of a phytase and
optionally an alpha-amylase to the slurry. In an embodiment, the pH
of the slurry will be in the range of about pH 4.5 to less than
about 6.0 (e.g., pH 4.5 to 5.8, pH 4.5 to 5.6, pH 4.8 to 5.8, pH
5.0 to 5.8, pH 5.0 to 5.4, pH 5.2 to 5.5 and pH 5.2 to 5.9). The pH
of the slurry may be between about pH 4.5 and 5.2 depending on the
amount of thin stillage added to the slurry and the type of
material comprising the thin stillage. For example, the pH of the
thin stillage may be between pH 3.8 and pH 4.5.
[0384] During ethanol production, acids can be added to lower the
pH in the beer well, to reduce the risk of microbial contamination
prior to distillation.
[0385] In some embodiments, a phytase is added to the slurry. In
other embodiments, in addition to a phytase, an alpha-amylase is
added to the slurry. In some embodiments, a phytase and
alpha-amylase are added to the slurry sequentially. In other
embodiments, a phytase and alpha-amylase are added simultaneously.
In some embodiments, the slurry comprising a phytase and
optionally, an alpha-amylase, are incubated (pretreated) for a
period of about 5 minutes to about 8 hours (e.g., 5 minutes to 6
hours, 5 minutes to 4 hours, 5 minutes to 2 hours, and 15 minutes
to 4 hours). In other embodiments, the slurry is incubated at a
temperature in the range of about 40 to 115.degree. C. (e.g., 45 to
80.degree. C., 50 to 70.degree. C., 50 to 75.degree. C., 60 to
110.degree. C., 60 to 95.degree. C., 70 to 110.degree. C., 70 to
85.degree. C. and 77 to 86.degree. C.).
[0386] In other embodiments, the slurry is incubated at a
temperature of about 0 to about 30.degree. C. (e.g., 0 to
25.degree. C., 0 to 20.degree. C., 0 to 15.degree. C., 0 to
10.degree. C. and 0 to 5.degree. C.) below the starch
gelatinization temperature of the starch-containing material. In
some embodiments, the temperature is below about 68.degree. C.,
below about 65.degree. C., below about 62.degree. C., below about
60.degree. C. and below about 55.degree. C. In some embodiments,
the temperature is above about 45.degree. C., above about
50.degree. C., above about 55.degree. C. and above about 60.degree.
C. In some embodiments, the incubation of the slurry comprising a
phytase and an alpha-amylase at a temperature below the starch
gelatinization temperature is referred to as a primary (1.degree.)
liquefaction.
[0387] In one embodiment, the milled starch-containing material is
corn or milo. The slurry comprises 25 to 40% DS, the pH is in the
range of 4.8 to 5.2, and the slurry is incubated with a phytase and
optionally an alpha-amylase for 5 minutes to 2 hours, at a
temperature range of 60 to 75.degree. C.
[0388] Currently, it is believed that commercially-available
microbial alpha-amylases used in the liquefaction process are
generally not stable enough to produce liquefied starch substrate
from a dry mill process using whole ground grain at a temperature
above about 80.degree. C. at a pH level that is less than pH 5.6.
The stability of many commercially available alpha-amylases is
reduced at a pH of less than about 4.0.
[0389] In a further liquefaction step, the incubated or pretreated
starch-containing material is exposed to an increase in temperature
such as about 0 to about 45.degree. C. above the starch
gelatinization temperature of the starch-containing material (e.g.,
70.degree. C. to 120.degree. C., 70.degree. C. to 110.degree. C.,
and 70.degree. C. to 90.degree. C.) for a period of time of about 2
minutes to about 6 hours (e.g., 2 minutes to 4 hrs, 90 minutes, 140
minutes and 90 to 140 minutes) at a pH of about 4.0 to 5.5 more
preferably between 1 hour to 2 hours. The temperature can be
increased by a conventional high temperature jet cooking system for
a short period of time, for example, for 1 to 15 minutes. Then the
starch maybe further hydrolyzed at a temperature ranging from about
75.degree. C. to 95.degree. C. (e.g., 80.degree. C. to 90.degree.
C. and 80.degree. C. to 85.degree. C.) for a period of about 15 to
150 minutes (e.g., 30 to 120 minutes). In a preferred embodiment,
the pH is not adjusted during these process steps and the pH of the
liquefied mash is in the range of about pH 4.0 to pH 5.8 (e.g., pH
4.5 to 5.8, pH 4.8 to 5.4, and pH 5.0 to 5.2). In some embodiments,
a second dose of thermostable alpha-amylase is added to the
secondary liquefaction step, but in other embodiments there is no
additional dosage of alpha-amylase.
[0390] The incubation and liquefaction steps may be followed by
saccharification and fermentation steps well known in the art.
Distillation
[0391] Optionally, following fermentation, an alcohol (e.g.,
ethanol) may be extracted by, for example, distillation and
optionally followed by one or more process steps.
[0392] In some embodiments, the yield of ethanol produced by the
methods provided herein is at least 8%, at least 10%, at least 12%,
at least 14%, at least 15%, at least 16%, at least 17% and at least
18% (v/v) and at least 23% v/v. The ethanol obtained according to
the process provided herein may be used as, for example, fuel
ethanol, drinking ethanol, i.e., potable neutral spirits, or
industrial ethanol.
By-Products
[0393] Left over from the fermentation is the grain, which is
typically used for animal feed either in liquid or dried form. In
further embodiments, the end product may include the fermentation
co-products such as distiller's dried grains (DDG) and distiller's
dried grain plus solubles (DDGS), which may be used, for example,
as an animal feed.
[0394] Further details on how to carry out liquefaction,
saccharification, fermentation, distillation, and recovery of
ethanol are well known to the skilled person.
[0395] According to the process provided herein, the
saccharification and fermentation may be carried out simultaneously
or separately.
Pulp and Paper Production
[0396] The alpha-amylase variants may also be used in the
production of lignocellulosic materials, such as pulp, paper and
cardboard, from starch reinforced waste paper and cardboard,
especially where re-pulping occurs at pH above 7 and where amylases
facilitate the disintegration of the waste material through
degradation of the reinforcing starch. The alpha-amylase variants
are especially useful in a process for producing a papermaking pulp
from starch-coated printed-paper. The process may be performed as
described in WO 95/14807, comprising the following steps:
[0397] a) disintegrating the paper to produce a pulp,
[0398] b) treating with a starch-degrading enzyme before, during or
after step a), and
[0399] c) separating ink particles from the pulp after steps a) and
b).
[0400] The alpha-amylase variants may also be useful in modifying
starch where enzymatically modified starch is used in papermaking
together with alkaline fillers such as calcium carbonate, kaolin
and clays. With the alpha-amylase variants it is possible to modify
the starch in the presence of the filler thus allowing for a
simpler integrated process.
[0401] Desizing of Textiles, Fabrics and Garments
[0402] The alpha-amylase variants may also be very useful in
textile, fabric or garment desizing. In the textile processing
industry, alpha-amylases are traditionally used as auxiliaries in
the desizing process to facilitate the removal of starch-containing
size, which has served as a protective coating on weft yarns during
weaving. Complete removal of the size coating after weaving is
important to ensure optimum results in the subsequent processes, in
which the fabric is scoured, bleached and dyed. Enzymatic starch
breakdown is preferred because it does not involve any harmful
effect on the fiber material. In order to reduce processing cost
and increase mill throughput, the desizing process is sometimes
combined with the scouring and bleaching steps. In such cases,
non-enzymatic auxiliaries such as alkali or oxidation agents are
typically used to break down the starch, because traditional
alpha-amylases are not very compatible with high pH levels and
bleaching agents. The non-enzymatic breakdown of the starch size
leads to some fiber damage because of the rather aggressive
chemicals used. Accordingly, it would be desirable to use the
alpha-amylase variants as they have an improved performance in
alkaline solutions. The alpha-amylase variants may be used alone or
in combination with a cellulase when desizing cellulose-containing
fabric or textile.
[0403] Desizing and bleaching processes are well known in the art.
For instance, such processes are described in, e.g., WO 95/21247,
U.S. Pat. No. 4,643,736, EP 119920, which are hereby incorporated
by reference.
Cleaning Processes and Detergent Compositions
[0404] The alpha-amylase variants may be added as a component of a
detergent composition for various cleaning or washing processes,
including laundry and dishwashing. For example, the variants may be
used in the detergent compositions described in WO 96/23874 and WO
97/07202.
[0405] The alpha-amylase variants may be incorporated in detergents
at conventionally employed concentrations. For example, a variant
of the invention may be incorporated in an amount corresponding to
0.00001-10 mg (calculated as pure, active enzyme protein) of
alpha-amylase per liter of wash/dishwash liquor using conventional
dosing levels of detergent.
[0406] The detergent composition may for example be formulated as a
hand or machine laundry detergent composition, including a laundry
additive composition suitable for pretreatment 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 hand or machine
dishwashing operations.
[0407] The detergent composition may further comprise one or more
other enzymes, such as a lipase, peroxidase, protease, another
amylolytic enzyme, e.g., another alpha-amylase, glucoamylase,
maltogenic amylase, CGTase, cellulase, mannanase (such as
Mannaway.TM. from Novozymes, Denmark)), pectinase, pectin lyase,
cutinase, and/or laccase.
[0408] In general the properties of the chosen enzyme(s) should be
compatible with the selected detergent (i.e., pH-optimum,
compatibility with other enzymatic and non-enzymatic ingredients,
etc.), and the enzyme(s) should be present in effective
amounts.
[0409] 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, e.g., a separate additive or a
combined additive, can be formulated, e.g., granulate, a liquid, a
slurry, etc. Preferred detergent additive formulations are
granulates, in particular non-dusting granulates, liquids, in
particular stabilized liquids, or slurries.
[0410] 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 1000 to 20000; ethoxylated
nonyl-phenols 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 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 238216.
[0411] 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, or non-aqueous.
[0412] The detergent composition comprises one or more surfactants,
which may be non-ionic including semi-polar and/or anionic and/or
cationic and/or zwitterionic. The surfactants are typically present
at a level of from about 0.1% to 60% by weight.
[0413] When included therein the detergent will usually 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.
[0414] When included therein the detergent will usually contain
from about 0.2% to about 40% of a non-ionic surfactant such as
alcohol ethoxylate, nonyl-phenol ethoxylate, alkylpolyglycoside,
alkyldimethylamine-oxide, ethoxylated fatty acid monoethanol-amide,
fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or
N-acyl N-alkyl derivatives of glucosamine ("glucamides").
[0415] 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,
diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid,
soluble silicates or layered silicates (e.g., SKS-6 from
Hoechst).
[0416] The detergent may comprise one or more polymers. Examples
are carboxymethylcellulose, poly(vinyl-pyrrolidone), poly (ethylene
glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide),
poly(vinylimidazole), polycarboxylates such as polyacrylates,
maleiclacrylic acid copolymers and lauryl methacrylate/acrylic acid
co-polymers.
[0417] The detergent may contain a bleaching system, which may
comprise a H.sub.2O.sub.2 source such as perborate or percarbonate
which may be combined with a peracid-forming bleach activator such
as tetraacetylethylenediamine or nonanoyloxyben-zenesul-fonate.
Alternatively, the bleaching system may comprise peroxy acids of,
e.g., the amide, imide, or sulfone type.
[0418] The enzyme(s) 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, e.g., an aromatic
borate ester, or a phenyl boronic acid derivative such as
4-formylphenyl boronic acid, and the composition may be formulated
as described in, e.g., WO 92/19708 and WO 92/19709.
[0419] 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 re-deposition agents, dyes,
bactericides, optical brighteners, hydrotropes, tarnish inhibitors,
or perfumes.
[0420] The detergent compositions may comprise any enzyme in an
amount corresponding to 0.01-100 mg of enzyme protein per liter of
wash liquor, preferably 0.055 mg of enzyme protein per liter of
wash liquor, in particular 0.1-1 mg of enzyme protein per liter of
wash liquor.
[0421] One or more of the variant enzymes described herein may
additionally be incorporated in the detergent formulations
disclosed in WO 97/07202, which is hereby incorporated as
reference.
[0422] This disclosure includes further detail in the following
examples, which are not in any way intended to limit the scope of
what is claimed. The following examples are thus offered to
illustrate, but not to limit what is claimed.
EXAMPLES
Materials and Methods
Fermentation of Alpha-Amylases and Variants
[0423] Fermentation may be performed by methods well known in the
art or as follows:
[0424] A B. subtilis strain harboring the relevant expression
plasmid is streaked on a LB-agar plate with a relevant antibiotic,
and grown overnight at 37.degree. C.
[0425] The colonies are transferred to 100 ml BPX media
supplemented with a relevant antibiotic (for instance 10 mg/l
chloroamphinicol) in a 500 ml shaking flask.
Composition of BPX medium:
TABLE-US-00004 Potato starch 100 g/l Barley flour 50 g/l BAN 5000
SKB 0.1 g/l Sodium caseinate 10 g/l Soy Bean Meal 20 g/l
Na.sub.2HPO.sub.4, 12H.sub.2O 9 g/l Pluronic .TM. 0.1 g/l BAN is
Bacillus amyloliquefaciens alpha-amylase sold by Novozymes.
[0426] The culture is shaken at 37.degree. C. at 270 rpm for 4 to 5
days.
[0427] Cells and cell debris are removed from the fermentation
broth by centrifugation at 4500 rpm in 20-25 minutes. Afterwards
the supernatant is filtered to obtain a completely clear solution.
The filtrate is concentrated and washed on an UF-filter (10000 cut
off membrane) and the buffer is changed to 20 mM acetate pH 5.5.
The UF-filtrate is applied on an S-sepharose F.F. and elution is
carried out by step elution with 0.2 M NaCl in the same buffer. The
eluate is dialyzed against 10 mM Tris, pH 9.0 and applied on a
Q-sepharose F.F. and eluted with a linear gradient from 0-0.3 M
NaCl over 6 column volumes. The fractions, which contain the
activity (measured by the Phadebas assay) are pooled, pH is
adjusted to 7.5 and remaining color is removed by a treatment with
0.5% w/vol active coal in 5 minutes.
Phadebas Assay
[0428] Alpha-amylase activity is determined by a method employing
Phadebas.RTM. tablets as substrate. Phadebas tablets (Phadebas.RTM.
Amylase Test, supplied by Magle Life Sciences, Lund, Sweden)
contain a cross-linked insoluble blue-colored starch polymer, which
has been mixed with bovine serum albumin and a buffer substance and
tabletted.
[0429] For every single measurement one tablet is suspended in a
tube containing 5 mL 50 mM Britton-Robinson buffer (50 mM acetic
acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl.sub.2,
0.01% TRITON.RTM. X100, pH adjusted to the value of interest with
NaOH). This is the substrate solution. The alpha-amylase to be
tested is diluted in 50 mM Britton-Robinson buffer. This is the
amylase solution. The test is performed at constant temperature,
e.g., at room temperature, 37.degree. C. or 50.degree. C. The
insoluble blue-colored starch polymer is hydrolyzed by the
alpha-amylase giving soluble blue fragments. The absorbance of the
resulting blue solution, measured spectrophotometrically at 620 nm,
is a function of the alpha-amylase activity.
[0430] 575 microliters substrate solution is equilibrated at the
selected temperature for 5 minutes. The hydrolysis is started by
adding 25 microliters amylase solution to the substrate solution
and incubating the sample under gentle mixing for 15 minutes at the
selected temperature. The reaction is stopped by adding 100
microliters 1 M NaOH and immediately cooling on an ice bath after
mixing. After centrifugation at 500 g.sub.av for 3 minutes, 200
microliters of the supernatant is transferred to a microtiter
plate, and the absorbance at 620 nm is read (A.sub.amyl). The blind
is prepared as described but where the 25 microliters amylase
solution is replaced by 25 microliters 50 mM Britton-Robison
buffer. The absorbance of the blind at 620 nm is A.sub.b. The
standard curve is prepared similarly by making a dilution series of
Termamyl with a known activity and measuring the release of blue
color to the solution as described above. The absorbance of the
standards at 620 nm is A.sub.s. The standard curve is a plot of
A.sub.s-A.sub.b against the Termamyl activity in the sample. The
activity of the amylase of interest can be determined by comparing
A.sub.amyl-A.sub.b to the Termamyl standard curve.
[0431] It is important that the measured 620 nm absorbance after 15
minutes of incubation (testing time) is in the range of 0.2 to 2.0
absorbance units at 620 nm. In this absorbance range there is
linearity between activity and absorbance (Lambert-Beer law). The
dilution of the enzyme (both amylase of interest and standard) must
therefore be adjusted to fit this criterion. Under a specified set
of conditions (temperature, pH, reaction time, buffer conditions),
1 mg of a given alpha-amylase will hydrolyze a certain amount of
substrate and a blue color will be produced.
G7-pNP Amylase Assay
[0432] Alpha-amylase activity may also be determined by a method
employing the PNP-G7 substrate. PNP-G7 which is an abbreviation for
p-nitrophenyl-alpha-D-maltoheptaoside, is a blocked oligosaccharide
which can be cleaved by an endo-amylase. Following the cleavage,
the alpha-glucosidase included in the kit digests the substrate to
liberate a free PNP molecule which has a yellow color and thus can
be measured by visible spectophometry at .lamda.=405 nm (400-420
nm). Kits containing PNP-G7 substrate and alpha-glucosidase is
manufactured by Roche/Hitachi (cat. no. 11876473). The G7-PNP
substrate from this kit contains 22 mmol/L 4,6-ethylidene-G7-PNP
and 52.4 mmol/L HEPES
(2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid), pH 7.0)
and the alpha-glucosidase contains 52.4 mmol/L HEPES, 87 mmol/L
NaCl, 12.6 mmol/L MgCl.sub.2, 0.075 mmol/L CaCl.sub.2, .gtoreq.4
kU/L alpha-glucosidase).
[0433] The amylase sample to be analyzed is diluted in 50 mM EPPS
(4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (Sigma,
E9502)), 0.01% TRITON.RTM. X100, 1 mM CaCl.sub.2, pH 7.0. Before
use substrate working solution was made by mixing 1 mL of the
alpha-glucosidase containing reagent with 0.2 mL
4,6-ethylidene-G7-PNP containing reagent from kit. Immediately
after incubation of samples in PCR machine the samples are diluted
10 times in residual activity buffer (50 mM EPPS, 0.01% TRITON.RTM.
X100, 1 mM CaCl.sub.2, pH7.0). The assay is performed by
transferring 20 microliters diluted enzyme samples to a 96 well
microtiter plate and adding 80 microliters substrate working
solution. The solution is mixed and pre-incubated 1 minute at room
temperature and absorption is measured every 20 sec. over 5 minutes
at OD 405 nm.
[0434] The slope (absorbance per minute) of the time dependent
absorption-curve is directly proportional to the specific activity
(activity per mg enzyme) of the alpha-amylase in question under the
given set of conditions. The amylase sample should be diluted to a
level where the slope is below 0.4 absorbance units per minute.
Enzchek.RTM. Amylase Activity Assay
[0435] Alpha-amylase activity may also be determined by a method
employing the EnzChek.RTM. substrate. The substrate in the
EnzChek.RTM. Ultra Amylase Assay Kit (E33651, Invitrogen, La Jolla,
Calif., USA) is a corn starch derivative, DQ.TM. starch, which is
corn starch labeled with BODIPY.RTM. FL dye to such a degree that
fluorescence is quenched.
[0436] One vial containing approx. 1 mg lyophilized substrate is
dissolved in 100 microliters of 50 mM sodium acetate (pH 4.0). The
vial is vortexed for 20 seconds and left at room temperature, in
the dark, with occasional mixing until dissolved. Then 900
microliters of 100 mM acetate, 0.01% (w/v) TRITON.RTM. X100, 0.12
mM CaCl.sub.2, pH 5.5 is added, vortexed thoroughly and stored at
room temperature, in the dark until ready to use. The substrate
working solution is prepared by diluting 10-fold in residual
activity buffer (100 mM acetate, 0.01% (w/v) TRITON.RTM. X100, 0.12
mM CaCl.sub.2, pH 5.5) giving a substrate concentration of 100
micrograms/ml. Immediately after incubation the enzyme is diluted
to a concentration of 20 ng enzyme protein/mL in 100 mM acetate,
0.01% (W/v) TRITON.RTM. X100, 0.12 mM CaCl.sub.2, pH 5.5.
[0437] For the assay, 25 microliters of the substrate working
solution is mixed for 10 second with 25 microliters of the diluted
enzyme in a black 384 well microtiter plate. The fluorescence
intensity is measured (excitation: 485 nm, emission: 555 nm) once
every minute for 15 minutes in each well at 25.degree. C. and the
V.sub.max is calculated as the slope of the plot of fluorescence
intensity against time. The plot should be linear and the residual
activity assay has been adjusted so that the diluted reference
enzyme solution is within the linear range of the activity
assay.
Example 1
Preparation of Hybrids
[0438] The following hybrids of the calcium-sensitive alpha-amylase
having the sequence shown in SEQ ID NO: 7 and the
calcium-insensitive alpha-amylase having the sequence shown in SEQ
ID NO: 13 were prepared.
Hybrid 1: the amino acid residues 106-215 of SEQ ID NO: 7 were
removed and replaced with the amino acid residues 103-211 of SEQ ID
NO: 13, which results in SEQ ID NO: 17, and the following
alterations were introduced: E182*, N183*, E188W, N189E and D192T
(using SEQ ID NO: 17 numbering), which correspond to E181*, N182*,
E187W, N188E and D191T using SEQ ID NO: 27 numbering. The sequence
of this hybrid is shown in SEQ ID NO: 18. Hybrid 2: the amino acid
residues 106-214 of SEQ ID NO: 7 were removed and replaced with the
amino acid residues 103-210 of SEQ ID NO: 13, which results in SEQ
ID NO: 19, and the following alterations were introduced: E182*,
N183*, E188W, N189E and D192T (using SEQ ID NO: 19 numbering),
which correspond to E181*, N182*, E187W, N188E and D191T using SEQ
ID NO: 27 numbering. The sequence of this hybrid is shown in SEQ ID
NO: 20. Hybrid 3: the amino acid residues 106-213 of SEQ ID NO: 7
were removed and replaced with the amino acid residues 103-209 of
SEQ ID NO: 13, which results in SEQ ID NO: 21, and the following
alterations were introduced: E182*, N183*, E188W, N189E and D192T
(using SEQ ID NO: 21 numbering), which correspond to E181*, N182*,
E187W, N188E and D191T using SEQ ID NO: 27 numbering. The sequence
of this hybrid is shown in SEQ ID NO: 22.
Example 2
Stability in the Presence of Chelator
[0439] Enzyme samples were incubated in buffer pH 8.0 (50 mM EPPS,
0.01% TRITON.RTM.X100, pH 8.0) with 1.5% final concentration of
DTPA at 49.degree. C. for 1 hour and reference samples were
incubated at 4.degree. C. for 1 hour. In addition, enzyme samples
were incubated in buffer pH 10.0 (50 mM EPPS, 0.01% TRITON.RTM.
X100, pH 10.0) with 1.5% final concentration of DTPA at 42.degree.
C. for 1 hour and reference samples were incubated at 4.degree. C.
for 1 hour.
[0440] For the determination of amylase stability in buffer pH 8
and pH 10 with DTPA the enzymes to be tested were adjusted to 0.25
and 0.5 mg enzyme protein/mL by diluting in 5 mM EPPS, 0.01%
TRITON.RTM. X100, pH 8.0.
[0441] 160 microliters stability buffer (50 mM EPPS, 0.01%
TRITON.RTM. X100, 1.875% DTPA, pH 8.0 or pH 10.0) and 40
microliters of the amylase solution were transferred to a 96-well
PCR microtiter plate in duplicate and the content was mixed for 1
minute. Final concentration of DTPA was 1.5% in each well. 20
microliters from each well was transferred to a new PCR microtiter
plate, which was placed at 4.degree. C. (reference sample). The PCR
MTP was incubated in PCR machine for 1 hour at 49.degree. C. when
buffer had pH 8.0 (pH 8, 49.degree. C. samples) and for 1 hour at
42.degree. C. when buffer had pH 10.0 (pH 10, 42.degree. C.
samples).
[0442] Immediately after incubation, the samples on PCR plates were
analyzed for amylase activity as described in the G7-pNP Amylase
assay. It should be noted that in order to reduce interference from
DTPA on the assay, both reference and pH 8, 49.degree. C.
samples/pH 10, 42.degree. C. samples were diluted to the same
concentration before being analyzed for residual activity. The
activity of both the reference samples and the pH 8, 49.degree. C.
samples or pH 10, 42.degree. C. samples were determined on the same
96 well plate. The residual activity was calculated as
100*V.sub.max(pH 8, 42.degree. C. or pH 10, 49.degree. C.
sample)/V.sub.max(reference sample).
TABLE-US-00005 Residual activity Residual activity in % after 1 in
% after 1 hour 49.degree. C., pH hour 42.degree. C., pH 8.0 and
1.5% 10.0 and 1.5% Enzyme DTPA DTPA SEQ ID NO: 7 with the 20 18
deletions D183* + T184* (SEQ ID NO: 7 numbering) SEQ ID NO: 7 1 9
SP707 (SEQ ID NO: 8) 1 3 Hybrid 1 100 102 Hybrid 2 102 102 Hybrid 3
103 103
[0443] Hybrids 1, 2 and 3 are highly stable and have 100% residual
activity after incubation for 1 hour at both pH 8, 49.degree. C.
and pH 10, 42.degree. C. In comparison SEQ ID NO: 7 with the
deletions D183*+T184* has less than 20% residual activity at these
conditions and SEQ ID NO: 7 and SP707 have even less residual
activity.
Example 3
Additional Alpha-Amylases
[0444] The following alpha-amylases were prepared:
Hybrid 4: the amino acid residues 106-212 of SEQ ID NO: 5 were
removed and replaced with the amino acid residues 103-208 of SEQ ID
NO: 13, which results in SEQ ID NO: 23, and the following
alterations were introduced: E182*, N183*, E188W, N189E and D192T
(using SEQ ID NO: 23 numbering), which correspond to E181*, N182*,
E187W, N188E and D191T in SEQ ID NO: 27 numbering. The sequence of
this hybrid is shown in SEQ ID NO: 24. Hybrid 5: the amino acid
residues 106-212 of SEQ ID NO: 8 were removed and replaced with the
amino acid residues 103-208 of SEQ ID NO: 13, which results in SEQ
ID NO: 25, and the following alterations were introduced: E182*,
N183*, E188W, N189E and D192T (using SEQ ID NO: 25 numbering),
which correspond to E181*, N182*, E187W, N188E and D191T in SEQ ID
NO: 27 numbering. The sequence of this hybrid is shown in SEQ ID
NO: 26. Hybrids 4 and 5 (SEQ ID NOS: 24 and 26), a variant of SEQ
ID NO: 5 with the alterations E182*, N183*, E188W, N189E and D192T
(using SEQ ID NO: 5 numbering), which correspond to E181*, N182*,
E187W, N188E and D191T in SEQ ID NO: 27 numbering, and the
alpha-amylase of SEQ ID NO: 8 were incubated with DTPA as described
in Example 2. The results show that hybrids 4 and 5 had almost 100%
residual activity after the incubations, whereas the other
alpha-amylases lost most of their activity during the
incubations.
Example 4
Stability of Alpha-Amylase Variants
[0445] The stability of a reference alpha-amylase with the amino
acid sequence of SEQ ID NO: 28 (a hybrid of Bacillus
stearothermophilus and Bacillus circulans alpha-amylases (SEQ ID
NO: 27) with the alterations
E181*+G182*+E187W+N188E+D191T+D407N+D430N+P432D) and alpha-amylase
variants thereof was determined by incubating the reference
alpha-amylase and variants at pH 4.5 and 5.5 and temperatures of
75.degree. C. and 85.degree. C. with 0.12 mM CaCl.sub.2 followed by
residual activity determination using the EnzChek.RTM. substrate
(EnzChek.RTM. Ultra Amylase assay kit, E33651, Molecular Probes,
Invitrogen, La Jolla, Calif., USA).
[0446] Purified enzyme samples were diluted to working
concentrations of 0.5 and 1 or 5 and 10 ppm (micrograms/ml) in
enzyme dilution buffer (10 mM acetate, 0.01% TRITON.RTM. X100, 0.12
mM CaCl.sub.2, pH 5.0). Twenty microliters enzyme sample was
transferred to 48-well PCR MTP and 180 microliters stability buffer
(150 mM acetate, 150 mM MES, 0.01% TRITON.RTM. X100, 0.12 mM
CaCl.sub.2, pH 4.5 or 5.5) was added to each well and mixed. The
assay was performed using two concentrations of enzyme in
duplicates. Before incubation at 75.degree. C. or 85.degree. C., 40
microliters was withdrawn and stored on ice as reference samples.
Incubation was performed in a PCR machine for 30/45 minutes (pH 4.5
and 75.degree. C.), 45/60 minutes (pH 5.5 and 75.degree. C.), 5/10
minutes (pH 4.5 and 85.degree. C.) and 10 minutes (pH 5.5 and
85.degree. C.).
[0447] After incubation, the reference samples and samples from the
PCR machine were diluted to 20 ng/ml in residual activity buffer
(100 mM acetate, 0.01% TRITON.RTM. X100, 0.12 mM CaCl.sub.2, pH
5.5) and 25 microliters diluted enzyme was transferred to black
384-MTP. Residual activity was determined using the EnzChek.RTM.
substrate as described in the section for the Enzchek.RTM. amylase
activity assay. In brief, 25 microliters substrate working solution
(100 micrograms/ml) is added to each well with diluted enzyme.
Fluorescence was determined every minute for 15 minutes using
excitation filter at 485-P nm and emission filter at 555 nm
(fluorescence reader is Polarstar, BMG). The residual activity was
normalized to control samples for each setup.
[0448] Assuming logarithmic decay the half life time (T1/2(min))
was calculated using the equation: T1/2(min)=T(min)*LN(0.5)/LN(%
RA/100), where T is the assay incubation time in minutes, and % RA
is the % residual activity determined in the assay.
[0449] Using this assay setup the half life time was determined for
the reference alpha-amylase and variants thereof as shown in Table
1.
TABLE-US-00006 TABLE 1 T1/2 (min) T1/2 (min) T1/2 (min) T1/2 (min)
(pH 4.5, 75.degree. C., (pH 5.5, 75.degree. C., (pH 4.5, 85.degree.
C., (pH 5.5, 85.degree. C., Mutations (SEQ ID NO: 27 0.12 mM 0.12
mM 0.12 mM 0.12 mM numbering) CaCl.sub.2) CaCl.sub.2) CaCl.sub.2)
CaCl.sub.2) Reference Alpha-Amylase 14 .+-. 2 33 .+-. 4 1.8 .+-.
0.2 4.5 .+-. 0.2 Reference Alpha-Amylase 49 4.2 13 with the
substitutions M8L + N105D + K184A Reference Alpha-Amylase 58 4.7 15
with the substitutions A27Q + Q86S + A90S + N105D + K184A Reference
Alpha-Amylase 59 6.8 28 with the substitutions S34K + N105D + K184A
+ S242Q Reference Alpha-Amylase 50 5.2 15 with the substitutions
R52G + S53Y + N105D + K184A Reference Alpha-Amylase 91 11.3 29 with
the substitutions V59A + A100G + N105D + T164V + K184A + M307L
Reference Alpha-Amylase >120 14.1 39 with the substitutions V59A
+ N105D + Q150T + T164V + K184A + S242Q + M307L Reference
Alpha-Amylase 108 9.4 24 with the substitutions T80D + N105D +
T164V + K184A + M307L Reference Alpha-Amylase 51 6.3 19 with the
substitutions A91L + N105D + K184A Reference Alpha-Amylase 69 7.3
25 with the substitutions A100L + N105D + T164V + K184A + Y222V +
M307L Reference Alpha-Amylase 32 129 4 <15 with the substitution
N105D Reference Alpha-Amylase 45 5.5 21 with the substitutions
N105D + K117D + Q150T + K184A + S301K + G303R + A304D Reference
Alpha-Amylase 20 3 6 with the substitutions N105D + E129V + R177L +
A179E Reference Alpha-Amylase 64 5 19 with the substitutions N105D
+ E132D + K184A Reference Alpha-Amylase 33 3.3 9 with the
substitutions N105D + F134E + K184A Reference Alpha-Amylase 44 3.9
16 with the substitutions N105D + E135N + A179N + K184A Reference
Alpha-Amylase 27 117 4 10 with the substitutions N105D + Q150T
Reference Alpha-Amylase 59 7.3 20 with the substitutions N105D +
Q150T + T164V + F166W + A168E + E171K + K184A + N407D + N430D +
D432P Reference Alpha-Amylase >120 13.6 >40 with the
substitutions N105D + Q150T + T164V + K184A + S242Q + M284T + M307L
Reference Alpha-Amylase 114 13.4 >40 with the substitutions
N105D + Q150T + T164V + K184A + S242Q + M284T + N407D Reference
Alpha-Amylase 37 141 5 13 with the substitutions N105D + Q150T +
F166W + A168E + E171K Reference Alpha-Amylase 68 7.9 21 with the
substitutions N105D + Q150T + F166W + A168E + E171K + K184A
Reference Alpha-Amylase 46 207 5.1 15 with the substitutions N105D
+ Q150T + K184A Reference Alpha-Amylase 42 4.7 14 with the
substitutions N105D + Q150T + K184A + Y206M Reference Alpha-Amylase
41 111 4.6 16 with the substitutions N105D + Q150T + K184A + S301K
+ G303R + A304D Reference Alpha-Amylase 50 5.2 15 with the
substitutions N105D + N157Y + E159Y + H160Y + K184A + H208Y + D210Y
Reference Alpha-Amylase 38 4.3 13 with the substitutions N105D +
H160Y + K184A Reference Alpha-Amylase 71 7.5 18 with the
substitutions N105D + T164V + K184A + Y222V + M307L Reference
Alpha-Amylase >120 11.7 39 with the substitutions N105D + T164V
+ K184A + S242Q + M284T + M307L Reference Alpha-Amylase 68 5.7 19
with the substitutions N105D + T164V + K184A + F244Y + M284T +
M307L Reference Alpha-Amylase 105 12 >40 with the substitutions
N105D + T164V + K184A + M284Q + M307L Reference Alpha-Amylase
>120 14.6 >40 with the substitutions N105D + T164V + K184A +
M284V + M307L Reference Alpha-Amylase 70 230 8.7 23 with the
substitutions N105D + T164V + K184A + M307L Reference Alpha-Amylase
49 140 4 13 with the substitutions N105D + F166W + A168E + E171K
Reference Alpha-Amylase 33 112 4.8 14 with the substitutions N105D
+ F166W + A168E + E171K + S301K + G303R + A304D Reference
Alpha-Amylase 68 5.8 19 with the substitutions N105D + A179D +
K184A Reference Alpha-Amylase 56 5.3 >40 with the substitutions
N105D + A179N + K184A Reference Alpha-Amylase 56 6 28 with the
substitutions N105D + A179Q + K184A Reference Alpha-Amylase 40 153
5.6 14 with the substitutions N105D + K184A Reference Alpha-Amylase
55 5.8 15 with the substitutions N105D + K184A + D210V Reference
Alpha-Amylase 14 37 1.4 4 with the substitutions N105D + K184A +
A235T Reference Alpha-Amylase 24 83 2.9 9 with the substitutions
N105D + K184A + S242E Reference Alpha-Amylase 60 183 7 19 with the
substitutions N105D + K184A + S242Q Reference Alpha-Amylase 91 10.8
30 with the substitutions N105D + K184A + S242Q + A235W Reference
Alpha-Amylase 91 11 30 with the substitutions N105D + K184A + S242Q
+ G282W Reference Alpha-Amylase 64 5.2 16 with the substitutions
N105D + K184A + P245A Reference Alpha-Amylase 46 5.5 16 with the
substitutions N105D + K184A + P245K Reference Alpha-Amylase 38 110
4.7 15 with the substitutions N105D + K184A + S301K + G303R + A304D
Reference Alpha-Amylase 46 5.5 16 with the substitutions N105D +
K184A + P348T Reference Alpha-Amylase 47 5.2 18 with the
substitutions N105D + K184A + P386E Reference Alpha-Amylase 46 5.4
18 with the substitutions N105D + K184A + P386Q Reference
Alpha-Amylase 49 5.5 17 with the substitutions N105D + K184A +
P386T Reference Alpha-Amylase 44 3.9 16 with the substitutions
N105D + K184A + P386V Reference Alpha-Amylase 52 5.7 17 with the
substitutions N105D + K184A + L388I Reference Alpha-Amylase 64 5.2
16 with the substitutions N105D + K184A + L388V Reference
Alpha-Amylase 45 5.6 17 with the substitutions N105D + K184A +
N407D + N430D + D432P Reference Alpha-Amylase 52 5.5 17 with the
substitutions N105D + K184A + D432P Reference Alpha-Amylase 47 5.2
18 with the substitutions N105D + K184A + T459P Reference
Alpha-Amylase 8 27 4 with the substitutions N105D + Y206K Reference
Alpha-Amylase 18 81 2.2 11 with the substitutions N105D + Y206M
Reference Alpha-Amylase 37 4.1 11 with the substitutions N105D +
K220P + N224L Reference Alpha-Amylase 23 116 2.9 10 with the
substitutions N105D + S301K + G303R + A304D Reference Alpha-Amylase
23 2 6 with the substitutions V115W + F134Y + E135Q + K169S + G170R
+ R172L + G174R + F176Y Reference Alpha-Amylase 17 37 2 5 with the
substitution K117D Reference Alpha-Amylase 9 21 with the
substitutions E129V + Q150T Reference Alpha-Amylase 21 78 2.2 7
with the substitutions F134Y + E135Q + K169S + G170R + R172L +
G174R + F176Y Reference Alpha-Amylase 32 90 4 10 with the
substitution Q150T Reference Alpha-Amylase 21 49 3 7 with the
substitution T164V Reference Alpha-Amylase 30 52 3.7 <15
with the substitution K184A Reference Alpha-Amylase with the
substitutions K184A + I204L Reference Alpha-Amylase 8 with the
substitutions K184A + I270L Reference Alpha-Amylase 23 74 3 9 with
the substitution Y206M Reference Alpha-Amylase 8 18 3 with the
substitution S242E Reference Alpha-Amylase 8 18 3 with the
substitutions S301K + G303R + A304D Reference Alpha-Amylase 8 20 3
with the substitution G475K Reference Alpha-Amylase 15 with the
substitution G475Q
[0450] The results demonstrate that the alpha-amylase variants have
a significantly greater half-life and stability than the reference
alpha-amylase.
Example 5
Production of Ethanol Using Alpha-Amylase Variants
[0451] Three small scale mashes of a Bacillus stearothermophilus
alpha-amylase variant sold by Novozymes under the name LIQUOZYME
SC.RTM. and two alpha-amylase variants described in Example 4 were
prepared as follows: about 54 g corn ground, about 51 g tap water,
and about 45 g backset were mixed in a 250 mL plastic bottle to a
total slurry weight of 150 g. The pH of the corn slurry was
adjusted to 4.5. The enzymes were added to the mashes at 2
micrograms of amylase per gram of dry solids. For liquefaction, the
alpha-amylases were added to the bottles and the bottles were mixed
thoroughly and placed into a preheated 85.degree. C. water bath.
The samples were held in the water bath for 2 hours at pH 4.5 while
being shaken every 10 minutes for the first 30 minutes and every 30
minutes thereafter for the remainder of the 2 hour liquefaction.
The samples were then cooled in an ice bath; pH was adjusted to
5.0, and 0.75 mL urea and 0.45 mL penicillin were added to reach
final concentrations of 1000 and 3 ppm in the mashes, respectively.
The samples were then subjected to simultaneous saccharification
and fermentation (SSF) with Spirizyme Fuel.RTM. (a glucoamylase
product sold by Novozymes).
[0452] Five gram aliquots of the mashes were transferred into
pre-weighed conical centrifuge tubes, using 5 replicates per mash.
SSF was then performed on these mashes in a 32.degree. C. water
bath for 54 hours using Spirizyme Fuel.RTM.. The glucoamylase dose
was 0.50 AGU/g DS for all fermentations. The CO.sub.2 weight loss
during SSF was measured and ethanol was quantified using HPLC after
54 hours of SSF. The average 54 hour HPLC SSF data are provided in
Table 2 below.
TABLE-US-00007 TABLE 2 Ethanol Yields After 54 Hours Fermentation
Ethanol, Alpha-Amylase g/L Std dev. LIQUOZYME SC .RTM. 105.5946
0.3708 Reference Alpha-Amylase 115.6339 0.5562 disclosed in Example
4 with the substitution N105D Reference Alpha-Amylase 116.7224
0.8226 disclosed in Example 4 with the substitution K184A
[0453] The results demonstrate that the use of the alpha-amylase
variants resulted in a significantly greater yield of ethanol
relative to LIQUOZYME SC.RTM..
Example 6
Wash Performance in a Detergent
[0454] In order to assess the wash performance of alpha-amylases in
a detergent, washing experiments were performed. The performance of
hybrids 1, 2 and 3 of Example 1 was tested using the Mini Wash
Assay and compared to the alpha-amylase having the amino acid
sequence of SEQ ID NO: 7 with the deletions D183*+T184* (SEQ ID NO:
7 numbering). In this test, the wash performance of
enzyme-detergent solutions can be examined at several enzyme
dosages simultaneously.
Description of the Mini Wash Assay
[0455] The Mini Wash has a number of beakers with each beaker able
to hold up to 80 ml enzyme-detergent solution. Water hardness is
adjusted to 10.degree. dH by addition of CaCl.sub.2, MgCl.sub.2,
and NaHCO.sub.3 to the test system. A textile sample, in this case
CS-28, is attached to a frame designed to dip the textile into the
enzyme-detergent solution with a frequency of 40 submersions per
min. The temperature of the enzyme-detergent solution is controlled
during wash. After wash the textile is rinsed in running tap water
and subsequently dried in the dark. The wash performance of the
enzyme-detergent solution is evaluated by measuring the remission
at 460 nm with a ZEISS MCS 521 VIS Spectrophotometer.
Textiles:
[0456] CS-28 is a technical rice starch stained cotton textile that
can be obtained from Center For Test materials BV, P.O. Box 120,
3133 KT Vlaardingen, the Netherlands.
[0457] The experiment was conducted under the experimental
conditions specified below:
TABLE-US-00008 Detergent Commercial Tide 2X Ultra, inactivated by
boiling for 15 minutes Detergent dosage 0.78 g/L Test solution
volume 60 mL pH After wash pH was measured to 8.3 Wash time 20
minutes followed by 5 minutes rinse Temperature 40.degree. C. Water
hardness 10.degree.dH, Ca/Mg 3:1 Enzyme concentration 0; 0.03;
0.06; 0.12; 0.20; 1.0 mg purified in test solution enzyme protein/L
Enzymes SEQ ID NO: 7 with the deletions D183* + T184* Hybrid 1
Hybrid 2 Hybrid 3 Test material CS-28 (Rice starch on cotton)
Results and Discussion:
[0458] The wash performance of the alpha-amylases was normalized to
the wash performance of the alpha-amylase having the amino acid
sequence of SEQ ID NO: 7 with the deletions D183*+T184*.
TABLE-US-00009 SEQ ID NO: 7 Dose (mg with the enzyme deletions
protein/L) D183* + T184* Hybrid 1 Hybrid 2 Hybrid 3 0 100.0 100.0
100.0 100.0 0.03 100.0 108.8 105.6 93.7 0.06 100.0 109.4 103.4 92.9
0.12 100.0 108.7 104.5 90.4 0.2 100.0 106.2 99.6 94.4 1 100.0 101.2
99.2 96.7
Example 7
Preparation of Hybrids
[0459] The following hybrids were prepared.
Hybrid 6: the amino acid residues 106-213 in a variant of SEQ ID
NO: 5 having the substitutions
M9L+R118K+G149A+G182T+G186A+D183*+G184*+N195F+M202L+V214V+T257I+Y295F+N29-
9Y+R320K+M323T+A339S+E345R+R458K (using SEQ ID NO: 5 numbering)
were removed and replaced with the amino acid residues 103-209 of
SEQ ID NO: 13, and the following alterations were introduced:
E182*, N183*, E188W, N189E and D192T, which correspond to E181*,
N182*, E187W, N188E and D191T using SEQ ID NO: 27 numbering. The
sequence of this hybrid is shown in SEQ ID NO: 32. Hybrid 7: the
amino acid residues 106-213 in a variant of SEQ ID NO: 8 (using SEQ
ID NO: 8 numbering) were removed and replaced with the amino acid
residues 103-209 of SEQ ID NO: 13. The sequence of this hybrid is
shown in SEQ ID NO: 33. Hybrid 8: the amino acid residues 104-208
in a variant of SEQ ID NO: 3 having amino acids 1-35 replaced by
amino acids 1-33 of SEQ ID NO: 1 and having the substitutions
G48A+T49I+G107A+H156Y+A181T+N190F+1201F+A209V+Q264S (using SEQ ID
NO: 3 numbering) were removed and replaced with the amino acid
residues 103-209 of SEQ ID NO: 13, and the following alterations
were introduced: N102D, Q147T, E178*, N179*, K181A, E184W, N185E
and D188T, which correspond to N105D, Q150T, E181*, N182*, K184A,
E187W, N188E and D191T using SEQ ID NO: 27 numbering. The sequence
of this hybrid is shown in SEQ ID NO: 34.
[0460] The invention described and claimed herein is not to be
limited in scope by the specific aspects herein disclosed, since
these aspects are intended as illustrations of several aspects of
the invention. Any equivalent aspects are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims. In the case of conflict, the
present disclosure including definitions will control.
[0461] The invention is further defined by the following
paragraphs:
Paragraph 1. An isolated alpha-amylase, comprising an A-domain of a
calcium-sensitive alpha-amylase which has at least 70% and less
than 100% sequence identity with any of the A-domains of SEQ ID
NOS: 1-12, 29, and 30, a B-domain of a calcium-insensitive
alpha-amylase, and a C-domain of a calcium-sensitive alpha-amylase,
wherein the alpha-amylase has alpha-amylase activity. Paragraph 2.
An isolated alpha-amylase, comprising an A-domain of a
calcium-sensitive alpha-amylase, a B-domain of a
calcium-insensitive alpha-amylase, and a C-domain of a
calcium-sensitive alpha-amylase which has at least 70% and less
than 100% sequence identity with any of the C-domains of SEQ ID
NOS: 1-12, 29, and 30, wherein the alpha-amylase has alpha-amylase
activity. Paragraph 3. The alpha-amylase of paragraph 2, wherein
the A-domain has at least 60% sequence identity, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, at least
99%, or at least 100% sequence identity with the A-domain of SEQ ID
NO: 1. Paragraph 4. The alpha-amylase of paragraph 1 or 2, wherein
the A-domain has at least 60% sequence identity, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 1. Paragraph
5. The alpha-amylase of paragraph 1 or 2, wherein the A-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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 at least 99% sequence
identity with the A-domain of SEQ ID NO: 2. Paragraph 6. The
alpha-amylase of paragraph 1 or 2, wherein the A-domain has at
least 60% sequence identity, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, 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 at least 99% sequence identity
with the A-domain of SEQ ID NO: 3. Paragraph 7. The alpha-amylase
of paragraph 1 or 2, wherein the A-domain has at least 60% sequence
identity, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, 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 at least 99% sequence identity with the A-domain
of SEQ ID NO: 4. Paragraph 8. The alpha-amylase of paragraph 1 or
2, wherein the A-domain has at least 60% sequence identity, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, 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 at least 99% sequence identity with the A-domain of SEQ ID NO:
5. Paragraph 9. The alpha-amylase of paragraph 1 or 2, wherein the
A-domain has at least 60% sequence identity, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, 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 at least 99%
sequence identity with the A-domain of SEQ ID NO: 6. Paragraph 10.
The alpha-amylase of paragraph 1 or 2, wherein the A-domain has at
least 60% sequence identity, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, 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 at least 99% sequence identity
with the A-domain of SEQ ID NO: 7. Paragraph 11. The alpha-amylase
of paragraph 1 or 2, wherein the A-domain has at least 60% sequence
identity, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, 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 at least 99% sequence identity with the A-domain
of SEQ ID NO: 8. Paragraph 12. The alpha-amylase of paragraph 1 or
2, wherein the A-domain has at least 60% sequence identity, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, 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 at least 99% sequence identity with the A-domain of SEQ ID NO:
9. Paragraph 13. The alpha-amylase of paragraph 1 or 2, wherein the
A-domain has at least 60% sequence identity, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, 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 at least 99%
sequence identity with the A-domain of SEQ ID NO: 10. Paragraph 14.
The alpha-amylase of paragraph 1 or 2, wherein the A-domain has at
least 60% sequence identity, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, 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 at least 99% sequence identity
with the A-domain of SEQ ID NO: 11. Paragraph 15. The alpha-amylase
of paragraph 1 or 2, wherein the A-domain has at least 60% sequence
identity, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, 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 at least 99% sequence identity with the A-domain
of SEQ ID NO: 12. Paragraph 16. The alpha-amylase of paragraph 1 or
2, wherein the A-domain has at least 60% sequence identity, e.g.,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, 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 at least 99% sequence identity with the A-domain of SEQ ID NO:
29. Paragraph 17. The alpha-amylase of paragraph 1 or 2, wherein
the A-domain has at least 60% sequence identity, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the A-domain of SEQ ID NO: 30. Paragraph
18. The alpha-amylase of any of paragraphs 1-17, wherein the
A1-domain has at least 60% sequence identity, e.g., at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, 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%, at least 99%,
or 100% sequence identity with the sequence starting at a position
in the range of positions 1-5 and ending a position in the range of
positions 91-111, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
96-101 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 101-106, in
particular positions 1-101 of SEQ ID NO: 1. Paragraph 19. The
alpha-amylase of any of paragraphs 1-17, wherein the A1-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity with the sequence starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 95-115, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
100-105 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 105-110, in
particular positions 1-105 of SEQ ID NO: 2. Paragraph 20. The
alpha-amylase of any of paragraphs 1-17, wherein the A1-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity with the sequence starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 93-113, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
98-103 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 103-108, in
particular positions 1-103 of SEQ ID NO: 3. Paragraph 21. The
alpha-amylase of any of paragraphs 1-17, wherein the A1-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity with the sequence starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 94-114, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
99-104 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 104-109, in
particular positions 1-104 of SEQ ID NO: 4. Paragraph 22. The
alpha-amylase of any of paragraphs 1-17, wherein the A1-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity with the sequence starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 95-115, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
100-105 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 105-110, in
particular positions 1-105 of SEQ ID NO: 5. Paragraph 23. The
alpha-amylase of any of paragraphs 1-17, wherein the A1-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity with the sequence starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 95-115, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
100-105 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 105-110, in
particular positions 1-105 of SEQ ID NO: 6. Paragraph 24. The
alpha-amylase of any of paragraphs 1-17, wherein the A1-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity with the sequence starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 95-115, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
100-105 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 105-110, in
particular positions 1-105 of SEQ ID NO: 7. Paragraph 25. The
alpha-amylase of any of paragraphs 1-17, wherein the A1-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity with the sequence starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 95-115, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
100-105 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 105-110, in
particular positions 1-105 of SEQ ID NO: 8. Paragraph 26. The
alpha-amylase of any of paragraphs 1-17, wherein the A1-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity with the sequence starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 95-115, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
100-105 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 105-110, in
particular positions 1-105 of SEQ ID NO: 9. Paragraph 27. The
alpha-amylase of any of paragraphs 1-17, wherein the A1-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity with the sequence starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 95-115, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
100-105 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 105-110, in
particular positions 1-105 of SEQ ID NO: 10. Paragraph 28. The
alpha-amylase of any of paragraphs 1-17, wherein the A1-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity with the sequence starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 95-115, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
100-105 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 105-110, in
particular positions 1-105 of SEQ ID NO: 11. Paragraph 29. The
alpha-amylase of any of paragraphs 1-17, wherein the A1-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity with the sequence starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 95-115, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
100-105 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 105-110, in
particular positions 1-105 of SEQ ID NO: 12. Paragraph 30. The
alpha-amylase of any of paragraphs 1-17, wherein the A1-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity with the sequence starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 94-114, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
99-104 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 104-109, in
particular positions 1-104 of SEQ ID NO: 29. Paragraph 31. The
alpha-amylase of any of paragraphs 1-17, wherein the A1-domain has
at least 60% sequence identity, e.g., at least 65%, at least 70%,
at least 75%, at least 80%, at least 85%, 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%, at least 99%, or 100%
sequence identity with the sequence starting at a position in the
range of positions 1-5 and ending a position in the range of
positions 92-112, e.g., starting at a position in the range of
positions 1-3 and ending at a position in the range of positions
97-102 or starting at a position in the range of positions 1-3 and
ending at a position in the range of positions 102-107, in
particular positions 1-102 of SEQ ID NO: 30. Paragraph 32. The
alpha-amylase of any of paragraphs 1-31, wherein the amino acid at
the position corresponding to position 8 is Ala, Arg, Asn, Asp,
Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp,
Tyr, or Val, e.g., Leu. Paragraph 33. The alpha-amylase of any of
paragraphs 1-32, wherein the amino acid at the position
corresponding to position 27 is Arg, Asn, Asp, Cys, Gln, Glu, Gly,
His, Ile, Leu, Lys,
Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Gln. Paragraph 34.
The alpha-amylase of any of paragraphs 1-33, wherein the amino acid
at the position corresponding to position 34 is Ala, Arg, Asn, Asp,
Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Thr, Trp, Tyr, or
Val, e.g., Lys. Paragraph 35. The alpha-amylase of any of
paragraphs 1-34, wherein the amino acid at the position
corresponding to position 52 is any amino acid except Ala, Asn,
Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Gly. Paragraph 36. The alpha-amylase
of any of paragraphs 1-35, wherein the amino acid at the position
corresponding to position 53 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val,
e.g., Tyr. Paragraph 37. The alpha-amylase of any of paragraphs
1-36, wherein the amino acid at the position corresponding to
position 59 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile,
Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, e.g., Ala.
Paragraph 38. The alpha-amylase of any of paragraphs 1-37, wherein
the amino acid at the position corresponding to position 80 is Ala,
Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,
Pro, Ser, Trp, Tyr, or Val, e.g., Asp. Paragraph 39. The
alpha-amylase of any of paragraphs 1-38, wherein the amino acid at
the position corresponding to position 86 is Ala, Arg, Asn, Asp,
Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,
Tyr, or Val, e.g., Ser. Paragraph 40. The alpha-amylase of any of
paragraphs 1-39, wherein the amino acid at the position
corresponding to position 90 is Arg, Asn, Asp, Cys, Gln, Glu, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Ser. Paragraph 41. The alpha-amylase of any of paragraphs
1-40, wherein the amino acid at the position corresponding to
position 91 is Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Leu.
Paragraph 42. The alpha-amylase of any of paragraphs 1-41, wherein
the amino acid at the position corresponding to position 100 is
Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,
Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Gly or Leu. Paragraph 43.
The alpha-amylase of any of paragraphs 1-42, wherein the amino acid
at the position corresponding to position 204 is Ala, Arg, Asn,
Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr,
Trp, Tyr, or Val, e.g., Leu. Paragraph 44. The alpha-amylase of any
of paragraphs 1-43, wherein the amino acid at the position
corresponding to position 206 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val,
e.g., Met. Paragraph 45. The alpha-amylase of any of paragraphs
1-44, wherein the amino acid at the position corresponding to
position 220 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile,
Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Pro.
Paragraph 46. The alpha-amylase of any of paragraphs 1-45, wherein
the amino acid at the position corresponding to position 222 is
Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,
Phe, Pro, Ser, Thr, Trp, or Val, e.g., Val. Paragraph 47. The
alpha-amylase of any of paragraphs 1-46, wherein the amino acid at
the position corresponding to position 224 is Ala, Arg, Asp, Cys,
Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,
Tyr, or Val, e.g., Leu. Paragraph 48. The alpha-amylase of any of
paragraphs 1-47, wherein the amino acid at the position
corresponding to position 235 is Arg, Asn, Asp, Cys, Gln, Glu, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Thr or Trp. Paragraph 49. The alpha-amylase of any of
paragraphs 1-48, wherein the amino acid at the position
corresponding to position 242 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val,
e.g., Gln or Glu. Paragraph 50. The alpha-amylase of any of
paragraphs 1-49, wherein the amino acid at the position
corresponding to position 244 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Tyr. Paragraph 51. The alpha-amylase of any of paragraphs
1-50, wherein the amino acid at the position corresponding to
position 245 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile,
Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val, e.g., Ala or Lys.
Paragraph 52. The alpha-amylase of any of paragraphs 1-51, wherein
the amino acid at the position corresponding to position 270 is
Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe,
Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Leu. Paragraph 53. The
alpha-amylase of any of paragraphs 1-52, wherein the amino acid at
the position corresponding to position 282 is Ala, Arg, Asn, Asp,
Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,
Tyr, or Val, e.g., Trp. Paragraph 54. The alpha-amylase of any of
paragraphs 1-53, wherein the amino acid at the position
corresponding to position 284 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Gln, Thr, or Val. Paragraph 55. The alpha-amylase of any of
paragraphs 1-54, wherein the amino acid at the position
corresponding to position 301 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val,
e.g., Lys. Paragraph 56. The alpha-amylase of any of paragraphs
1-55, wherein the amino acid at the position corresponding to
position 303 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Arg.
Paragraph 57. The alpha-amylase of any of paragraphs 1-56, wherein
the amino acid at the position corresponding to position 304 is
Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,
Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp. Paragraph 58. The
alpha-amylase of any of paragraphs 1-57, wherein the amino acid at
the position corresponding to position 307 is Ala, Arg, Asn, Asp,
Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp,
Tyr, or Val, e.g., Leu. Paragraph 59. The alpha-amylase of any of
paragraphs 1-58, wherein the amino acid at the position
corresponding to position 348 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val,
e.g., Thr. Paragraph 60. The alpha-amylase of any of paragraphs
1-59, wherein the amino acid at the position corresponding to
position 386 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile,
Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val, e.g., Gln, Glu,
Thr, or Val. Paragraph 61. The alpha-amylase of any of paragraphs
1-60, wherein the amino acid at the position corresponding to
position 388 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ile or Val.
Paragraph 62. The alpha-amylase of any of paragraphs 1-61, wherein
the A-domain of the variant is different from any of the A-domains
of SEQ ID NOS: 1-12, 29, and 30 at the positions corresponding to
positions 52 and 53, e.g., the amino acids at the positions
corresponding to positions 52 and 53 are Gly and Tyr, respectively.
Paragraph 63. The alpha-amylase of any of paragraphs 1-62, wherein
the A-domain of the variant is different from any of the A-domains
of SEQ ID NOS: 1-12, 29, and 30 at the positions corresponding to
positions 59, 242, and 307, e.g., the amino acids at the positions
corresponding to positions 59, 242, and 307 are Ala; Gln or Glu;
and Leu, respectively. Paragraph 64. The alpha-amylase of any of
paragraphs 1-63, wherein the A-domain of the variant is different
from any of the A-domains of SEQ ID NOS: 1-12, 29, and 30 at the
positions corresponding to positions 220 and 224, e.g., the amino
acids at the positions corresponding to positions 220 and 224 are
Pro and Leu, respectively. Paragraph 65. The alpha-amylase of any
of paragraphs 1-64, wherein the A-domain of the variant is
different from any of the A-domains of SEQ ID NOS: 1-12, 29, and 30
at the positions corresponding to positions 242 and 282, e.g., the
amino acids at the positions corresponding to positions 242 and 282
are Gln or Glu; and Trp, respectively. Paragraph 66. The
alpha-amylase of any of paragraphs 1-65, wherein the A-domain of
the variant is different from any of the A-domains of SEQ ID NOS:
1-12, 29, and 30 at the positions corresponding to positions 242
and 284, e.g., the amino acids at the positions corresponding to
positions 242 and 284 are Gln or Glu; and Gln, Thr, or Val,
respectively. Paragraph 67. The alpha-amylase of any of paragraphs
1-66, wherein the A-domain of the variant is different from any of
the A-domains of SEQ ID NOS: 1-12, 29, and 30 at the positions
corresponding to positions 242, 284, and 307, e.g., the amino acids
at the positions corresponding to positions 242, 284, and 307 are
Gln or Glu; Gln, Thr, or Val; and Leu, respectively. Paragraph 68.
The alpha-amylase of any of paragraphs 1-67, wherein the A-domain
of the variant is different from any of the A-domains of SEQ ID
NOS: 1-12, 29, and 30 at the positions corresponding to positions
242 and 307, e.g., the amino acids at the positions corresponding
to positions 242 and 307 are Gln or Glu; and Leu, respectively.
Paragraph 69. The alpha-amylase of any of paragraphs 1-68, wherein
the A-domain of the variant is different from any of the A-domains
of SEQ ID NOS: 1-12, 29, and 30 at the positions corresponding to
positions 244 and 284, e.g., the amino acids at the positions
corresponding to positions 244 and 284 are Tyr; and Gln, Thr, or
Val, respectively. Paragraph 70. The alpha-amylase of any of
paragraphs 1-69, wherein the A-domain of the variant is different
from any of the A-domains of SEQ ID NOS: 1-12, 29, and 30 at the
positions corresponding to positions 244, 284, and 307, e.g., the
amino acids at the positions corresponding to positions 244, 284,
and 307 are Tyr; Gln, Thr, or Val; and Leu, respectively. Paragraph
71. The alpha-amylase of any of paragraphs 1-70, wherein the
A-domain of the variant is different from any of the A-domains of
SEQ ID NOS: 1-12, 29, and 30 at the positions corresponding to
positions 244 and 307, e.g., the amino acids at the positions
corresponding to positions 244 and 307 are Tyr and Leu,
respectively. Paragraph 72. The alpha-amylase of any of paragraphs
1-71, wherein the A-domain of the variant is different from any of
the A-domains of SEQ ID NOS: 1-12, 29, and 30 at the positions
corresponding to positions 284 and 307, e.g., the amino acids at
the positions corresponding to positions 284 and 307 are Gln, Thr,
or Val; and Leu, respectively. Paragraph 73. The alpha-amylase of
any of paragraphs 1-72, wherein the A-domain of the variant is
different from any of the A-domains of SEQ ID NOS: 1-12, 29, and 30
at the positions corresponding to positions 301, 303, and 304,
e.g., the amino acids at the positions corresponding to positions
301, 303, and 304 are Lys, Arg, and Asp, respectively. Paragraph
74. The alpha-amylase of any of paragraphs 1-73, wherein the
B-domain has at least 60% sequence identity, e.g., at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, 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%, at least 99%, or
100% sequence identity with the B-domain of SEQ ID NO: 13.
Paragraph 75. The alpha-amylase of any of paragraphs 1-74, wherein
the B-domain has at least 60% sequence identity, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, at least
99%, or 100% sequence identity with the B-domain of SEQ ID NO: 14.
Paragraph 76. The alpha-amylase of any of paragraphs 1-75, wherein
the B-domain has at least 60% sequence identity, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, at least
99%, or 100% sequence identity with the B-domain of SEQ ID NO: 15.
Paragraph 77. The alpha-amylase of any of paragraphs 1-76, wherein
the B-domain has at least 60% sequence identity, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, at least
99%, or 100% sequence identity with the B-domain of SEQ ID NO: 16.
Paragraph 78. The alpha-amylase of any of paragraphs 1-77, wherein
the B-domain has at least 60% sequence identity, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, at least
99%, or 100% sequence identity with the B-domain of SEQ ID NO: 31.
Paragraph 79. The alpha-amylase of any of paragraphs 1-78, wherein
the B-domain has at least 60% sequence identity, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, at least
99%, or 100% sequence identity with the sequence starting at a
position in the range of positions 93-113 and ending at a position
in the range of positions 195-215, e.g., starting at a position in
the range of positions 97-109 and ending at a position in the range
of positions 199-211 or starting at a position in the range of
positions 100-106 and ending at a position in the range of
positions 202-208, in particular positions 103-208 of SEQ ID NO:
13. Paragraph 80. The alpha-amylase of any of paragraphs 1-79,
wherein the B-domain has at least 60% sequence identity, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
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%, at
least 99%, or 100% sequence identity with the sequence starting at
a position in the range of positions 93-113 and ending at a
position in the range of positions 195-215, e.g., starting at a
position in the range of positions 97-109 and ending at a position
in the range of positions 199-211 or starting at a position in the
range of positions 100-106 and ending at a position in the range of
positions 202-208, in particular positions 104-207 of SEQ ID NO:
14. Paragraph 81. The alpha-amylase of any of paragraphs 1-80,
wherein the B-domain has at least 60% sequence identity, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
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%, at
least 99%, or 100% sequence identity with the sequence starting at
a position in the range of positions 93-113 and ending at a
position in the range of positions 195-215, e.g., starting at a
position in the range of positions 97-109 and ending at a position
in the range of positions 199-211 or starting at a position in the
range of positions 100-106 and ending at a position in the range of
positions 202-208, in particular positions 104-207 of SEQ ID NO:
15. Paragraph 82. The alpha-amylase of any of paragraphs 1-81,
wherein the B-domain has at least 60% sequence identity, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
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%, at
least 99%, or 100% sequence identity with the sequence starting at
a position in the range of positions 100-120 and ending at a
position in the range of positions 161-181, e.g., starting at a
position in the range of positions 105-115 and ending at a position
in the range of positions 166-171 or starting at a position in the
range of positions 107-113 and ending at a position in the range of
positions 171-176, in particular positions 110-171 of SEQ ID NO:
16. Paragraph 83. The alpha-amylase of any of paragraphs 1-82,
wherein the B-domain has at least 60% sequence identity, e.g., at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%,
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%, at
least 99%, or 100% sequence identity with the sequence starting at
a position in the range of positions 100-120 and ending at a
position in the range of positions 161-181, e.g., starting at a
position in the range of positions 105-115 and ending at a position
in the range of positions 166-171 or starting at a position in the
range of positions 107-113 and ending at a position in the range of
positions 171-176, in particular positions 110-171 of SEQ ID NO:
31. Paragraph 84. The alpha-amylase of any of paragraphs 1-83,
wherein the B-domain has at least 55% and less than 100% sequence
identity with the B-domain of SEQ ID NO: 13. Paragraph 85. The
alpha-amylase of any of paragraphs 1-84, wherein the amino acid at
the position corresponding to position 105 is Ala, Arg, Asn, Asp,
Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,
Trp, Tyr, or Val, e.g., Asp. Paragraph 86. The alpha-amylase of any
of paragraphs 1-85, wherein the amino acid at the position
corresponding to position 115 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr,
e.g., Trp. Paragraph 87. The alpha-amylase of any of paragraphs
1-86, wherein the amino acid at the position corresponding to
position 117 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile,
Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp.
Paragraph 88. The alpha-amylase of
any of paragraphs 1-87, wherein the amino acid at the position
corresponding to position 129 is Ala, Arg, Asn, Asp, Cys, Gln, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Val. Paragraph 89. The alpha-amylase of any of paragraphs
1-88, wherein the amino acid at the position corresponding to
position 132 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp.
Paragraph 90. The alpha-amylase of any of paragraphs 1-89, wherein
the amino acid at the position corresponding to position 134 is
Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,
Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Glu or Tyr. Paragraph 91.
The alpha-amylase of any of paragraphs 1-90, wherein the amino acid
at the position corresponding to position 135 is Ala, Arg, Asn,
Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,
Trp, Tyr, or Val, e.g., Asn or Gln. Paragraph 92. The alpha-amylase
of any of paragraphs 1-91, wherein the amino acid at the position
corresponding to position 150 is Ala, Arg, Asn, Asp, Cys, Glu, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Thr. Paragraph 93. The alpha-amylase of any of paragraphs
1-92, wherein the amino acid at the position corresponding to
position 157 is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr.
Paragraph 94. The alpha-amylase of any of paragraphs 1-93, wherein
the amino acid at the position corresponding to position 159 is
Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe,
Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr. Paragraph 95. The
alpha-amylase of any of paragraphs 1-94, wherein the amino acid at
the position corresponding to position 160 is Ala, Arg, Asn, Asp,
Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,
Tyr, or Val, e.g., Tyr. Paragraph 96. The alpha-amylase of any of
paragraphs 1-95, wherein the amino acid at the position
corresponding to position 164 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val,
e.g., Val. Paragraph 97. The alpha-amylase of any of paragraphs
1-96, wherein the amino acid at the position corresponding to
position 166 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile,
Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Trp.
Paragraph 98. The alpha-amylase of any of paragraphs 1-97, wherein
the amino acid at the position corresponding to position 168 is
Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,
Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Glu. Paragraph 99. The
alpha-amylase of any of paragraphs 1-98, wherein the amino acid at
the position corresponding to position 169 is Ala, Arg, Asn, Asp,
Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp,
Tyr, or Val, e.g., Ser. Paragraph 100. The alpha-amylase of any of
paragraphs 1-99, wherein the amino acid at the position
corresponding to position 170 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Arg. Paragraph 101. The alpha-amylase of any of paragraphs
1-100, wherein the amino acid at the position corresponding to
position 171 is Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Lys.
Paragraph 102. The alpha-amylase of any of paragraphs 1-101,
wherein the amino acid at the position corresponding to position
172 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,
Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Leu. Paragraph 103. The
alpha-amylase of any of paragraphs 1-102, wherein the amino acid at
the position corresponding to position 174 is Ala, Arg, Asn, Asp,
Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,
Tyr, or Val, e.g., Arg. Paragraph 104. The alpha-amylase of any of
paragraphs 1-103, wherein the amino acid at the position
corresponding to position 176 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Tyr. Paragraph 105. The alpha-amylase of any of paragraphs
1-104, wherein the amino acid at the position corresponding to
position 177 is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Leu.
Paragraph 106. The alpha-amylase of any of paragraphs 1-105,
wherein the amino acid at the position corresponding to position
179 is Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,
Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asn, Asp, Gln, or Glu,
or is absent. Paragraph 107. The alpha-amylase of any of paragraphs
1-106, wherein the amino acid at the position corresponding to
position 180 is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Ala, Pro, or
Ser, or is absent. Paragraph 108. The alpha-amylase of any of
paragraphs 1-107, wherein the amino acid at the position
corresponding to position 181 is Ala, Arg, Asn, Asp, Cys, Gln, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Ala, Asp, Cys, Leu, or Pro, or is absent. Paragraph 109. The
alpha-amylase of any of paragraphs 1-108, wherein the amino acid at
the position corresponding to position 182 is Ala, Arg, Asp, Cys,
Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,
Tyr, or Val, e.g., Ala, Pro, or Ser, or is absent. Paragraph 110.
The alpha-amylase of any of paragraphs 1-109, wherein the amino
acid at the position corresponding to position 184 is Ala, Arg,
Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, or Val, e.g., Ala. Paragraph 111. The alpha-amylase
of any of paragraphs 1-110, wherein the amino acid at the position
corresponding to position 187 is Ala, Arg, Asn, Asp, Cys, Gln, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Trp. Paragraph 112. The alpha-amylase of any of paragraphs
1-111, wherein the amino acid at the position corresponding to
position 188 is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Glu.
Paragraph 113. The alpha-amylase of any of paragraphs 1-112,
wherein the amino acid at the position corresponding to position
191 is Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,
Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Thr. Paragraph 114. The
alpha-amylase of any of paragraphs 1-113, wherein the amino acid at
the position corresponding to position 206 is Ala, Arg, Asn, Asp,
Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,
Trp, or Val, e.g., Lys or Met. Paragraph 115. The alpha-amylase of
any of paragraphs 1-114, wherein the amino acid at the position
corresponding to position 208 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Tyr. Paragraph 116. The alpha-amylase of any of paragraphs
1-115, wherein the amino acid at the position corresponding to
position 210 is Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Tyr or Val.
Paragraph 117. The alpha-amylase of any of paragraphs 1-116,
wherein the B-domain has at least one difference at a position
corresponding to position 105, 115, 117, 129, 132, 134, 135, 150,
157, 159, 160, 164, 166, 168, 169, 170, 171, 172, 174, 176, 177,
179, 184, 187, 188, 191, 202, 204, 206, 208, or 210, and the amino
acids at at least two positions corresponding to positions 179-182
are absent. Paragraph 118. The alpha-amylase of any of paragraphs
1-117, wherein the B-domain is different from the B-domain of SEQ
ID NO: 13 at the positions corresponding to positions 105, 117,
150, and 184, e.g., the amino acids at the positions corresponding
to positions 105, 117, 150, and 184 are Asp, Asp, Thr, and Ala,
respectively. Paragraph 119. The alpha-amylase of any of paragraphs
1-118, wherein the B-domain is different from the B-domain of SEQ
ID NO: 3 at the positions corresponding to positions 105, 129, 177,
and 179, e.g., the amino acids at the positions corresponding to
positions 105, 129, 177, and 179 are Asp; Val; Leu; and Asn, Asp,
Gln, or Glu, or is absent; and Ala, respectively. Paragraph 120.
The alpha-amylase of any of paragraphs 1-119, wherein the B-domain
is different from the B-domain of SEQ ID NO: 3 at the positions
corresponding to positions 105, 132, and 184, e.g., the amino acids
at the positions corresponding to positions 105, 132, and 184 are
Asp, Asp, and Ala, respectively. Paragraph 121 The alpha-amylase of
any of paragraphs 1-120, wherein the B-domain is different from the
B-domain of SEQ ID NO: 3 at the positions corresponding to
positions 105, 134, and 184, e.g., the amino acids at the positions
corresponding to positions 105, 134, and 184 are Asp, Glu, and Ala,
respectively. Paragraph 122. The alpha-amylase of any of paragraphs
1-121, wherein the B-domain is different from the B-domain of SEQ
ID NO: 3 at the positions corresponding to positions 105, 135, 179,
and 184, e.g., the amino acids at the positions corresponding to
positions 105, 135, 179, and 184 are Asp; Asn; Asn, Asp, Gln, or
Glu, or is absent; and Ala, respectively. Paragraph 123. The
alpha-amylase of any of paragraphs 1-122, wherein the B-domain is
different from the B-domain of SEQ ID NO: 3 at the positions
corresponding to positions 105 and 150, e.g., the amino acids at
the positions corresponding to positions 105 and 150 are Asp and
Thr, respectively. Paragraph 124. The alpha-amylase of any of
paragraphs 1-123, wherein the B-domain is different from the
B-domain of SEQ ID NO: 3 at the positions corresponding to
positions 105, 150, 164, 166, 168, 171, and 184, e.g., the amino
acids at the positions corresponding to positions 105, 150, 164,
166, 168, 171, and 184 are Asp, Thr, Val, Trp, Glu, Lys, and Ala,
respectively. Paragraph 125. The alpha-amylase of any of paragraphs
1-124, wherein the B-domain is different from the B-domain of SEQ
ID NO: 3 at the positions corresponding to positions 105, 150, 164,
and 184, e.g., the amino acids at the positions corresponding to
positions 105, 150, 164, and 184 are Asp, Thr, Val, and Ala,
respectively. Paragraph 126. The alpha-amylase of any of paragraphs
1-125, wherein the B-domain is different from the B-domain of SEQ
ID NO: 3 at the positions corresponding to positions 105, 150, 166,
168, and 171, e.g., the amino acids at the positions corresponding
to positions 105, 150, 166, 168, and 171 are Asp, Thr, Trp, Glu,
and Lys, respectively. Paragraph 127. The alpha-amylase of any of
paragraphs 1-126, wherein the B-domain is different from the
B-domain of SEQ ID NO: 3 at the positions corresponding to
positions 105, 150, 166, 168, 171, and 184, e.g., the amino acids
at the positions corresponding to positions 105, 150, 166, 168,
171, and 184 are Asp, Thr, Trp, Glu, Lys, and Ala, respectively.
Paragraph 128. The alpha-amylase of any of paragraphs 1-127,
wherein the B-domain is different from the B-domain of SEQ ID NO: 3
at the positions corresponding to positions 105, 150, and 184,
e.g., the amino acids at the positions corresponding to positions
105, 150, and 184 are Asp, Thr, and Ala, respectively. Paragraph
129. The alpha-amylase of any of paragraphs 1-128, wherein the
B-domain is different from the B-domain of SEQ ID NO: 3 at the
positions corresponding to positions 105, 150, 184, and 206, e.g.,
the amino acids at the positions corresponding to positions 105,
150, 184, and 206 are Asp, Thr, Ala, and Met, respectively.
Paragraph 130. The alpha-amylase of any of paragraphs 1-129,
wherein the B-domain is different from the B-domain of SEQ ID NO: 3
at the positions corresponding to positions 105, 157, 159, 160,
184, 208, and 210, e.g., the amino acids at the positions
corresponding to positions 105, 157, 159, 160, 184, 208, and 210
are Asp; Tyr; Tyr; Tyr; Ala; Tyr; and Tyr or Val, respectively.
Paragraph 131. The alpha-amylase of any of paragraphs 1-130,
wherein the B-domain is different from the B-domain of SEQ ID NO: 3
at the positions corresponding to positions 105, 160, and 184,
e.g., the amino acids at the positions corresponding to positions
105, 160, and 184 are Asp, Tyr, and Ala, respectively. Paragraph
132. The alpha-amylase of any of paragraphs 1-131, wherein the
B-domain is different from the B-domain of SEQ ID NO: 3 at the
positions corresponding to positions 105, 164, and 184, e.g., the
amino acids at the positions corresponding to positions 105, 164,
and 184 are Asp, Val, and Ala, respectively. Paragraph 133. The
alpha-amylase of any of paragraphs 1-132, wherein the B-domain is
different from the B-domain of SEQ ID NO: 3 at the positions
corresponding to positions 105, 166, 168, and 171, e.g., the amino
acids at the positions corresponding to positions 105, 166, 168,
and 171 are Asp, Trp, Glu, and Lys, respectively. Paragraph 134.
The alpha-amylase of any of paragraphs 1-133, wherein the B-domain
is different from the B-domain of SEQ ID NO: 3 at the positions
corresponding to positions 105, 179, and 184, e.g., the amino acids
at the positions corresponding to positions 105, 179, and 184 are
Asp; Asn, Asp, Gln, or Glu, or is absent; and Ala, respectively.
Paragraph 135. The alpha-amylase of any of paragraphs 1-134,
wherein the B-domain is different from the B-domain of SEQ ID NO: 3
at the positions corresponding to positions 105 and 184, e.g., the
amino acids at the positions corresponding to positions 105 and 184
are Asp and Ala, respectively. Paragraph 136. The alpha-amylase of
any of paragraphs 1-135, wherein the B-domain is different from the
B-domain of SEQ ID NO: 3 at the positions corresponding to
positions 105, 184, and 210, e.g., the amino acids at the positions
corresponding to positions 105, 184, and 210 are Asp; Ala; and Tyr
or Val, respectively. Paragraph 137. The alpha-amylase of any of
paragraphs 1-136, wherein the B-domain is different from the
B-domain of SEQ ID NO: 3 at the positions corresponding to
positions 105 and 206, e.g., the amino acids at the positions
corresponding to positions 105 and 206 are Asp and Met,
respectively. Paragraph 138. The alpha-amylase of any of paragraphs
1-137, wherein the B-domain is different from the B-domain of SEQ
ID NO: 3 at the positions corresponding to positions 166, 168, and
171, e.g., the amino acids at the positions corresponding to
positions 166, 168, and 171 are Trp, Glu, and Lys, respectively.
Paragraph 139. The alpha-amylase of any of paragraphs 1-138,
wherein the B-domain is different from the B-domain of SEQ ID NO: 3
at the positions corresponding to positions 187, 188, and 191,
e.g., the amino acids at the positions corresponding to positions
187, 188, and 191 are Trp, Glu, and Thr, respectively. Paragraph
140. The alpha-amylase of any of paragraphs 1-139, wherein the
C-domain has at least 70% sequence identity, e.g., at least 75%, at
least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity with the
C-domain of SEQ ID NO: 1. Paragraph 141. The alpha-amylase of any
of paragraphs 1-139, wherein the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 1. Paragraph
142. The alpha-amylase of any of paragraphs 1-139, wherein the
C-domain has at least 70% sequence identity, e.g., at least 75%, at
least 80%, at least 85%, 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 at least 99% sequence identity with the
C-domain of SEQ ID NO: 2. Paragraph 143. The alpha-amylase of any
of paragraphs 1-139, wherein the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 3. Paragraph
144. The alpha-amylase of any of paragraphs 1-139, wherein the
C-domain has at least 70% sequence identity, e.g., at least 75%, at
least 80%, at least 85%, 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 at least 99% sequence identity with the
C-domain of SEQ ID NO: 4. Paragraph 145. The alpha-amylase of any
of paragraphs 1-139, wherein the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 5. Paragraph
146. The alpha-amylase of any of paragraphs 1-139, wherein the
C-domain has at least 70% sequence identity, e.g., at least 75%, at
least 80%, at least 85%, 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 at least 99% sequence identity with the
C-domain of SEQ ID NO: 6. Paragraph 147. The alpha-amylase of any
of paragraphs 1-139, wherein the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 7. Paragraph
148. The alpha-amylase of any of paragraphs 1-139, wherein the
C-domain has at least 70% sequence identity, e.g., at least 75%, at
least 80%, at least 85%, 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 at least 99%
sequence identity with the C-domain of SEQ ID NO: 8. Paragraph 149.
The alpha-amylase of any of paragraphs 1-139, wherein the C-domain
has at least 70% sequence identity, e.g., at least 75%, at least
80%, at least 85%, 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 at least 99% sequence identity with the C-domain
of SEQ ID NO: 9. Paragraph 150. The alpha-amylase of any of
paragraphs 1-139, wherein the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 10. Paragraph
151. The alpha-amylase of any of paragraphs 1-139, wherein the
C-domain has at least 70% sequence identity, e.g., at least 75%, at
least 80%, at least 85%, 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 at least 99% sequence identity with the
C-domain of SEQ ID NO: 11. Paragraph 152. The alpha-amylase of any
of paragraphs 1-139, wherein the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 12. Paragraph
153. The alpha-amylase of any of paragraphs 1-139, wherein the
C-domain has at least 70% sequence identity, e.g., at least 75%, at
least 80%, at least 85%, 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 at least 99% sequence identity with the
C-domain of SEQ ID NO: 29. Paragraph 154. The alpha-amylase of any
of paragraphs 1-139, wherein the C-domain has at least 70% sequence
identity, e.g., at least 75%, at least 80%, at least 85%, 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 at least
99% sequence identity with the C-domain of SEQ ID NO: 30. Paragraph
155. The alpha-amylase of any of paragraphs 1-155, wherein the A2
and C-domains have at least 60% sequence identity, e.g., at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, at least
99%, or 100% sequence identity with the sequence starting at a
position in the range of positions 198-218 and ending at a position
in the range of positions 478-483, e.g., starting at a position in
the range of positions 203-208 and ending at a position in the
range of positions 480-483 or starting at a position in the range
of positions 208-213 and ending at a position in the range of
positions 480-483, in particular positions 208-483 of SEQ ID NO: 1.
Paragraph 156. The alpha-amylase of any of paragraphs 1-155,
wherein the A2 and C-domains have at least 60% sequence identity,
e.g., at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, 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%, at least 99%, or 100% sequence identity with the sequence
starting at a position in the range of positions 202-222 and ending
at a position in the range of positions 479-484, e.g., starting at
a position in the range of positions 207-212 and ending at a
position in the range of positions 481-484 or starting at a
position in the range of positions 212-217 and ending at a position
in the range of positions 481-484, in particular positions 212-484
of SEQ ID NO: 2. Paragraph 157. The alpha-amylase of any of
paragraphs 1-155, wherein the A2 and C-domains have at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity
with the sequence starting at a position in the range of positions
198-218 and ending at a position in the range of positions 478-483,
e.g., starting at a position in the range of positions 203-208 and
ending at a position in the range of positions 480-483 or starting
at a position in the range of positions 208-213 and ending at a
position in the range of positions 480-483, in particular positions
208-483 of SEQ ID NO: 3. Paragraph 158. The alpha-amylase of any of
paragraphs 1-155, wherein the A2 and C-domains have at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity
with the sequence starting at a position in the range of positions
201-221 and ending at a position in the range of positions 478-483,
e.g., starting at a position in the range of positions 206-211 and
ending at a position in the range of positions 480-483 or starting
at a position in the range of positions 211-216 and ending at a
position in the range of positions 480-483, in particular positions
211-483 of SEQ ID NO: 4. Paragraph 159. The alpha-amylase of any of
paragraphs 1-155, wherein the A2 and C-domains have at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity
with the sequence starting at a position in the range of positions
193-223 and ending at a position in the range of positions 482-485,
e.g., starting at a position in the range of positions 208-213 and
ending at a position in the range of positions 482-485 or starting
at a position in the range of positions 213-218 and ending at a
position in the range of positions 482-485, in particular positions
213-485 of SEQ ID NO: 5. Paragraph 160. The alpha-amylase of any of
paragraphs 1-155, wherein the A2 and C-domains have at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity
with the sequence starting at a position in the range of positions
203-223 and ending at a position in the range of positions 482-485,
e.g., starting at a position in the range of positions 208-213 and
ending at a position in the range of positions 482-485 or starting
at a position in the range of positions 213-218 and ending at a
position in the range of positions 482-485, in particular positions
213-485 of SEQ ID NO: 6. Paragraph 161. The alpha-amylase of any of
paragraphs 1-155, wherein the A2 and C-domains have at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity
with the sequence starting at a position in the range of positions
203-223 and ending at a position in the range of positions 482-485,
e.g., starting at a position in the range of positions 208-213 and
ending at a position in the range of positions 482-485 or starting
at a position in the range of positions 213-218 and ending at a
position in the range of positions 482-485, in particular positions
213-485 of SEQ ID NO: 7. Paragraph 162. The alpha-amylase of any of
paragraphs 1-155, wherein the A2 and C-domains have at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity
with the sequence starting at a position in the range of positions
203-223 and ending at a position in the range of positions 482-485,
e.g., starting at a position in the range of positions 208-213 and
ending at a position in the range of positions 482-485 or starting
at a position in the range of positions 213-218 and ending at a
position in the range of positions 482-485, in particular positions
213-485 of SEQ ID NO: 8. Paragraph 163. The alpha-amylase of any of
paragraphs 1-155, wherein the A2 and C-domains have at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity
with the sequence starting at a position in the range of positions
203-223 and ending at a position in the range of positions 481-484,
e.g., starting at a position in the range of positions 208-213 and
ending at a position in the range of positions 482-484 or starting
at a position in the range of positions 213-218 and ending at a
position in the range of positions 482-484, in particular positions
213-484 of SEQ ID NO: 9. Paragraph 164. The alpha-amylase of any of
paragraphs 1-155, wherein the A2 and C-domains have at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity
with the sequence starting at a position in the range of positions
203-223 and ending at a position in the range of positions 482-484,
e.g., starting at a position in the range of positions 208-213 and
ending at a position in the range of positions 482-484 or starting
at a position in the range of positions 213-218 and ending at a
position in the range of positions 482-484, in particular positions
213-484 of SEQ ID NO: 10. Paragraph 165. The alpha-amylase of any
of paragraphs 1-155, wherein the A2 and C-domains have at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity
with the sequence starting at a position in the range of positions
203-223 and ending at a position in the range of positions 482-485,
e.g., starting at a position in the range of positions 208-213 and
ending at a position in the range of positions 482-485 or starting
at a position in the range of positions 213-218 and ending at a
position in the range of positions 482-485, in particular positions
213-485 of SEQ ID NO: 11. Paragraph 166. The alpha-amylase of any
of paragraphs 1-155, wherein the A2 and C-domains have at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity
with the sequence starting at a position in the range of positions
203-223 and ending at a position in the range of positions 482-485,
e.g., starting at a position in the range of positions 208-213 and
ending at a position in the range of positions 482-485 or starting
at a position in the range of positions 213-218 and ending at a
position in the range of positions 482-485, in particular positions
213-485 of SEQ ID NO: 12. Paragraph 167. The alpha-amylase of any
of paragraphs 1-155, wherein the A2 and C-domains have at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity
with the sequence starting at a position in the range of positions
201-221 and ending at a position in the range of positions 478-483,
e.g., starting at a position in the range of positions 206-211 and
ending at a position in the range of positions 480-483 or starting
at a position in the range of positions 211-216 and ending at a
position in the range of positions 480-483, in particular positions
211-483 of SEQ ID NO: 29. Paragraph 168. The alpha-amylase of any
of paragraphs 1-155, wherein the A2 and C-domains have at least 60%
sequence identity, e.g., at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, 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%, at least 99%, or 100% sequence identity
with the sequence starting at a position in the range of positions
199-219 and ending at a position in the range of positions 479-484,
e.g., starting at a position in the range of positions 204-209 and
ending at a position in the range of positions 481-484 or starting
at a position in the range of positions 209-214 and ending at a
position in the range of positions 481-484, in particular positions
209-484 of SEQ ID NO: 30. Paragraph 169. The alpha-amylase of any
of paragraphs 1-168, wherein the amino acid at the position
corresponding to position 407 is Ala, Arg, Asp, Cys, Gln, Glu, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val,
e.g., Asp. Paragraph 170. The alpha-amylase of any of paragraphs
1-169, wherein the amino acid at the position corresponding to
position 430 is Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Asp.
Paragraph 171. The alpha-amylase of any of paragraphs 1-170,
wherein the amino acid at the position corresponding to position
432 is Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,
Phe, Pro, Ser, Thr, Trp, Tyr, or Val, e.g., Pro. Paragraph 172. The
alpha-amylase of any of paragraphs 1-171, wherein the amino acid at
the position corresponding to position 459 is Ala, Arg, Asn, Asp,
Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp,
Tyr, or Val, e.g., Pro. Paragraph 173. The alpha-amylase of any of
paragraphs 1-172, wherein the amino acid at the position
corresponding to position 475 is Ala, Arg, Asn, Asp, Cys, Gln, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val,
e.g., Gln or Lys. Paragraph 174. The alpha-amylase of any of
paragraphs 1-173, wherein the C-domain is different from any of the
C-domains of SEQ ID NOS: 1-12, 29, and 30 at the positions
corresponding to positions 407 and 430, e.g., the amino acids at
the positions corresponding to positions 407 and 430 are Asp.
Paragraph 175. The alpha-amylase of any of paragraphs 1-174,
wherein the C-domain is different from any of the C-domains of SEQ
ID NOS: 1-12, 29, and 30 at the positions corresponding to
positions 407, 430, and 432, e.g., the amino acids at the positions
corresponding to positions 407, 430, and 432 are Asp, Asp, and Pro,
respectively. Paragraph 176. The alpha-amylase of any of paragraphs
1-175, wherein the C-domain is different from any of the C-domains
of SEQ ID NOS: 1-12, 29, and 30 at the positions corresponding to
positions 407 and 432, e.g., the amino acids at the positions
corresponding to positions 407 and 432 are Asp and Pro,
respectively. Paragraph 177. The alpha-amylase of any of paragraphs
1-176, wherein the C-domain is different from any of the C-domains
of SEQ ID NOS: 1-12, 29, and 30 at the positions corresponding to
positions 430 and 432, e.g., the amino acids at the positions
corresponding to positions 430 and 432 are Asp and Pro,
respectively. Paragraph 178. The alpha-amylase of any of paragraphs
1-177, wherein the alpha-amylase consists of 481 to 515, 481 to
493, or 481 to 486 amino acids. Paragraph 179. The alpha-amylase of
any of paragraphs 1-178, which is more thermostable than the
alpha-amylase of any of SEQ ID NOS: 1-12, 29 and 30. Paragraph 180.
The alpha-amylase of any of paragraphs 1-179, which has reduced
calcium sensitivity than the alpha-amylase of any of SEQ ID NOS:
1-12, 29 and 30.
Sequence CWU 1
1
341483PRTBacillus amyloliquefaciens 1Val Asn Gly Thr Leu Met Gln
Tyr Phe Glu Trp Tyr Thr Pro Asn Asp1 5 10 15Gly Gln His Trp Lys Arg
Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30Ile Gly Ile Thr Ala
Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser 35 40 45Gln Ser Asp Asn
Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu 50 55 60Phe Gln Gln
Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu65 70 75 80Leu
Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr 85 90
95Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp
100 105 110Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu
Thr Ser 115 120 125Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg
Phe Pro Gly Arg 130 135 140Gly Asn Thr Tyr Ser Asp Phe Lys Trp His
Trp Tyr His Phe Asp Gly145 150 155 160Ala Asp Trp Asp Glu Ser Arg
Lys Ile Ser Arg Ile Phe Lys Phe Arg 165 170 175Gly Glu Gly Lys Ala
Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr Asp Tyr
Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val 195 200 205Val
Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser 210 215
220Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser
Phe225 230 235 240Leu Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr
Gly Lys Glu Met 245 250 255Phe Thr Val Ala Glu Tyr Trp Gln Asn Asn
Ala Gly Lys Leu Glu Asn 260 265 270Tyr Leu Asn Lys Thr Ser Phe Asn
Gln Ser Val Phe Asp Val Pro Leu 275 280 285His Phe Asn Leu Gln Ala
Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met 290 295 300Arg Arg Leu Leu
Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala305 310 315 320Val
Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330
335Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu
340 345 350Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met
Tyr Gly 355 360 365Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu
Lys Asp Asn Ile 370 375 380Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr
Ala Tyr Gly Pro Gln His385 390 395 400Asp Tyr Ile Asp His Pro Asp
Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Ser Ala Ala Lys
Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430Gly Gly Ser
Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr 435 440 445Trp
Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser 450 455
460Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile
Tyr465 470 475 480Val Gln Lys2584PRTBacillus flavothermus 2Val Pro
Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu Pro1 5 10 15Asp
Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Asn Ala Gln Ser Leu 20 25
30Ala Asn Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys Gly
35 40 45Thr Ser Ser Ser Asp Val Gly Tyr Gly Val 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 80Thr Gln Tyr Ile Gln Ala Ile Gln Ala Ala His Thr
Ala Gly Met Gln 85 90 95Val Tyr Ala Asp Val Val Phe Asn His Lys Ala
Gly Ala Asp Gly Thr 100 105 110Glu Leu Val Asp Ala Val Glu Val Asn
Pro Ser Asp Arg Asn Gln Glu 115 120 125Ile Ser Gly Thr Tyr Gln Ile
Gln Ala Trp Thr Lys Phe Asp Phe Pro 130 135 140Gly Arg Gly Asn Thr
Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His Phe145 150 155 160Asp Gly
Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170
175Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu Asn
180 185 190Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp
His Pro 195 200 205Glu Val Val Ser Glu Leu Lys Asn Trp Gly Lys Trp
Tyr Val Thr 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
Ser Tyr Val Arg Thr Gln Thr Gln Lys 245 250 255Pro Leu Phe Ala Val
Gly Glu Phe Trp Ser Tyr Asp Ile Ser 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 Ile Ala Ser Lys Ser Gly Gly Tyr Phe 290 295
300Asp Met Arg Thr Leu Leu Asn Asn Thr Leu Met Lys Asp Gln Pro
Thr305 310 315 320Leu Ala Val Thr Leu Val Asp Asn His Asp Thr Glu
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 Ala Leu Lys Ser Lys Leu Asp 370 375 380Pro Leu Leu Ile
Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His Asp385 390 395 400Tyr
Ile Asp Ser Ala Asp Ile Ile Gly Trp Thr Arg Glu Gly Val Ala 405 410
415Glu Lys Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly
420 425 430Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys
Thr 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 480Pro Lys Ile Ser Thr Thr Ser
Gln Ile Thr Phe Thr Val Asn Asn Ala 485 490 495Thr Thr Val Trp Gly
Gln Asn Val Tyr Val Val Gly Asn Ile Ser Gln 500 505 510Leu Gly Asn
Trp Asp Pro Val His Ala Val Gln Met Thr Pro Ser Ser 515 520 525Tyr
Pro Thr Trp Thr Val Thr Ile Pro Leu Leu Gln Gly Gln Asn Ile 530 535
540Gln Phe Lys Phe Ile Lys Lys Asp Ser Ala Gly Asn Val Ile Trp
Glu545 550 555 560Asp Ile Ser Asn Arg Thr Tyr Thr Val Pro Thr Ala
Ala Ser Gly Ala 565 570 575Tyr Thr Ala Ser Trp Asn Val Pro
5803483PRTBacillus licheniformis 3Ala Asn Leu Asn Gly Thr Leu Met
Gln Tyr Phe Glu Trp Tyr Met Pro1 5 10 15Asn Asp Gly Gln His Trp Arg
Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30Ala Glu His Gly Ile Thr
Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser Gln Ala Asp
Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60Gly Glu Phe His
Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75 80Gly Glu
Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn 85 90 95Val
Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr 100 105
110Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
115 120 125Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His
Phe Pro 130 135 140Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His
Trp Tyr His Phe145 150 155 160Asp Gly Thr Asp Trp Asp Glu Ser Arg
Lys Leu Asn Arg Ile Tyr Lys 165 170 175Phe Gln Gly Lys Ala Trp Asp
Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190Tyr Asp Tyr Leu Met
Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200 205Ala Ala Glu
Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215 220Leu
Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe225 230
235 240Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu
Met 245 250 255Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala
Leu Glu Asn 260 265 270Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val
Phe Asp Val Pro Leu 275 280 285His Tyr Gln Phe His Ala Ala Ser Thr
Gln Gly Gly Gly Tyr Asp Met 290 295 300Arg Lys Leu Leu Asn Gly Thr
Val Val Ser Lys His Pro Leu Lys Ser305 310 315 320Val Thr Phe Val
Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335Ser Thr
Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345
350Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His
Lys Ile 370 375 380Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr
Gly Ala Gln His385 390 395 400Asp Tyr Phe Asp His His Asp Ile Val
Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Ser Val Ala Asn Ser Gly
Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430Gly Gly Ala Lys Arg
Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445Trp His Asp
Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455 460Glu
Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr465 470
475 480Val Gln Arg4515PRTBacillus stearothermophilus 4Ala Ala Pro
Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu1 5 10 15Pro Asp
Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30Leu
Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40
45Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
50 55 60Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
Thr65 70 75 80Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala
Ala Gly Met 85 90 95Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly
Gly Ala Asp Gly 100 105 110Thr Glu Trp Val Asp Ala Val Glu Val Asn
Pro Ser Asp Arg Asn Gln 115 120 125Glu Ile Ser Gly Thr Tyr Gln Ile
Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140Pro Gly Arg Gly Asn Thr
Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His145 150 155 160Phe Asp Gly
Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175Lys
Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185
190Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His
195 200 205Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr
Val Asn 210 215 220Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val
Lys His Ile Lys225 230 235 240Phe Ser Phe Phe Pro Asp Trp Leu Ser
Tyr Val Arg Ser Gln Thr Gly 245 250 255Lys Pro Leu Phe Thr Val Gly
Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 260 265 270Leu His Asn Tyr Ile
Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp 275 280 285Ala Pro Leu
His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala 290 295 300Phe
Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro305 310
315 320Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly
Gln 325 330 335Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu
Ala Tyr Ala 340 345 350Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys
Val Phe Tyr Gly Asp 355 360 365Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile
Pro Ser Leu Lys Ser Lys Ile 370 375 380Asp Pro Leu Leu Ile Ala Arg
Arg Asp Tyr Ala Tyr Gly Thr Gln His385 390 395 400Asp Tyr Leu Asp
His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly 405 410 415Thr Glu
Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425
430Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val
435 440 445Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile
Asn Ser 450 455 460Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser
Val Ser Val Trp465 470 475 480Val Pro Arg Lys Thr Thr Val Ser Thr
Ile Ala Arg Pro Ile Thr Thr 485 490 495Arg Pro Trp Thr Gly Glu Phe
Val Arg Trp Thr Glu Pro Arg Leu Val 500 505 510Ala Trp Pro
5155485PRTBacillus 5His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr
Phe Glu Trp Tyr1 5 10 15Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu
Arg Ser Asp Ala Ser 20 25 30Asn Leu Lys Asp Lys Gly Ile Ser Ala Val
Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly Ala Ser Gln Asn Asp Val Gly
Tyr Gly Ala 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 Arg Asn Gln Leu Gln
Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95Ile Gln Val Tyr Gly
Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110Ala Thr Glu
Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125Gln
Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135
140Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp
Tyr145 150 155 160His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys
Leu Asn Asn Arg 165 170 175Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly
Trp Asp Trp Glu Val Asp 180 185 190Thr Glu Asn Gly Asn Tyr Asp Tyr
Leu Met Tyr Ala Asp Ile Asp Met 195 200 205Asp His Pro Glu Val Val
Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220Thr Asn Thr Leu
Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225 230 235 240Ile
Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245 250
255Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His
Ser Val 275 280 285Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala
Ser Lys Ser Gly 290 295 300Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn
Gly Thr Val Val Gln Arg305 310 315 320His Pro Met His Ala Val Thr
Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335Glu Glu Ala Leu Glu
Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350Tyr Ala Leu
Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365Gly
Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375
380Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly
Arg385 390
395 400Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg
Glu 405 410 415Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile
Met Ser Asp 420 425 430Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly
Arg Asn Lys Ala Gly 435 440 445Gln Val Trp Thr Asp Ile Thr Gly Asn
Arg Ala Gly Thr Val Thr Ile 450 455 460Asn Ala Asp Gly Trp Gly Asn
Phe Ser Val Asn Gly Gly Ser Val Ser465 470 475 480Ile Trp Val Asn
Lys 4856484PRTBacillus 6His His Asn Gly Thr Asn Gly Thr Met Met Gln
Tyr Phe Glu Trp Tyr1 5 10 15Leu Pro Asn Asp Gly Asn His Trp Asn Arg
Leu Arg Asp Asp Ala Ala 20 25 30Asn Leu Lys Ser Lys Gly Ile Thr Ala
Val Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly Thr Ser Gln Asn Asp Val
Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln
Lys Gly Thr Val Arg Thr Lys Tyr Gly65 70 75 80Thr Arg Asn Gln Leu
Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95Ile Gln Val Tyr
Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110Gly Thr
Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120
125Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg
Trp Tyr145 150 155 160His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg
Gln Leu Gln Asn Lys 165 170 175Ile Tyr Lys Phe Arg Gly Thr Gly Lys
Ala Trp Asp Trp Glu Val Asp 180 185 190Thr Glu Asn Gly Asn Tyr Asp
Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205Asp His Pro Glu Val
Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220Thr Asn Thr
Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225 230 235
240Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr
245 250 255Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn
Asp Leu 260 265 270Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp
Asn His Ser Val 275 280 285Phe Asp Val Pro Leu His Tyr Asn Leu Tyr
Asn Ala Ser Asn Ser Gly 290 295 300Gly Tyr Tyr Asp Met Arg Asn Ile
Leu Asn Gly Ser Val Val Gln Lys305 310 315 320His Pro Thr His Ala
Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335Gly Glu Ala
Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala 340 345 350Tyr
Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360
365Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser
370 375 380Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr
Gly Thr385 390 395 400Gln His Asp Tyr Phe Asp His His Asp Ile Ile
Gly Trp Thr Arg Glu 405 410 415Gly Asn Ser Ser His Pro Asn Ser Gly
Leu Ala Thr Ile Met Ser Asp 420 425 430Gly Pro Gly Gly Asn Lys Trp
Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445Gln Val Trp Arg Asp
Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460Asn Ala Asp
Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser465 470 475
480Val Trp Val Lys7485PRTBacillus 7His His Asn Gly Thr Asn Gly Thr
Met Met Gln Tyr Phe Glu Trp His1 5 10 15Leu Pro Asn Asp Gly Asn His
Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30Asn Leu Arg Asn Arg Gly
Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly Thr Ser Gln
Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu
Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65 70 75 80Thr Arg
Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95Val
Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105
110Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn
115 120 125Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys
Phe Asp 130 135 140Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys
Trp Arg Trp Tyr145 150 155 160His Phe Asp Gly Val Asp Trp Asp Gln
Ser Arg Gln Phe Gln Asn Arg 165 170 175Ile Tyr Lys Phe Arg Gly Asp
Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190Ser Glu Asn Gly Asn
Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205Asp His Pro
Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr 210 215 220Thr
Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225 230
235 240Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn
Ala 245 250 255Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys
Asn Asp Leu 260 265 270Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn
Trp Asn His Ser Val 275 280 285Phe Asp Val Pro Leu His Tyr Asn Leu
Tyr Asn Ala Ser Asn Ser Gly 290 295 300Gly Asn Tyr Asp Met Ala Lys
Leu Leu Asn Gly Thr Val Val Gln Lys305 310 315 320His Pro Met His
Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335Gly Glu
Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala 340 345
350Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met
Lys Ala 370 375 380Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe
Ala Tyr Gly Thr385 390 395 400Gln His Asp Tyr Phe Asp His His Asn
Ile Ile Gly Trp Thr Arg Glu 405 410 415Gly Asn Thr Thr His Pro Asn
Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430Gly Pro Gly Gly Glu
Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly 435 440 445Gln Val Trp
His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile 450 455 460Asn
Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser465 470
475 480Ile Trp Val Lys Arg 4858485PRTBacillus 8His His Asn Gly Thr
Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr1 5 10 15Leu Pro Asn Asp
Gly Asn His Trp Asn Arg Leu Asn Ser Asp Ala Ser 20 25 30Asn Leu Lys
Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly
Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60Asp
Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65 70 75
80Thr Arg Ser Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly
85 90 95Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala
Asp 100 105 110Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn
Asn Arg Asn 115 120 125Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala
Trp Thr Arg Phe Asp 130 135 140Phe Pro Gly Arg Gly Asn Thr His Ser
Ser Phe Lys Trp Arg Trp Tyr145 150 155 160His Phe Asp Gly Val Asp
Trp Asp Gln Ser Arg Arg Leu Asn Asn Arg 165 170 175Ile Tyr Lys Phe
Arg Gly His Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190Thr Glu
Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200
205Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr
210 215 220Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val
Lys His225 230 235 240Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn
His Val Arg Ser Ala 245 250 255Thr Gly Lys Asn Met Phe Ala Val Ala
Glu Phe Trp Lys Asn Asp Leu 260 265 270Gly Ala Ile Glu Asn Tyr Leu
Gln Lys Thr Asn Trp Asn His Ser Val 275 280 285Phe Asp Val Pro Leu
His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295 300Gly Asn Tyr
Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg305 310 315
320His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro
Leu Ala 340 345 350Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro
Ser Val Phe Tyr 355 360 365Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly
Val Pro Ala Met Arg Ser 370 375 380Lys Ile Asp Pro Ile Leu Glu Ala
Arg Gln Lys Tyr Ala Tyr Gly Lys385 390 395 400Gln Asn Asp Tyr Leu
Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415Gly Asn Thr
Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430Gly
Ala Gly Gly Ser Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440
445Gln Val Trp Ser Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile
450 455 460Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser
Val Ser465 470 475 480Ile Trp Val Asn Lys 4859586PRTBacillus 9Gly
Ser Val Pro Val Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr1 5 10
15Leu Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Asn Ala Gln
20 25 30Ser Leu Ala Asn Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala
Tyr 35 40 45Lys Gly Thr Ser Ser Ser Asp Val Gly Tyr Gly Val Tyr Asp
Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr
Lys Tyr Gly65 70 75 80Thr Lys Thr Gln Tyr Ile Gln Ala Ile Gln Ala
Ala His Thr 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 Leu Val Asp Ala Val
Glu Val Asn Pro Ser Asp Arg Asn 115 120 125Gln Glu Ile 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 Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Thr
180 185 190Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp
Met Asp 195 200 205His Pro Glu Val Val Ser Glu Leu Lys Asn Trp Gly
Lys Trp Tyr Val 210 215 220Ile 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 Ser Tyr Leu Arg Thr Gln Thr 245 250 255Gln Lys Pro Leu Phe
Ala Val Gly Glu Phe Trp Ser Tyr Asp Ile 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 Ile Ala Ser Lys Ser Gly Gly 290 295
300Tyr Phe Asp Met Arg Thr Leu Leu Asn Asn Thr Leu Met Lys Glu
Gln305 310 315 320Pro Thr Leu Ser Val Thr Leu Val Asp Asn His Asp
Thr Glu 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 Ala Leu Lys Ser Lys 370 375 380Leu Asp Pro Leu
Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln385 390 395 400His
Asp Tyr Ile Asp Asn Ala Asp Ile Ile Gly Trp Thr Arg Glu Gly 405 410
415Val Ala Glu Lys Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly
420 425 430Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala
Gly Lys 435 440 445Thr 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 Pro Lys Thr Ser Thr
Thr Ser Gln Ile Thr Phe Thr Val Asn 485 490 495Asn Ala Thr Thr Val
Trp Gly Gln Asn Val Tyr Val Val Gly Asn Ile 500 505 510Ser Gln Leu
Gly Asn Trp Asp Pro Val Asn Ala Val Gln Met Thr Pro 515 520 525Ser
Ser Tyr Pro Thr Trp Val Val Thr Val Pro Leu Pro Gln Ser Gln 530 535
540Asn Ile Gln Phe Lys Phe Ile Lys Lys Asp Gly Ser Gly Asn Val
Ile545 550 555 560Trp Glu Asn Ile Ser Asn Arg Thr Tyr Thr Val Pro
Thr Ala Ala Ser 565 570 575Gly Ala Tyr Thr Ala Asn Trp Asn Val Pro
580 58510583PRTBacillus 10Asn 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 Lys Thr Ser Asn Val Thr Phe Thr Val
Asn Asn Ala Thr 485 490 495Thr Thr Ser Gly Gln Asn Val Tyr Val Val
Ala Asn Ile Pro Glu Leu 500 505 510Gly Asn Trp Asn Thr Ala Asn Ala
Ile Lys Met Asn Pro Ser Ser Tyr 515 520 525Pro Thr Trp Lys Ala Thr
Ile Ala Leu Pro Gln Gly Lys Ala Ile Glu 530 535 540Phe Lys Phe Ile
Lys Lys Asp Gln Ala Gly Asn Val Ile Trp Glu Ser545 550 555 560Thr
Ser Asn Arg Thr Tyr Thr Val Pro Phe Ser Ser Thr Gly Ser Tyr 565 570
575Thr Ala Ser Trp Asn Val Pro 58011485PRTBacillus 11His His Asn
Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His1 5 10 15Leu Pro
Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30Asn
Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40
45Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly65 70 75 80Thr Arg Ser Gln Leu Gln Gly Ala Val Thr Ser Leu Lys
Asn Asn Gly 85 90 95Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys
Gly Gly Ala Asp 100 105 110Gly Thr Glu Met Val Asn Ala Val Glu Val
Asn Arg Ser Asn Arg Asn 115 120 125Gln Glu Ile Ser Gly Glu Tyr Thr
Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140Phe Pro Gly Arg Gly Asn
Thr His Ser Asn Phe Lys Trp Arg Trp Tyr145 150 155 160His Phe Asp
Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys 165 170 175Ile
Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185
190Ile Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met
195 200 205Asp His Pro Glu Val Ile Asn Glu Leu Arg Asn Trp Gly Val
Trp Tyr 210 215 220Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp
Ala Val Lys His225 230 235 240Ile Lys Tyr Ser Tyr Thr Arg Asp Trp
Leu Thr His Val Arg Asn Thr 245 250 255Thr Gly Lys Pro Met Phe Ala
Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270Ala Ala Ile Glu Asn
Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val 275 280 285Phe Asp Val
Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300Gly
Tyr Phe Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys305 310
315 320His Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln
Pro 325 330 335Gly Glu Ala Leu Glu Ser Phe Val Gln Ser Trp Phe Lys
Pro Leu Ala 340 345 350Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr
Pro Ser Val Phe Tyr 355 360 365Gly Asp Tyr Tyr Gly Ile Pro Thr His
Gly Val Pro Ser Met Lys Ser 370 375 380Lys Ile Asp Pro Leu Leu Gln
Ala Arg Gln Thr Tyr Ala Tyr Gly Thr385 390 395 400Gln His Asp Tyr
Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415Gly Asp
Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425
430Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys His Lys Ala Gly
435 440 445Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val
Thr Ile 450 455 460Asn Ala Asp Gly Trp Gly Asn Phe Thr Val Asn Gly
Gly Ala Val Ser465 470 475 480Val Trp Val Lys Gln
48512485PRTBacillus 12His His Asn Gly Thr Asn Gly Thr Met Met Gln
Tyr Phe Glu Trp Tyr1 5 10 15Leu Pro Asn Asp Gly Asn His Trp Asn Arg
Leu Arg Ser Asp Ala Ser 20 25 30Asn Leu Lys Asp Lys Gly Ile Thr Ala
Val Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly Ala Ser Gln Asn Asp Val
Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln
Lys Gly Thr Val Arg Thr Lys Tyr Gly65 70 75 80Thr Arg Asn Gln Leu
Gln Ala Ala Val Thr Ala Leu Lys Ser Asn Gly 85 90 95Ile Gln Val Tyr
Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110Ala Thr
Glu Trp Val Arg Ala Val Glu Val Asn Pro Ser Asn Arg Asn 115 120
125Gln Glu Val Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg
Trp Tyr145 150 155 160His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg
Gln Leu Gln Asn Arg 165 170 175Ile Tyr Lys Phe Arg Gly Asp Gly Lys
Gly Trp Asp Trp Glu Val Asp 180 185 190Thr Glu Asn Gly Asn Tyr Asp
Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205Asp His Pro Glu Val
Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220Thr Asn Thr
Leu Gly Leu Asp Gly Phe Arg Ile Gly Ala Val Lys His225 230 235
240Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr
245 250 255Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn
Asp Ile 260 265 270Gly Ala Ile Glu Asn Tyr Leu Ser Lys Thr Asn Trp
Asn His Ser Val 275 280 285Phe Asp Val Pro Leu His Tyr Asn Leu Tyr
Asn Ala Ser Arg Ser Gly 290 295 300Gly Asn Tyr Asp Met Arg Gln Ile
Phe Asn Gly Thr Val Val Gln Arg305 310 315 320His Pro Thr His Ala
Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335Glu Glu Ala
Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350Cys
Ala Leu Thr Leu Thr Arg Asp Gln Gly Tyr Pro Ser Val Phe Tyr 355 360
365Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser
370 375 380Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr
Gly Lys385 390 395 400Gln Asn Asp Tyr Leu Asp His His Asn Met Ile
Gly Trp Thr Arg Glu 405 410 415Gly Asn Thr Ala His Pro Asn Ser Gly
Leu Ala Thr Ile Met Ser Asp 420 425 430Gly Pro Gly Gly Asn Lys Trp
Met Tyr Val Gly Arg Asn Lys Ala Gly 435 440 445Gln Val Trp Arg Asp
Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile 450 455 460Asn Ala Asp
Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser465 470 475
480Ile Trp Val Asn Asn 48513492PRTBacillus circulans 13Lys Arg Asn
His Thr Met Met Gln Phe Phe Glu Trp His Leu Ala Ala1 5 10 15Asp Gly
Asp His Trp Lys Arg Leu Ala Glu Met Ala Pro Glu Leu Lys 20 25 30Ala
Lys Gly Ile Asp Thr Val Trp Val Pro Pro Val Thr Lys Ala Val 35 40
45Ser Ala Glu Asp Thr Gly Tyr Gly Val Tyr Asp Leu Tyr Asp Leu Gly
50 55 60Glu Phe Asp Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys
Gln65 70 75 80Glu Leu Ile Glu Ala Ile Ala Glu Cys Gln Lys Asn Gly
Ile Ala Val 85 90 95Tyr Val Asp Leu Val Met Asn His Lys Ala Gly Ala
Asp Glu Thr Glu 100 105 110Val Phe Lys Val Ile Glu Val Asp Pro Asn
Asp Arg Thr Lys Glu Ile 115 120 125Ser Glu Pro Phe Glu Ile Glu Gly
Trp Thr Lys Phe Thr Phe Pro Gly 130 135 140Arg Gly Asp Gln Tyr Ser
Ser Phe Lys Trp Asn Ser Glu His Phe Asn145 150 155 160Gly Thr Asp
Phe Asp Ala Arg Glu Glu Arg Thr Gly Val Phe Arg Ile 165 170 175Ala
Gly Glu Asn Lys Lys Trp Asn Glu Asn Val Asp Asp Glu Phe Gly 180 185
190Asn Tyr Asp Tyr Leu Met Phe Ala Asn Ile Asp Tyr Asn His Pro Asp
195 200 205Val Arg Arg Glu Met Ile Asp Trp Gly Lys Trp Leu Ile Asp
Thr Leu 210 215 220Gln Cys Gly Gly Phe Arg Leu Asp Ala Ile Lys His
Ile Asn His Glu225 230 235 240Phe Ile Lys Glu Phe Ala Ala Glu Met
Ile Arg Lys Arg Gly Gln Asp 245 250 255Phe Tyr Ile Val Gly Glu Phe
Trp Asn Ser Asn Leu Asp Ala Cys Arg 260 265 270Glu Phe Leu Asp Thr
Val Asp Tyr Gln Ile Asp Leu Phe Asp Val Ser 275 280 285Leu His Tyr
Lys Leu His Glu Ala Ser Leu Lys Gly Arg Asp Phe Asp 290 295 300Leu
Ser Lys Ile Phe Asp Asp Thr Leu Val Gln Thr His Pro Thr His305 310
315 320Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro His Glu Ala
Leu 325 330 335Glu Ser Trp Ile Gly Asp Trp Phe Lys Pro Ser Ala Tyr
Ala Leu Thr 340 345 350Leu Leu Arg Arg Asp Gly Tyr Pro Val Val Phe
Tyr Gly Asp Tyr Tyr 355 360 365Gly Ile Gly Gly Pro Glu Pro Val Asp
Gly Lys Lys Glu Ile Leu Asp 370 375 380Ile Leu Leu Ser Ala Arg Cys
Asn Lys Ala Tyr Gly Glu Gln Glu Asp385 390 395 400Tyr Phe Asp His
Ala Asn Thr Ile Gly Trp Val Arg Arg Gly Val Glu 405 410 415Glu Ile
Glu Gly Ser Gly Cys Ala Val Val Ile Ser Asn Gly Asp Asp 420 425
430Gly Glu Lys Arg Met Phe Ile Gly Glu His Arg Ala Gly Glu Val Trp
435 440 445Val Asp Leu Thr Lys Ser Cys Asp Asp Gln Ile Thr Ile Glu
Glu Asp 450 455 460Gly Trp Ala Thr Phe His Val Cys Gly Gly Gly Val
Ser Val Trp Ala465 470 475 480Leu Pro Glu Gln Asn Glu Asp Cys Ala
Asp Ala Glu 485 49014480PRTBacillus 14Asp Gly Leu Asn Gly Thr Met
Met Gln Tyr Tyr Glu Trp His Leu Glu1 5 10 15Asn Asp Gly Gln His Trp
Asn Arg Leu His Asp Asp Ala Glu Ala Leu 20 25 30Ser Asn Ala Gly Ile
Thr Ala Ile Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45Asn Ser Gln Ala
Asp Val Gly Tyr Gly Ala 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 80Ala
Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys Ser Asn Asp Ile Asn 85 90
95Val Tyr Gly Asp Val Val Met Asn His Lys Leu Gly Ala Asp Phe Thr
100 105 110Glu Ala Val Gln Ala Val Gln Val Asn Pro Ser Asn Arg Trp
Gln Asp 115 120 125Ile Ser Gly Val Tyr Thr Ile Asp Ala Trp Thr Gly
Phe Asp Phe Pro 130 135 140Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys
Trp Arg Trp Phe His Phe145 150 155 160Asn Gly Val Asp Trp Asp Gln
Arg Tyr Gln Glu Asn His Leu Phe Arg 165 170 175Phe Ala Asn Thr Asn
Trp Asn Trp Arg Val Asp Glu Glu Asn Gly Asn 180 185 190Tyr Asp Tyr
Leu Leu Gly Ser Asn Ile Asp Phe Ser His Pro Glu Val 195 200 205Gln
Glu Glu Leu Lys Asp Trp Gly Ser Trp Phe Thr Asp Glu Leu Asp 210 215
220Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His Ile Pro Phe Trp
Tyr225 230 235 240Thr Ser Asp Trp Val Arg His Gln Arg Ser Glu Ala
Asp Gln Asp Leu 245 250 255Phe Val Val Gly Glu Tyr Trp Lys Asp Asp
Val Gly Ala Leu Glu Phe 260 265 270Tyr Leu Asp Glu Met Asn Trp Glu
Met Ser Leu Phe Asp Val Pro Leu 275 280 285Asn Tyr Asn Phe Tyr Arg
Ala Ser Lys Gln Gly Gly Ser Tyr Asp Met 290 295 300Arg Asn Ile Leu
Arg Gly Ser Leu Val Glu Ala His Pro Ile His Ala305 310 315 320Val
Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Glu Ser Leu Glu 325 330
335Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala Tyr Ala Thr Ile Leu
340 345 350Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr Gly Asp Tyr
Tyr Gly 355 360 365Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp Met
Ile Asp Glu Leu 370 375 380Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly
Thr Gln His Asp Tyr Phe385 390 395 400Asp His Trp Asp Ile Val Gly
Trp Thr Arg Glu Gly Thr Ser Ser Arg 405 410 415Pro Asn Ser Gly Leu
Ala Thr Ile Met Ser Asn Gly Pro Gly Gly Ser 420 425 430Lys Trp Met
Tyr Val Gly Gln Gln His Ala Gly Gln Thr Trp Thr Asp 435 440 445Leu
Thr Gly Asn His Ala Ala Ser Val Thr Ile Asn Gly Asp Gly Trp 450 455
460Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser Val Tyr Val Asn
Gln465 470 475 48015480PRTBacillus 15Asp Gly Leu Asn Gly Thr Met
Met Gln Tyr Tyr Glu Trp His Leu Glu1 5 10 15Asn Asp Gly Gln His Trp
Asn Arg Leu His Asp Asp Ala Ala Ala Leu 20 25 30Ser Asp Ala Gly Ile
Thr Ala Ile Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45Asn Ser Gln Ala
Asp Val Gly Tyr Gly Ala 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 80Ala
Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys Ser Asn Asp Ile Asn 85 90
95Val Tyr Gly Asp Val Val Met Asn His Lys Met Gly Ala Asp Phe Thr
100 105 110Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr Asn Arg Trp
Gln Asp 115 120 125Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr Gly
Phe Asp Phe Ser 130 135 140Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys
Trp Arg Trp Phe His Phe145 150 155 160Asn Gly Val Asp Trp Asp Gln
Arg Tyr Gln Glu Asn His Ile Phe Arg 165 170 175Phe Ala Asn Thr Asn
Trp Asn Trp Arg Val Asp Glu Glu Asn Gly Asn 180 185 190Tyr Asp Tyr
Leu Leu Gly Ser Asn Ile Asp Phe Ser His Pro Glu Val 195 200 205Gln
Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe Thr Asp Glu Leu Asp 210 215
220Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His Ile Pro Phe Trp
Tyr225 230 235 240Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu Ala
Asp Gln Asp Leu 245 250 255Phe Val Val Gly Glu Tyr Trp Lys Asp Asp
Val Gly Ala Leu Glu Phe 260 265 270Tyr
Leu Asp Glu Met Asn Trp Glu Met Ser Leu Phe Asp Val Pro Leu 275 280
285Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly Gly Ser Tyr Asp Met
290 295 300Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala His Pro Met
His Ala305 310 315 320Val Thr Phe Val Asp Asn His Asp Thr Gln Pro
Gly Glu Ser Leu Glu 325 330 335Ser Trp Val Ala Asp Trp Phe Lys Pro
Leu Ala Tyr Ala Thr Ile Leu 340 345 350Thr Arg Glu Gly Gly Tyr Pro
Asn Val Phe Tyr Gly Asp Tyr Tyr Gly 355 360 365Ile Pro Asn Asp Asn
Ile Ser Ala Lys Lys Asp Met Ile Asp Glu Leu 370 375 380Leu Asp Ala
Arg Gln Asn Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe385 390 395
400Asp His Trp Asp Val Val Gly Trp Thr Arg Glu Gly Ser Ser Ser Arg
405 410 415Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn Gly Pro Gly
Gly Ser 420 425 430Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly Gln
Thr Trp Thr Asp 435 440 445Leu Thr Gly Asn Asn Gly Ala Ser Val Thr
Ile Asn Gly Asp Gly Trp 450 455 460Gly Glu Phe Phe Thr Asn Gly Gly
Ser Val Ser Val Tyr Val Asn Gln465 470 475 48016435PRTPyrococcus
woesei 16Ala Lys Tyr Leu Glu Leu Glu Glu Gly Gly Val Ile Met Gln
Ala Phe1 5 10 15Tyr Trp Asp Val Pro Gly Gly Gly Ile Trp Trp Asp His
Ile Arg Ser 20 25 30Lys Ile Pro Glu Trp Tyr Glu Ala Gly Ile Ser Ala
Ile Trp Leu Pro 35 40 45Pro Pro Ser Lys Gly Met Ser Gly Gly Tyr Ser
Met Gly Tyr Asp Pro 50 55 60Tyr Asp Tyr Phe Asp Leu Gly Glu Tyr Tyr
Gln Lys Gly Thr Val Glu65 70 75 80Thr Arg Phe Gly Ser Lys Glu Glu
Leu Val Arg Leu Ile Gln Thr Ala 85 90 95His Ala Tyr Gly Ile Lys Val
Ile Ala Asp Val Val Ile Asn His Arg 100 105 110Ala Gly Gly Asp Leu
Glu Trp Asn Pro Phe Val Gly Asp Tyr Thr Trp 115 120 125Thr Asp Phe
Ser Lys Val Ala Ser Gly Lys Tyr Thr Ala Asn Tyr Leu 130 135 140Asp
Phe His Pro Asn Glu Leu His Cys Cys Asp Glu Gly Thr Phe Gly145 150
155 160Gly Phe Pro Asp Ile Cys His His Lys Glu Trp Asp Gln Tyr Trp
Leu 165 170 175Trp Lys Ser Asn Glu Ser Tyr Ala Ala Tyr Leu Arg Ser
Ile Gly Phe 180 185 190Asp Gly Trp Arg Phe Asp Tyr Val Lys Gly Tyr
Gly Ala Trp Val Val 195 200 205Arg Asp Trp Leu Asn Trp Trp Gly Gly
Trp Ala Val Gly Glu Tyr Trp 210 215 220Asp Thr Asn Val Asp Ala Leu
Leu Ser Trp Ala Tyr Glu Ser Gly Ala225 230 235 240Lys Val Phe Asp
Phe Pro Leu Tyr Tyr Lys Met Asp Glu Ala Phe Asp 245 250 255Asn Asn
Asn Ile Pro Ala Leu Val Tyr Ala Leu Gln Asn Gly Gln Thr 260 265
270Val Val Ser Arg Asp Pro Phe Lys Ala Val Thr Phe Val Ala Asn His
275 280 285Asp Thr Asp Ile Ile Trp Asn Lys Tyr Pro Ala Tyr Ala Phe
Ile Leu 290 295 300Thr Tyr Glu Gly Gln Pro Val Ile Phe Tyr Arg Asp
Phe Glu Glu Trp305 310 315 320Leu Asn Lys Asp Lys Leu Ile Asn Leu
Ile Trp Ile His Asp His Leu 325 330 335Ala Gly Gly Ser Thr Thr Ile
Val Tyr Tyr Asp Asn Asp Glu Leu Ile 340 345 350Phe Val Arg Asn Gly
Asp Ser Arg Arg Pro Gly Leu Ile Thr Tyr Ile 355 360 365Asn Leu Ser
Pro Asn Trp Val Gly Arg Trp Val Tyr Val Pro Lys Phe 370 375 380Ala
Gly Ala Cys Ile His Glu Tyr Thr Gly Asn Leu Gly Gly Trp Val385 390
395 400Asp Lys Arg Val Asp Ser Ser Gly Trp Val Tyr Leu Glu Ala Pro
Pro 405 410 415His Asp Pro Ala Asn Gly Tyr Tyr Gly Tyr Ser Val Trp
Ser Tyr Cys 420 425 430Gly Val Gly 43517484PRTArtificial
sequenceSynthetic construct 17His His Asn Gly Thr Asn Gly Thr Met
Met Gln Tyr Phe Glu Trp His1 5 10 15Leu Pro Asn Asp Gly Asn His Trp
Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30Asn Leu Arg Asn Arg Gly Ile
Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly Thr Ser Gln Asn
Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe
Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65 70 75 80Thr Arg Ser
Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95Val Gln
Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp 100 105
110Glu Thr Glu Val Phe Lys Val Ile Glu Val Asp Pro Asn Asp Arg Thr
115 120 125Lys Glu Ile Ser Glu Pro Phe Glu Ile Glu Gly Trp Thr Lys
Phe Thr 130 135 140Phe Pro Gly Arg Gly Asp Gln Tyr Ser Ser Phe Lys
Trp Asn Ser Glu145 150 155 160His Phe Asn Gly Thr Asp Phe Asp Ala
Lys Gly Glu Arg Thr Gly Val 165 170 175Phe Arg Ile Ala Gly Glu Asn
Lys Lys Trp Asn Glu Asn Val Asp Asp 180 185 190Glu Phe Gly Asn Tyr
Asp Tyr Leu Met Phe Ala Asn Ile Asp Tyr Asn 195 200 205His Pro Asp
Val Arg Arg Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr 210 215 220Asn
Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile225 230
235 240Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala
Thr 245 250 255Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn
Asp Leu Gly 260 265 270Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp
Asn His Ser Val Phe 275 280 285Asp Val Pro Leu His Tyr Asn Leu Tyr
Asn Ala Ser Asn Ser Gly Gly 290 295 300Asn Tyr Asp Met Ala Lys Leu
Leu Asn Gly Thr Val Val Gln Lys His305 310 315 320Pro Met His Ala
Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly 325 330 335Glu Ser
Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr 340 345
350Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly
355 360 365Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys
Ala Lys 370 375 380Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala
Tyr Gly Thr Gln385 390 395 400His Asp Tyr Phe Asp His His Asn Ile
Ile Gly Trp Thr Arg Glu Gly 405 410 415Asn Thr Thr His Pro Asn Ser
Gly Leu Ala Thr Ile Met Ser Asp Gly 420 425 430Pro Gly Gly Glu Lys
Trp Met Tyr Val Gly Gln Asn Lys Ala Gly Gln 435 440 445Val Trp His
Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile Asn 450 455 460Ala
Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile465 470
475 480Trp Val Lys Arg18482PRTArtificial sequenceSynthetic
construct 18His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu
Trp His1 5 10 15Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp
Asp Ala Ser 20 25 30Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile
Pro Pro Ala Trp 35 40 45Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly
Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr
Val Arg Thr Lys Tyr Gly65 70 75 80Thr Arg Ser Gln Leu Glu Ser Ala
Ile His Ala Leu Lys Asn Asn Gly 85 90 95Val Gln Val Tyr Gly Asp Val
Val Met Asn His Lys Ala Gly Ala Asp 100 105 110Glu Thr Glu Val Phe
Lys Val Ile Glu Val Asp Pro Asn Asp Arg Thr 115 120 125Lys Glu Ile
Ser Glu Pro Phe Glu Ile Glu Gly Trp Thr Lys Phe Thr 130 135 140Phe
Pro Gly Arg Gly Asp Gln Tyr Ser Ser Phe Lys Trp Asn Ser Glu145 150
155 160His Phe Asn Gly Thr Asp Phe Asp Ala Lys Gly Glu Arg Thr Gly
Val 165 170 175Phe Arg Ile Ala Gly Lys Lys Trp Asn Trp Glu Val Asp
Thr Glu Phe 180 185 190Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asn Ile
Asp Tyr Asn His Pro 195 200 205Asp Val Arg Arg Glu Leu Arg Arg Trp
Gly Glu Trp Tyr Thr Asn Thr 210 215 220Leu Asn Leu Asp Gly Phe Arg
Ile Asp Ala Val Lys His Ile Lys Tyr225 230 235 240Ser Phe Thr Arg
Asp Trp Leu Thr His Val Arg Asn Ala Thr Gly Lys 245 250 255Glu Met
Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala Leu 260 265
270Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe Asp Val
275 280 285Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly
Asn Tyr 290 295 300Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln
Lys His Pro Met305 310 315 320His Ala Val Thr Phe Val Asp Asn His
Asp Ser Gln Pro Gly Glu Ser 325 330 335Leu Glu Ser Phe Val Gln Glu
Trp Phe Lys Pro Leu Ala Tyr Ala Leu 340 345 350Ile Leu Thr Arg Glu
Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp Tyr 355 360 365Tyr Gly Ile
Pro Thr His Ser Val Pro Ala Met Lys Ala Lys Ile Asp 370 375 380Pro
Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr Gln His Asp385 390
395 400Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn
Thr 405 410 415Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
Gly Pro Gly 420 425 430Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys
Ala Gly Gln Val Trp 435 440 445His Asp Ile Thr Gly Asn Lys Pro Gly
Thr Val Thr Ile Asn Ala Asp 450 455 460Gly Trp Ala Asn Phe Ser Val
Asn Gly Gly Ser Val Ser Ile Trp Val465 470 475 480Lys
Arg19484PRTArtificial sequenceSynthetic construct 19His His Asn Gly
Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His1 5 10 15Leu Pro Asn
Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30Asn Leu
Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45Lys
Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55
60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65
70 75 80Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn
Gly 85 90 95Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly
Ala Asp 100 105 110Glu Thr Glu Val Phe Lys Val Ile Glu Val Asp Pro
Asn Asp Arg Thr 115 120 125Lys Glu Ile Ser Glu Pro Phe Glu Ile Glu
Gly Trp Thr Lys Phe Thr 130 135 140Phe Pro Gly Arg Gly Asp Gln Tyr
Ser Ser Phe Lys Trp Asn Ser Glu145 150 155 160His Phe Asn Gly Thr
Asp Phe Asp Ala Lys Gly Glu Arg Thr Gly Val 165 170 175Phe Arg Ile
Ala Gly Glu Asn Lys Lys Trp Asn Glu Asn Val Asp Asp 180 185 190Glu
Phe Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asn Ile Asp Tyr Asn 195 200
205His Pro Asp Val Arg Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr
210 215 220Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys
His Ile225 230 235 240Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His
Val Arg Asn Ala Thr 245 250 255Gly Lys Glu Met Phe Ala Val Ala Glu
Phe Trp Lys Asn Asp Leu Gly 260 265 270Ala Leu Glu Asn Tyr Leu Asn
Lys Thr Asn Trp Asn His Ser Val Phe 275 280 285Asp Val Pro Leu His
Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly 290 295 300Asn Tyr Asp
Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys His305 310 315
320Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly
325 330 335Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu
Ala Tyr 340 345 350Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser
Val Phe Tyr Gly 355 360 365Asp Tyr Tyr Gly Ile Pro Thr His Ser Val
Pro Ala Met Lys Ala Lys 370 375 380Ile Asp Pro Ile Leu Glu Ala Arg
Gln Asn Phe Ala Tyr Gly Thr Gln385 390 395 400His Asp Tyr Phe Asp
His His Asn Ile Ile Gly Trp Thr Arg Glu Gly 405 410 415Asn Thr Thr
His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly 420 425 430Pro
Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly Gln 435 440
445Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile Asn
450 455 460Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val
Ser Ile465 470 475 480Trp Val Lys Arg20482PRTArtificial
sequenceSynthetic construct 20His His Asn Gly Thr Asn Gly Thr Met
Met Gln Tyr Phe Glu Trp His1 5 10 15Leu Pro Asn Asp Gly Asn His Trp
Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30Asn Leu Arg Asn Arg Gly Ile
Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly Thr Ser Gln Asn
Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe
Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65 70 75 80Thr Arg Ser
Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95Val Gln
Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp 100 105
110Glu Thr Glu Val Phe Lys Val Ile Glu Val Asp Pro Asn Asp Arg Thr
115 120 125Lys Glu Ile Ser Glu Pro Phe Glu Ile Glu Gly Trp Thr Lys
Phe Thr 130 135 140Phe Pro Gly Arg Gly Asp Gln Tyr Ser Ser Phe Lys
Trp Asn Ser Glu145 150 155 160His Phe Asn Gly Thr Asp Phe Asp Ala
Lys Gly Glu Arg Thr Gly Val 165 170 175Phe Arg Ile Ala Gly Lys Lys
Trp Asn Trp Glu Val Asp Thr Glu Phe 180 185 190Gly Asn Tyr Asp Tyr
Leu Met Phe Ala Asn Ile Asp Tyr Asn His Pro 195 200 205Asp Val Arg
Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr Asn Thr 210 215 220Leu
Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys Tyr225 230
235 240Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala Thr Gly
Lys 245 250 255Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
Gly Ala Leu 260 265 270Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His
Ser Val Phe Asp Val 275 280 285Pro Leu His Tyr Asn Leu Tyr Asn Ala
Ser Asn Ser Gly Gly Asn Tyr 290 295 300Asp Met Ala Lys Leu Leu Asn
Gly Thr Val Val Gln Lys His Pro Met305 310 315 320His Ala Val Thr
Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu Ser 325 330 335Leu Glu
Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr Ala Leu 340 345
350Ile Leu Thr Arg Glu Gln
Gly Tyr Pro Ser Val Phe Tyr Gly Asp Tyr 355 360 365Tyr Gly Ile Pro
Thr His Ser Val Pro Ala Met Lys Ala Lys Ile Asp 370 375 380Pro Ile
Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr Gln His Asp385 390 395
400Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn Thr
405 410 415Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly
Pro Gly 420 425 430Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala
Gly Gln Val Trp 435 440 445His Asp Ile Thr Gly Asn Lys Pro Gly Thr
Val Thr Ile Asn Ala Asp 450 455 460Gly Trp Ala Asn Phe Ser Val Asn
Gly Gly Ser Val Ser Ile Trp Val465 470 475 480Lys
Arg21484PRTArtificial sequenceSynthetic construct 21His His Asn Gly
Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His1 5 10 15Leu Pro Asn
Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30Asn Leu
Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45Lys
Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55
60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65
70 75 80Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn
Gly 85 90 95Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly
Ala Asp 100 105 110Glu Thr Glu Val Phe Lys Val Ile Glu Val Asp Pro
Asn Asp Arg Thr 115 120 125Lys Glu Ile Ser Glu Pro Phe Glu Ile Glu
Gly Trp Thr Lys Phe Thr 130 135 140Phe Pro Gly Arg Gly Asp Gln Tyr
Ser Ser Phe Lys Trp Asn Ser Glu145 150 155 160His Phe Asn Gly Thr
Asp Phe Asp Ala Lys Gly Glu Arg Thr Gly Val 165 170 175Phe Arg Ile
Ala Gly Glu Asn Lys Lys Trp Asn Glu Asn Val Asp Asp 180 185 190Glu
Phe Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asn Ile Asp Tyr Asn 195 200
205His Pro Asp Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr
210 215 220Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys
His Ile225 230 235 240Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His
Val Arg Asn Ala Thr 245 250 255Gly Lys Glu Met Phe Ala Val Ala Glu
Phe Trp Lys Asn Asp Leu Gly 260 265 270Ala Leu Glu Asn Tyr Leu Asn
Lys Thr Asn Trp Asn His Ser Val Phe 275 280 285Asp Val Pro Leu His
Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly Gly 290 295 300Asn Tyr Asp
Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys His305 310 315
320Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly
325 330 335Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu
Ala Tyr 340 345 350Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser
Val Phe Tyr Gly 355 360 365Asp Tyr Tyr Gly Ile Pro Thr His Ser Val
Pro Ala Met Lys Ala Lys 370 375 380Ile Asp Pro Ile Leu Glu Ala Arg
Gln Asn Phe Ala Tyr Gly Thr Gln385 390 395 400His Asp Tyr Phe Asp
His His Asn Ile Ile Gly Trp Thr Arg Glu Gly 405 410 415Asn Thr Thr
His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly 420 425 430Pro
Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly Gln 435 440
445Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile Asn
450 455 460Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val
Ser Ile465 470 475 480Trp Val Lys Arg22482PRTArtificial
sequenceSynthetic construct 22His His Asn Gly Thr Asn Gly Thr Met
Met Gln Tyr Phe Glu Trp His1 5 10 15Leu Pro Asn Asp Gly Asn His Trp
Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30Asn Leu Arg Asn Arg Gly Ile
Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly Thr Ser Gln Asn
Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe
Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65 70 75 80Thr Arg Ser
Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95Val Gln
Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp 100 105
110Glu Thr Glu Val Phe Lys Val Ile Glu Val Asp Pro Asn Asp Arg Thr
115 120 125Lys Glu Ile Ser Glu Pro Phe Glu Ile Glu Gly Trp Thr Lys
Phe Thr 130 135 140Phe Pro Gly Arg Gly Asp Gln Tyr Ser Ser Phe Lys
Trp Asn Ser Glu145 150 155 160His Phe Asn Gly Thr Asp Phe Asp Ala
Lys Gly Glu Arg Thr Gly Val 165 170 175Phe Arg Ile Ala Gly Lys Lys
Trp Asn Trp Glu Val Asp Thr Glu Phe 180 185 190Gly Asn Tyr Asp Tyr
Leu Met Phe Ala Asn Ile Asp Tyr Asn His Pro 195 200 205Asp Val Val
Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr Thr Asn Thr 210 215 220Leu
Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys Tyr225 230
235 240Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala Thr Gly
Lys 245 250 255Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
Gly Ala Leu 260 265 270Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His
Ser Val Phe Asp Val 275 280 285Pro Leu His Tyr Asn Leu Tyr Asn Ala
Ser Asn Ser Gly Gly Asn Tyr 290 295 300Asp Met Ala Lys Leu Leu Asn
Gly Thr Val Val Gln Lys His Pro Met305 310 315 320His Ala Val Thr
Phe Val Asp Asn His Asp Ser Gln Pro Gly Glu Ser 325 330 335Leu Glu
Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala Tyr Ala Leu 340 345
350Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp Tyr
355 360 365Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala Lys
Ile Asp 370 375 380Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly
Thr Gln His Asp385 390 395 400Tyr Phe Asp His His Asn Ile Ile Gly
Trp Thr Arg Glu Gly Asn Thr 405 410 415Thr His Pro Asn Ser Gly Leu
Ala Thr Ile Met Ser Asp Gly Pro Gly 420 425 430Gly Glu Lys Trp Met
Tyr Val Gly Gln Asn Lys Ala Gly Gln Val Trp 435 440 445His Asp Ile
Thr Gly Asn Lys Pro Gly Thr Val Thr Ile Asn Ala Asp 450 455 460Gly
Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp Val465 470
475 480Lys Arg23484PRTArtificial sequenceSynthetic construct 23His
His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr1 5 10
15Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser
20 25 30Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala
Trp 35 40 45Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala 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 Arg Asn Gln Leu Gln Ala Ala Val Asn Ala
Leu Lys Ser Asn Gly 85 90 95Ile Gln Val Tyr Gly Asp Val Val Met Asn
His Lys Ala Gly Ala Asp 100 105 110Glu Thr Glu Val Phe Lys Val Ile
Glu Val Asp Pro Asn Asp Arg Thr 115 120 125Lys Glu Ile Ser Glu Pro
Phe Glu Ile Glu Gly Trp Thr Lys Phe Thr 130 135 140Phe Pro Gly Arg
Gly Asp Gln Tyr Ser Ser Phe Lys Trp Asn Ser Glu145 150 155 160His
Phe Asn Gly Thr Asp Phe Asp Ala Lys Gly Glu Arg Thr Gly Val 165 170
175Phe Arg Ile Ala Gly Glu Asn Lys Lys Trp Asn Glu Asn Val Asp Asp
180 185 190Glu Phe Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asn Ile Asp
Tyr Asn 195 200 205His Pro Asp Val Val Asn Glu Leu Arg Asn Trp Gly
Val Trp Tyr Thr 210 215 220Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile
Asp Ala Val Lys His Ile225 230 235 240Lys Tyr Ser Phe Thr Arg Asp
Trp Ile Asn His Val Arg Ser Ala Thr 245 250 255Gly Lys Asn Met Phe
Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly 260 265 270Ala Ile Glu
Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val Phe 275 280 285Asp
Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly Gly 290 295
300Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg
His305 310 315 320Pro Met His Ala Val Thr Phe Val Asp Asn His Asp
Ser Gln Pro Glu 325 330 335Glu Ala Leu Glu Ser Phe Val Glu Glu Trp
Phe Lys Pro Leu Ala Tyr 340 345 350Ala Leu Thr Leu Thr Arg Glu Gln
Gly Tyr Pro Ser Val Phe Tyr Gly 355 360 365Asp Tyr Tyr Gly Ile Pro
Thr His Gly Val Pro Ala Met Lys Ser Lys 370 375 380Ile Asp Pro Ile
Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg Gln385 390 395 400Asn
Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly 405 410
415Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly
420 425 430Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala
Gly Gln 435 440 445Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr
Val Thr Ile Asn 450 455 460Ala Asp Gly Trp Gly Asn Phe Ser Val Asn
Gly Gly Ser Val Ser Ile465 470 475 480Trp Val Asn
Lys24482PRTArtificial sequenceSynthetic construct 24His His Asn Gly
Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr1 5 10 15Leu Pro Asn
Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30Asn Leu
Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp 35 40 45Lys
Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala 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 Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn
Gly 85 90 95Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly
Ala Asp 100 105 110Glu Thr Glu Val Phe Lys Val Ile Glu Val Asp Pro
Asn Asp Arg Thr 115 120 125Lys Glu Ile Ser Glu Pro Phe Glu Ile Glu
Gly Trp Thr Lys Phe Thr 130 135 140Phe Pro Gly Arg Gly Asp Gln Tyr
Ser Ser Phe Lys Trp Asn Ser Glu145 150 155 160His Phe Asn Gly Thr
Asp Phe Asp Ala Lys Gly Glu Arg Thr Gly Val 165 170 175Phe Arg Ile
Ala Gly Lys Lys Trp Asn Trp Glu Val Asp Thr Glu Phe 180 185 190Gly
Asn Tyr Asp Tyr Leu Met Phe Ala Asn Ile Asp Tyr Asn His Pro 195 200
205Asp Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn Thr
210 215 220Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile
Lys Tyr225 230 235 240Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg
Ser Ala Thr Gly Lys 245 250 255Asn Met Phe Ala Val Ala Glu Phe Trp
Lys Asn Asp Leu Gly Ala Ile 260 265 270Glu Asn Tyr Leu Asn Lys Thr
Asn Trp Asn His Ser Val Phe Asp Val 275 280 285Pro Leu His Tyr Asn
Leu Tyr Asn Ala Ser Lys Ser Gly Gly Asn Tyr 290 295 300Asp Met Arg
Gln Ile Phe Asn Gly Thr Val Val Gln Lys His Pro Met305 310 315
320His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Glu Glu Ala
325 330 335Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala Tyr
Ala Leu 340 345 350Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe
Tyr Gly Asp Tyr 355 360 365Tyr Gly Ile Pro Thr His Gly Val Pro Ala
Met Lys Ser Lys Ile Asp 370 375 380Pro Ile Leu Glu Ala Arg Gln Lys
Tyr Ala Tyr Gly Arg Gln Asn Asp385 390 395 400Tyr Leu Asp His His
Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn Thr 405 410 415Ala His Pro
Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Ala Gly 420 425 430Gly
Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly Gln Val Trp 435 440
445Thr Asp Ile Thr Gly Asn Lys Ala Gly Thr Val Thr Ile Asn Ala Asp
450 455 460Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile
Trp Val465 470 475 480Asn Lys25484PRTArtificial sequenceSynthetic
construct 25His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu
Trp Tyr1 5 10 15Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser
Asp Ala Ser 20 25 30Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile
Pro Pro Ala Trp 35 40 45Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly
Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr
Val Arg Thr Lys Tyr Gly65 70 75 80Thr Arg Ser Gln Leu Gln Ala Ala
Val Thr Ser Leu Lys Asn Asn Gly 85 90 95Ile Gln Val Tyr Gly Asp Val
Val Met Asn His Lys Ala Gly Ala Asp 100 105 110Glu Thr Glu Val Phe
Lys Val Ile Glu Val Asp Pro Asn Asp Arg Thr 115 120 125Lys Glu Ile
Ser Glu Pro Phe Glu Ile Glu Gly Trp Thr Lys Phe Thr 130 135 140Phe
Pro Gly Arg Gly Asp Gln Tyr Ser Ser Phe Lys Trp Asn Ser Glu145 150
155 160His Phe Asn Gly Thr Asp Phe Asp Ala Lys Gly Glu Arg Thr Gly
Val 165 170 175Phe Arg Ile Ala Gly Glu Asn Lys Lys Trp Asn Glu Asn
Val Asp Asp 180 185 190Glu Phe Gly Asn Tyr Asp Tyr Leu Met Phe Ala
Asn Ile Asp Tyr Asn 195 200 205His Pro Asp Val Val Asn Glu Leu Arg
Asn Trp Gly Val Trp Tyr Thr 210 215 220Asn Thr Leu Gly Leu Asp Gly
Phe Arg Ile Asp Ala Val Lys His Ile225 230 235 240Lys Tyr Ser Phe
Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala Thr 245 250 255Gly Lys
Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly 260 265
270Ala Ile Glu Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val Phe
275 280 285Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser
Gly Gly 290 295 300Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val
Val Gln Arg His305 310 315 320Pro Ser His Ala Val Thr Phe Val Asp
Asn His Asp Ser Gln Pro Glu 325 330 335Glu Ala Leu Glu Ser Phe Val
Glu Glu Trp Phe Lys Pro Leu Ala Tyr 340 345 350Ala Leu Thr Leu Thr
Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly 355 360 365Asp Tyr Tyr
Gly Ile Pro Thr His Gly Val Pro Ala Met Arg Ser Lys 370 375 380Ile
Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys
Gln385 390 395 400Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp
Thr Arg Glu Gly 405 410 415Asn Thr Ala His Pro Asn Ser Gly Leu Ala
Thr Ile Met Ser Asp Gly 420 425 430Ala Gly Gly Ser Lys Trp Met Phe
Val Gly Arg Asn Lys Ala Gly Gln 435 440 445Val Trp Ser Asp Ile Thr
Gly Asn Arg Thr Gly Thr Val Thr Ile Asn 450 455 460Ala Asp Gly Trp
Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile465 470 475 480Trp
Val Asn Lys26482PRTArtificial sequenceSynthetic construct 26His His
Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr1 5 10 15Leu
Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser Asp Ala Ser 20 25
30Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp
35 40 45Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu
Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys
Tyr Gly65 70 75 80Thr Arg Ser Gln Leu Gln Ala Ala Val Thr Ser Leu
Lys Asn Asn Gly 85 90 95Ile Gln Val Tyr Gly Asp Val Val Met Asn His
Lys Ala Gly Ala Asp 100 105 110Glu Thr Glu Val Phe Lys Val Ile Glu
Val Asp Pro Asn Asp Arg Thr 115 120 125Lys Glu Ile Ser Glu Pro Phe
Glu Ile Glu Gly Trp Thr Lys Phe Thr 130 135 140Phe Pro Gly Arg Gly
Asp Gln Tyr Ser Ser Phe Lys Trp Asn Ser Glu145 150 155 160His Phe
Asn Gly Thr Asp Phe Asp Ala Lys Gly Glu Arg Thr Gly Val 165 170
175Phe Arg Ile Ala Gly Lys Lys Trp Asn Trp Glu Val Asp Thr Glu Phe
180 185 190Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asn Ile Asp Tyr Asn
His Pro 195 200 205Asp Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp
Tyr Thr Asn Thr 210 215 220Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala
Val Lys His Ile Lys Tyr225 230 235 240Ser Phe Thr Arg Asp Trp Ile
Asn His Val Arg Ser Ala Thr Gly Lys 245 250 255Asn Met Phe Ala Val
Ala Glu Phe Trp Lys Asn Asp Leu Gly Ala Ile 260 265 270Glu Asn Tyr
Leu Gln Lys Thr Asn Trp Asn His Ser Val Phe Asp Val 275 280 285Pro
Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly Gly Asn Tyr 290 295
300Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg His Pro
Ser305 310 315 320His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln
Pro Glu Glu Ala 325 330 335Leu Glu Ser Phe Val Glu Glu Trp Phe Lys
Pro Leu Ala Tyr Ala Leu 340 345 350Thr Leu Thr Arg Glu Gln Gly Tyr
Pro Ser Val Phe Tyr Gly Asp Tyr 355 360 365Tyr Gly Ile Pro Thr His
Gly Val Pro Ala Met Arg Ser Lys Ile Asp 370 375 380Pro Ile Leu Glu
Ala Arg Gln Lys Tyr Ala Tyr Gly Lys Gln Asn Asp385 390 395 400Tyr
Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly Asn Thr 405 410
415Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly Ala Gly
420 425 430Gly Ser Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly Gln
Val Trp 435 440 445Ser Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr
Ile Asn Ala Asp 450 455 460Gly Trp Gly Asn Phe Ser Val Asn Gly Gly
Ser Val Ser Ile Trp Val465 470 475 480Asn Lys27515PRTArtificial
sequenceSynthetic construct 27Ala Ala Pro Phe Asn Gly Thr Met Met
Gln Tyr Phe Glu Trp Tyr Leu1 5 10 15Pro Asp Asp Gly Thr Leu Trp Thr
Lys Val Ala Asn Glu Ala Asn Asn 20 25 30Leu Ser Ser Leu Gly Ile Thr
Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45Gly Thr Ser Arg Ser Asp
Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60Leu Gly Glu Phe Asn
Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr65 70 75 80Lys Ala Gln
Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met 85 90 95Gln Val
Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp Glu 100 105
110Thr Glu Val Phe Lys Val Ile Glu Val Asp Pro Asn Asp Arg Thr Lys
115 120 125Glu Ile Ser Glu Pro Phe Glu Ile Glu Gly Trp Thr Lys Phe
Thr Phe 130 135 140Pro Gly Arg Gly Asp Gln Tyr Ser Ser Phe Lys Trp
Asn Ser Glu His145 150 155 160Phe Asn Gly Thr Asp Phe Asp Ala Lys
Gly Glu Arg Thr Gly Val Phe 165 170 175Arg Ile Ala Gly Glu Asn Lys
Lys Trp Asn Glu Asn Val Asp Asp Glu 180 185 190Phe Gly Asn Tyr Asp
Tyr Leu Met Phe Ala Asn Ile Asp Tyr Asn His 195 200 205Pro Asp Val
Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn 210 215 220Thr
Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys225 230
235 240Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr
Gly 245 250 255Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp
Ile Asn Lys 260 265 270Leu His Asn Tyr Ile Thr Lys Thr Asn Gly Thr
Met Ser Leu Phe Asp 275 280 285Ala Pro Leu His Asn Lys Phe Tyr Thr
Ala Ser Lys Ser Gly Gly Ala 290 295 300Phe Asp Met Arg Thr Leu Met
Thr Asn Thr Leu Met Lys Asp Gln Pro305 310 315 320Thr Leu Ala Val
Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln 325 330 335Ala Leu
Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340 345
350Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp
355 360 365Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser
Lys Ile 370 375 380Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr
Gly Thr Gln His385 390 395 400Asp Tyr Leu Asp His Ser Asp Ile Ile
Gly Trp Thr Arg Glu Gly Val 405 410 415Thr Glu Lys Pro Gly Ser Gly
Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430Gly Gly Ser Lys Trp
Met Tyr Val Gly Lys Gln His Ala Gly Lys Val 435 440 445Phe Tyr Asp
Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser 450 455 460Asp
Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp465 470
475 480Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro Ile Thr
Thr 485 490 495Arg Pro Trp Thr Gly Glu Phe Val Arg Trp Thr Glu Pro
Arg Leu Val 500 505 510Ala Trp Pro 51528491PRTArtificial
sequenceSynthetic construct 28Ala Ala Pro Phe Asn Gly Thr Met Met
Gln Tyr Phe Glu Trp Tyr Leu1 5 10 15Pro Asp Asp Gly Thr Leu Trp Thr
Lys Val Ala Asn Glu Ala Asn Asn 20 25 30Leu Ser Ser Leu Gly Ile Thr
Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45Gly Thr Ser Arg Ser Asp
Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60Leu Gly Glu Phe Asn
Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr65 70 75 80Lys Ala Gln
Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met 85 90 95Gln Val
Tyr Ala Asp Val Val Phe Asn His Lys Ala Gly Ala Asp Glu 100 105
110Thr Glu Val Phe Lys Val Ile Glu Val Asp Pro Asn Asp Arg Thr Lys
115 120 125Glu Ile Ser Glu Pro Phe Glu Ile Glu Gly Trp Thr Lys Phe
Thr Phe 130 135 140Pro Gly Arg Gly Asp Gln Tyr Ser Ser Phe Lys Trp
Asn Ser Glu His145 150 155 160Phe Asn Gly Thr Asp Phe Asp Ala Lys
Gly Glu Arg Thr Gly Val Phe 165 170 175Arg Ile Ala Gly Lys Lys Trp
Asn Trp Glu Val Asp Thr Glu Phe Gly 180 185 190Asn Tyr Asp Tyr Leu
Met Phe Ala Asn Ile Asp Tyr Asn His Pro Asp 195 200 205Val Val Thr
Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn Thr Thr 210 215 220Asn
Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser225 230
235 240Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys
Pro 245 250 255Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn
Lys Leu His 260 265 270Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser
Leu Phe Asp Ala Pro 275 280 285Leu His Asn Lys Phe Tyr Thr Ala Ser
Lys Ser Gly Gly Ala Phe Asp 290 295 300Met Arg Thr Leu Met Thr Asn
Thr Leu Met Lys Asp Gln Pro Thr Leu305 310 315 320Ala Val Thr Phe
Val Asp Asn His Asp Thr Glu Pro Gly Gln Ala Leu 325 330 335Gln Ser
Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile 340 345
350Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp Tyr Tyr
355 360 365Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile
Asp Pro 370 375 380Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr
Gln His Asp Tyr385 390 395 400Leu Asp His Ser Asn Ile Ile Gly Trp
Thr Arg Glu Gly Gly Thr Glu 405 410 415Lys Pro Gly Ser Gly Leu Ala
Ala Leu Ile Thr Asn Gly Asp Gly Gly 420 425 430Ser Lys Trp Met Tyr
Val Gly Lys Gln His Ala Gly Lys Val Phe Tyr 435 440 445Asp Leu Thr
Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser Asp Gly 450 455 460Trp
Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp Val Pro465 470
475 480Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro 485
49029486PRTBacillus stearothermophilus 29Ala Ala Pro Phe Asn Gly
Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu1 5 10 15Pro Asp Asp Gly Thr
Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30Leu Ser Ser Leu
Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45Gly Thr Ser
Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60Leu Gly
Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr65 70 75
80Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met
85 90 95Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp
Gly 100 105 110Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp
Arg Asn Gln 115 120 125Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp
Thr Lys Phe Asp Phe 130 135 140Pro Gly Arg Gly Asn Thr Tyr Ser Ser
Phe Lys Trp Arg Trp Tyr His145 150 155 160Phe Asp Gly Val Asp Trp
Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175Lys Phe Arg Gly
Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185 190Asn Gly
Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His 195 200
205Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn
210 215 220Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His
Ile Lys225 230 235 240Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val
Arg Ser Gln Thr Gly 245 250 255Lys Pro Leu Phe Thr Val Gly Glu Tyr
Trp Ser Tyr Asp Ile Asn Lys 260 265 270Leu His Asn Tyr Ile Thr Lys
Thr Asn Gly Thr Met Ser Leu Phe Asp 275 280 285Ala Pro Leu His Asn
Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala 290 295 300Phe Asp Met
Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro305 310 315
320Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln
325 330 335Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala
Tyr Ala 340 345 350Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val
Phe Tyr Gly Asp 355 360 365Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro
Ser Leu Lys Ser Lys Ile 370 375 380Asp Pro Leu Leu Ile Ala Arg Arg
Asp Tyr Ala Tyr Gly Thr Gln His385 390 395 400Asp Tyr Leu Asp His
Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Val 405 410 415Thr Glu Lys
Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430Gly
Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val 435 440
445Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser
450 455 460Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser
Val Trp465 470 475 480Val Pro Arg Lys Thr Thr 48530485PRTCytophaga
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 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 48531435PRTArtificial sequenceSynthetic construct
31Ala Lys Tyr Ser Glu Leu Glu Lys Gly Gly Val Ile Met Gln Ala Phe1
5 10 15Tyr Trp Asp Val Pro Ser Gly Gly Ile Trp Trp Asp Thr Ile Arg
Gln 20 25 30Lys Ile Pro Glu Trp Tyr Asp Ala Gly Ile Ser Ala Ile Trp
Ile Pro 35 40 45Pro Ala Ser Lys Gly Met Gly Gly Ala Tyr Ser Met Gly
Tyr Asp Pro 50 55 60Tyr Asp Phe Phe Asp Leu Gly Glu Tyr Asp Gln Lys
Gly Thr Val Glu65 70 75 80Thr Arg Phe Gly Ser Lys Gln Glu Leu Val
Asn Met Ile Asn Thr Ala 85 90 95His Ala Tyr Gly Met Lys Val Ile Ala
Asp Ile Val Ile Asn His Arg 100 105 110Ala Gly Gly Asp Leu Glu Trp
Asn Pro Phe Val Asn Asp Tyr Thr Trp 115 120 125Thr Asp Phe Ser Lys
Val Ala Ser Gly Lys Tyr Thr Ala Asn Tyr Leu 130 135 140Asp Phe His
Pro Asn Glu Leu His Ala Gly Asp Ser Gly Thr Phe Gly145 150 155
160Gly Tyr Pro Asp Ile Cys His Asp Lys Ser Trp Asp Gln Tyr Trp Leu
165 170 175Trp Ala Ser Gln Glu Ser Tyr Ala Ala Tyr Leu Arg Ser Ile
Gly Ile 180 185 190Asp Ala Trp Arg Phe Asp Tyr Val Lys Gly Tyr Ala
Pro Trp Val Val 195 200 205Lys Asp Trp Leu Asn Trp Trp Gly Gly Trp
Ala Val Gly Glu Tyr Trp 210 215 220Asp Thr Asn Val Asp Ala Val Leu
Asn Trp Ala Tyr Ser Ser Gly Ala225 230 235 240Lys Val Phe Asp Phe
Ala Leu Tyr Tyr Lys Met Asp Glu Ala Phe Asp 245 250 255Asn Lys Asn
Ile Pro Ala Leu Val Ser Ala Leu Gln Asn Gly Gln Thr 260 265 270Val
Val Ser Arg Asp Pro Phe Lys Ala Val Thr Phe Val Ala Asn His 275 280
285Asp Thr Asp Ile Ile Trp Asn Lys Tyr Pro Ala Tyr Ala Phe Ile Leu
290 295 300Thr Tyr Glu Gly Gln Pro Thr Ile Phe Tyr Arg Asp Tyr Glu
Glu Trp305 310 315 320Leu Asn Lys Asp Lys Leu Lys Asn Leu Ile Trp
Ile His Glu Asn Leu 325 330 335Ala Gly Gly Ser Thr Asp Ile Val Tyr
Tyr Asp Asn Asp Glu Leu Ile 340 345 350Phe Val Arg Asn Gly Tyr Gly
Asp Lys Pro Gly Leu Ile Thr Tyr Ile 355 360 365Asn Leu Gly Ser Ser
Lys Ala Gly Arg Trp Val Tyr Val Pro Lys Phe 370 375 380Ala Gly Ala
Cys Ile His Glu Tyr Thr Gly Asn Leu Gly Gly Trp Val385 390 395
400Asp Lys Tyr Val Tyr Ser Ser Gly Trp Val Tyr Leu Glu Ala Pro Ala
405 410 415Tyr Asp Pro Ala Asn Gly Gln Tyr Gly Tyr Ser Val Trp Ser
Tyr Cys 420 425 430Gly Val Gly 43532482PRTArtificial
sequenceSynthetic construct 32His His Asn Gly Thr Asn Gly Thr Leu
Met Gln Tyr Phe Glu Trp Tyr1 5 10 15Leu Pro Asn Asp Gly Asn His Trp
Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30Asn Leu Lys Asp Lys Gly Ile
Ser Ala Val Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly Ala Ser Gln Asn
Asp Val Gly Tyr Gly Ala 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 Arg Asn
Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95Ile Gln
Val Tyr Gly Asp Val Val Met Asn His Lys Ala Gly Ala Asp 100 105
110Glu Thr Glu Val Phe Lys Val Ile Glu Val Asp Pro Asn Asp Arg Thr
115 120 125Lys Glu Ile Ser Glu Pro Phe Glu Ile Glu Gly Trp Thr Lys
Phe Thr 130 135 140Phe Pro Gly Arg Gly Asp Gln Tyr Ser Ser Phe Lys
Trp Asn Ser Glu145 150 155 160His Phe Asn Gly Thr Asp Phe Asp Ala
Lys Gly Glu Arg Thr Gly Val 165 170 175Phe Arg Ile Ala Gly Lys Lys
Trp Asn Trp Glu Val Asp Thr Glu Phe 180 185 190Gly Asn Tyr Asp Tyr
Leu Met Phe Ala Asn Ile Asp Tyr Asn His Pro 195 200 205Asp Val Val
Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn Thr 210 215 220Leu
Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile Lys Tyr225 230
235 240Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala Ile Gly
Lys 245 250 255Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
Gly Ala Ile 260 265 270Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His
Ser Val Phe Asp Val 275 280 285Pro Leu His Phe Asn Leu Tyr Tyr Ala
Ser Lys Ser Gly Gly Asn Tyr 290 295 300Asp Met Arg Gln Ile Phe Asn
Gly Thr Val Val Gln Lys His Pro Thr305 310 315 320His Ala Val Thr
Phe Val Asp Asn His Asp Ser Gln Pro Glu Glu Ser 325 330 335Leu Glu
Ser Phe Val Arg Glu Trp Phe Lys Pro Leu Ala Tyr Ala Leu 340 345
350Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp Tyr
355 360 365Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser Lys
Ile Asp 370 375 380Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly
Arg Gln Asn Asp385 390 395 400Tyr Leu Asp His His Asn Ile Ile Gly
Trp Thr Arg Glu Gly Asn Thr 405 410 415Ala His Pro Asn Ser Gly Leu
Ala Thr Ile Met Ser Asp Gly Ala Gly 420 425 430Gly Asn Lys Trp Met
Phe Val Gly Arg Asn Lys Ala Gly Gln Val Trp 435 440 445Thr Asp Ile
Thr Gly Asn Lys Ala Gly Thr Val Thr Ile Asn Ala Asp 450 455 460Gly
Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser Ile Trp Val465 470
475 480Asn Lys33484PRTArtificial sequenceSynthetic construct 33His
His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr1 5 10
15Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Asn Ser Asp Ala Ser
20 25 30Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala
Trp 35 40 45Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp
Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr
Lys Tyr Gly65 70 75 80Thr Arg Ser Gln Leu Gln Ala Ala Val Thr Ser
Leu Lys Asn Asn Gly 85 90 95Ile Gln Val Tyr Gly Asp Val Val Met Asn
His Lys Ala Gly Ala Asp 100 105 110Glu Thr Glu Val Phe Lys Val Ile
Glu Val Asp Pro Asn Asp Arg Thr 115 120 125Lys Glu Ile Ser Glu Pro
Phe Glu Ile Glu Gly Trp Thr Lys Phe Thr 130 135 140Phe Pro Gly Arg
Gly Asp Gln Tyr Ser Ser Phe Lys Trp Asn Ser Glu145 150 155 160His
Phe Asn Gly Thr Asp Phe Asp Ala Lys Gly Glu Arg Thr Gly Val 165 170
175Phe Arg Ile Ala Gly Glu Asn Lys Lys Trp Asn Glu Asn Val Asp Asp
180 185 190Glu Phe Gly Asn Tyr Asp Tyr Leu Met Phe Ala Asn Ile Asp
Tyr Asn 195 200 205His Pro Asp Val Val Asn Glu Leu Arg Asn Trp Gly
Val Trp Tyr Thr 210 215 220Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile
Asp Ala Val Lys His Ile225 230 235 240Lys Tyr Ser Phe Thr Arg Asp
Trp Ile Asn His Val Arg Ser Ala Thr 245 250 255Gly Lys Asn Met Phe
Ala Val Ala Glu Phe Trp Lys Asn Asp Leu Gly 260 265 270Ala Ile Glu
Asn Tyr Leu Gln Lys Thr Asn Trp Asn His Ser Val Phe 275 280 285Asp
Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly Gly 290 295
300Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg
His305 310 315 320Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp
Ser Gln Pro Glu 325 330 335Glu Ala Leu Glu Ser Phe Val Glu Glu Trp
Phe Lys Pro Leu Ala Tyr 340 345 350Ala Leu Thr Leu Thr Arg Glu Gln
Gly Tyr Pro Ser Val Phe Tyr Gly 355 360 365Asp Tyr Tyr Gly Ile Pro
Thr His Gly Val Pro Ala Met Arg Ser Lys 370 375 380Ile Asp Pro Ile
Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys Gln385 390 395 400Asn
Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu Gly 405 410
415Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly
420 425 430Ala Gly Gly Ser Lys Trp Met Phe Val Gly Arg Asn Lys Ala
Gly Gln 435 440 445Val Trp Ser Asp Ile Thr Gly Asn Arg Thr Gly Thr
Val Thr Ile Asn 450 455 460Ala Asp Gly Trp Gly Asn Phe Ser Val Asn
Gly Gly Ser Val Ser Ile465 470 475 480Trp Val Asn
Lys34483PRTArtificial sequenceSynthetic construct 34Ala Ala Val Asn
Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro1 5 10 15Asn Asp Gly
Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu 20 25 30Ser Asp
Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Ala 35 40 45Ile
Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55
60Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65
70 75 80Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile
Asn 85 90 95Val Tyr Gly Asp Val Val Ile Asp His Lys Ala Gly Ala Asp
Glu Thr 100 105 110Glu Val Phe Lys Val Ile Glu Val Asp Pro Asn Asp
Arg Thr Lys Glu 115 120 125Ile Ser Glu Pro Phe Glu Ile Glu Gly Trp
Thr Lys Phe Thr Phe Pro 130 135 140Gly Arg Gly Asp Thr Tyr Ser Ser
Phe Lys Trp Asn Ser Glu His Phe145 150 155 160Asn Gly Val Asp Phe
Asp Ala Lys Gly Glu Arg Thr Gly Val Phe Arg 165 170 175Ile Ala Gly
Lys Ala Trp Asn Trp Glu Val Asp Thr Glu Phe Gly Asn 180 185 190Tyr
Asp Tyr Leu Met Phe Ala Asn Ile Asp Tyr Asn His Pro Asp Val 195 200
205Val Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln
210 215 220Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe
Ser Phe225 230 235 240Leu Arg Asp Trp Val Asn His Val Arg Glu Lys
Thr Gly Lys Glu Met 245 250 255Phe Thr Val Ala Glu Tyr Trp Ser Asn
Asp Leu Gly Ala Leu Glu Asn 260 265 270Tyr Leu Asn Lys Thr Asn Phe
Asn His Ser Val Phe Asp Val Pro Leu 275 280 285His Tyr Gln Phe His
Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295 300Arg Lys Leu
Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser305 310 315
320Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe
Ile Leu 340 345 350Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly
Asp Met Tyr Gly 355 360 365Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro
Ala Leu Lys His Lys Ile 370 375 380Glu Pro Ile Leu Lys Ala Arg Lys
Gln Tyr Ala Tyr Gly Ala Gln His385 390 395 400Asp Tyr Phe Asp His
His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Ser Val
Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430Gly
Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440
445Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
450 455 460Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser
Ile Tyr465 470 475 480Val Gln Arg
* * * * *