Patent application title: Alpha-Amylase Mutants With Altered Properties
Inventors:
Thomas Thisted (Rungsted Kyst, DK)
Soren Kjaerulff (Vanlose, DK)
Carsten Andersen (Vaerlose, DK)
Claus Crone Fuglsang (Niva, DK)
Assignees:
Novozymes A/S
IPC8 Class: AC12N916FI
USPC Class:
435196
Class name: Enzyme (e.g., ligases (6. ), etc.), proenzyme; compositions thereof; process for preparing, activating, inhibiting, separating, or purifying enzymes hydrolase (3. ) acting on ester bond (3.1)
Publication date: 2010-04-22
Patent application number: 20100099161
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Patent application title: Alpha-Amylase Mutants With Altered Properties
Inventors:
Carsten Andersen
Claus Crone Fuglsang
Soren Kjaerulff
Thomas Thisted
Agents:
NOVOZYMES NORTH AMERICA, INC.
Assignees:
Novozymes A/S
Origin: NEW YORK, NY US
IPC8 Class: AC12N916FI
USPC Class:
435196
Patent application number: 20100099161
Abstract:
The present invention relates to variants (mutants) of parent
Termamyl-like alpha-amylases, which variant has alpha-amylase activity
and exhibits altered stability, in particular at high temperatures and/or
at low pH relative, and/or low Ca2+ to the parent alpha-amylase.Claims:
1. An isolated variant of a parent alpha-amylase, wherein:(a) the variant
has at least 90% sequence identity to SEQ ID NO: 6,(b) the variant
comprises a substitution of the amino acid at position 239 relative to
the parent alpha-amylase, using the amino acid sequence of SEQ ID NO: 8
for determining position numbering, and(c) the variant has alpha-amylase
activity.
2. The variant of claim 1, wherein the variant has at least 95% sequence identity to SEQ ID NO: 6.
3. The variant of claim 1, wherein the variant has at least 97% sequence identity to SEQ ID NO: 6.
4. The variant of claim 1, wherein the variant has at least 99% sequence identity to SEQ ID NO: 6.
5. The variant of claim 1, wherein the parent alpha-amylase is a Bacillus stearothermophilus alpha-amylase.
6. The variant of claim 5, wherein the Bacillus stearothermophilus alpha-amylase is the amino acid sequence of SEQ ID NO: 6.
7. The variant of claim 1, wherein the substitution at position 239 is a substitution of serine with a different amino acid.
8. The variant of claim 1, wherein the variant further comprises an alteration at one or more positions selected from the group consisting of 49, 60, 104, 132, 161, 170, 176, 179, 180, 181, 183, 200, 203, 204, 207, 212, 237, 250, 280, 298, 318, 374, 385, 393, 402, 406, 427, 430, 440, 444, 447, and 482, wherein the alteration(s) are independently selected from an insertion, a deletion, or a substitution.
9. A composition comprising the variant of claim 1 and (i) another alpha-amylase; or (ii) one or more enzymes selected from the group consisting of glucoamylase, phytase, and pullalanase.
10. An isolated variant of a parent alpha-amylase, wherein:(a) the variant has an amino acid sequence with 1-15 alteration(s) relative to the parent alpha-amylase, wherein(i) the 1-15 alteration(s) are independently selected from an insertion, a deletion, or a substitution, and(ii) the 1-15 alteration(s) include a substitution of the amino acid at position 239, and(b) the parent alpha-amylase has at least 90% sequence identity to SEQ ID NO: 6, and(c) the amino acid sequence of SEQ ID NO: 8 is used for determining position numbering; and(d) the variant has alpha-amylase activity.
11. The variant of claim 10, wherein the alteration(s) are substitution(s).
12. The variant of claim 10, wherein the variant has 1 alteration relative to the parent alpha-amylase which is the substitution of the amino acid at position 239.
13. The variant of claim 10, wherein the substitution at position 239 is a substitution of serine with a different amino acid.
14. The variant of claim 10, wherein the parent alpha-amylase has at least 95% sequence identity to SEQ ID NO: 6.
15. The variant of claim 10, wherein the parent alpha-amylase has at least 99% sequence identity to SEQ ID NO: 6.
16. The variant of claim 10, wherein one or more alteration(s) are at a position selected from the group consisting of 49, 60, 104, 132, 161, 170, 176, 179, 180, 181, 183, 200, 203, 204, 207, 212, 237, 250, 280, 298, 318, 374, 385, 393, 402, 406, 427, 430, 440, 444, 447, and 482.
17. A composition comprising the variant of claim 10 and (i) another alpha-amylase or (i) one or more enzymes selected from the group consisting of glucoamylase, phytase.
18. An isolated variant of a Bacillus stearothermophilus alpha-amylase, wherein the variant consists of a substitution of the amino acid at position 239 with a different amino acid, using the amino acid sequence of SEQ ID NO: 8 for determining position numbering.
19. The variant of claim 18, wherein the substitution at position 239 is a substitution of serine.
20. The variant of claim 18, wherein the Bacillus stearothermophilus alpha-amylase is the amino acid sequence of SEQ ID NO: 6.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]This application is a continuation of U.S. application Ser. No. 12/566,238 filed Sep. 24, 2009, which is a continuation of U.S. application Ser. No. 10/630,203 filed Jul. 29, 2003, which is a continuation of U.S. application Ser. No. 09/918,543 filed Jul. 31, 2001, now abandoned, which claims the benefit or priority under 35 U.S.C. 119 of Danish Application Nos. PA 2000 01160, PA 2000 01354, PA 2000 01687 and PA 2001 00655 filed Aug. 1, 2000, Sep. 12, 2000, Nov. 10, 2000, and Apr. 26, 2001, respectively, and U.S. Provisional Application Nos. 60/225,140, 60/233,986, 60/249,104 and 60/286,869 filed on Aug. 14, 2000, Sep. 20, 2000, Nov. 16, 2000, and Apr. 26, 2001, respectively, the contents of which are fully incorporated herein by reference.
SEQUENCE LISTING
[0002]The present application contains a Sequence Listing in the form of a text file, which is incorporated herein by reference.
FIELD OF THE INVENTION
[0003]The present invention relates to variants (mutants) of parent Termamyl-like alpha-amylases, which variant has alpha-amylase activity and exhibits an alteration in at least one of the following properties relative to said parent alpha-amylase: stability under, e.g., high temperature and/or low pH conditions, in particular at low calcium concentrations. The variant of the invention are suitable for starch conversion, ethanol production, laundry wash, dish wash, hard surface cleaning, textile desizing, and/or sweetener production.
BACKGROUND OF THE INVENTION
[0004]Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1) constitute a group of enzymes, which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.
BRIEF DISCLOSURE OF THE INVENTION
[0005]The object of the present invention is to provide Termamyl-like amylases which variants in comparison to the corresponding parent alpha-amylase, i.e., un-mutated alpha-amylase, has alpha-amylase activity and exhibits an alteration in at least one of the following properties relative to said parent alpha-amylase: stability under, e.g., high temperature and/or low pH conditions, in particular at low calcium concentrations.
Nomenclature
[0006]In the present description and claims, the conventional one-letter and three-letter codes for amino acid residues are used. For ease of reference, alpha-amylase variants of the invention are described by use of the following nomenclature: [0007]Original amino acid(s): position(s): substituted amino acid(s)
[0008]According to this nomenclature, for instance the substitution of alanine for asparagine in position 30 is shown as: [0009]Ala30Asn or A30Na deletion of alanine in the same position is shown as: [0010]Ala30* or A30*and an insertion of an additional amino acid residue, such as lysine, is shown as: [0011]Ala30AlaLys or A30AK
[0012]A deletion of a consecutive stretch of amino acid residues, such as amino acid residues 30-33, is indicated as (30-33)* or Δ(A30-N33).
[0013]Where a specific alpha-amylase contains a "deletion" in comparison with other alpha-amylases and an insertion is made in such a position this is indicated as: [0014]*36Asp or *36Dfor an insertion of an aspartic acid in position 36.
[0015]Multiple mutations are separated by plus signs, i.e.: [0016]Ala30Asp+Glu34Ser or A30N+E34Srepresenting mutations in positions 30 and 34 substituting alanine and glutamic acid for asparagine and serine, respectively.
[0017]When one or more alternative amino acid residues may be inserted in a given position it is indicated as [0018]A30N,E or A30N or A30E
[0019]Furthermore, when a position suitable for modification is identified herein without any specific modification being suggested, it is to be understood that any amino acid residue may be substituted for the amino acid residue present in the position. Thus, for instance, when a modification of an alanine in position 30 is mentioned, but not specified, it is to be understood that the alanine may be deleted or substituted for any other amino acid, i.e., any one of: R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.
[0020]Further, "A30X" means any one of the following substitutions:
A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A301, A30L, A30K, A30M, A30F, A30P, A30S, A30T, A30W, A30Y, or A30 V; or in short: A30R,N,D,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.
[0021]If the parent enzyme--used for the numbering--already has the amino acid residue in question suggested for substitution in that position the following nomenclature is used: [0022]"X30N" or "X30N,V"in the case where for instance one or N or V is present in the wildtype.
[0023]Thus, it means that other corresponding parent enzymes are substituted to an "Asn" or "Val" in position 30.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024]FIG. 1 is an alignment of the amino acid sequences of five parent Termamyl-like alpha-amylases. The numbers on the extreme left designate the respective amino acid sequences as follows:
1: SEQ ID NO: 4 (SP722)
2: SEQ ID NO: 2 (SP690)
3: SEQ ID NO: 10 (BAN)
4: SEQ ID NO: 8 (BLA)
5: SEQ ID NO: 6 (BSG).
DETAILED DISCLOSURE OF THE INVENTION
[0025]The object of the present invention is to provide Termamyl-like amylases, which variants have alpha-amylase activity and exhibits altered stability at high temperatures and/or at low pH, in particular at low calcium concentrations.
Termamyl-Like Alpha-Amylases
[0026]A number of alpha-amylases produced by Bacillus spp. are highly homologous (identical) on the amino acid level.
[0027]The identity of a number of known Bacillus alpha-amylases can be found in the below Table 1:
TABLE-US-00001 TABLE 1 Percent identity 707 AP1378 BAN BSG SP690 SP722 AA560 Termamyl 707 100.0 86.4 66.9 66.5 87.6 86.2 95.5 68.1 AP1378 86.4 100.0 67.1 68.1 95.1 86.6 86.0 69.4 BAN 66.9 67.1 100.0 65.6 67.1 68.8 66.9 80.7 BSG 66.5 68.1 65.6 100.0 67.9 67.1 66.3 65.4 SP690 87.6 95.1 67.1 67.9 100.0 87.2 87.0 69.2 SP722 86.2 86.6 68.8 67.1 87.2 100.0 86.8 70.8 AA560 95.5 86.0 66.9 66.3 87.0 86.8 100.0 68.3 Termamyl 68.1 69.4 80.7 65.4 69.2 70.8 68.3 100.0
[0028]For instance, the B. licheniformis alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 8 (commercially available as Termamyl®) has been found to be about 81% homologous with the B. amyloliquefaciens alpha-amylase comprising the amino acid sequence shown in SEQ ID NO: 10 and about 65% homologous with the B. stearothermophilus alpha-amylase (BSG) comprising the amino acid sequence shown in SEQ ID NO: 6. Further homologous alpha-amylases include SP690 and SP722 disclosed in WO 95/26397 and further depicted in SEQ ID NO: 2 and SEQ ID NO: 4, respectively, herein. Other amylases are the AA560 alpha-amylase derived from Bacillus sp. and shown in SEQ ID NO: 12, and the #707 alpha-amylase derived from Bacillus sp., shown in SEQ ID NO: 13 and described by Tsukamoto et al., 1988, Biochemical and Biophysical Research Communications 151: 25-31.
[0029]The KSM AP1378 alpha-amylase is disclosed in WO 97/00324 (from KAO Corporation).
[0030]Still further homologous alpha-amylases include the alpha-amylase produced by the B. licheniformis strain described in EP 0252666 (ATCC 27811), and the alpha-amylases identified in WO 91/00353 and WO 94/18314. Other commercial Termamyl-like alpha-amylases are comprised in the products sold under the following tradenames: Optitherm® and Takatherm® (Solvay); Maxamyl® (available from Gist-brocades/Genencor), Spezym AA® and Spezyme Delta AA® (available from Genencor), and Keistase® (available from Daiwa), Dex lo, GC 521 (available from Genencor) and Ultraphlow (from Enzyme Biosystems).
[0031]Because of the substantial homology found between these alpha-amylases, they are considered to belong to the same class of alpha-amylases, namely the class of "Termamyl-like alpha-amylases".
[0032]Accordingly, in the present context, the term "Termamyl-like" alpha-amylase" is intended to indicate an alpha-amylase, in particular Bacillus alpha-amylase, which, at the amino acid level, exhibits a substantial identity to Termamyl®, i.e., the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8, herein.
[0033]In other words, all of the following alpha-amylases, which have the amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12 and 13 herein, are considered to be "Termamyl-like alpha-amylase". Other Termamyl-like alpha-amylases are alpha-amylases i) which display at least 60%, such as at least 70%, e.g., at least 75%, or at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% homology (identity) with at least one of said amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, and 13, and/or are encoded by a DNA sequence which hybridizes to the DNA sequences encoding the above-specified alpha-amylases which are apparent from SEQ ID NOS: 1, 3, 5, 7, 9, and of the present specification (which encoding sequences encode the amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10 and 12 herein, respectively).
Homology
[0034]The homology may be determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (described above). Thus, Gap GCGv8 may be used with the default scoring matrix for identity and the following default parameters: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, respectively for nucleic acidic sequence comparison, and GAP creation penalty of 3.0 and GAP extension penalty of 0.1, respectively, for protein sequence comparison. GAP uses the method of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453, to make alignments and to calculate the identity.
[0035]A structural alignment between Termamyl (SEQ ID NO: 8) and, e.g., another alpha-amylase may be used to identify equivalent/corresponding positions in other Termamyl-like alpha-amylases. One method of obtaining said structural alignment is to use the Pile Up programme from the GCG package using default values of gap penalties, i.e., a gap creation penalty of 3.0 and gap extension penalty of 0.1. Other structural alignment methods include the hydrophobic cluster analysis (Gaboriaud et al., 1987, FEBS Letters 224: 149-155) and reverse threading (Huber and Torda, 1998, Protein Science 7(1): 142-149).
Hybridization
[0036]The oligonucleotide probe used in the characterization of the Termamyl-like alpha-amylase above may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the alpha-amylase in question.
[0037]Suitable conditions for testing hybridization involve pre-soaking in 5×SSC and prehybridizing for 1 hour at 40° C. in a solution of 20% formamide, 5×Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 mg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 100 mM ATP for 18 hours at 40° C., followed by three times washing of the filter in 2×SSC, 0.2% SDS at 40° C. for 30 minutes (low stringency), preferably at 50° C. (medium stringency), more preferably at 65° C. (high stringency), even more preferably at 75° C. (very high stringency). More details about the hybridization method can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.
[0038]In the present context, "derived from" is intended not only to indicate an alpha-amylase produced or producible by a strain of the organism in question, but also an alpha-amylase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence. Finally, the term is intended to indicate an alpha-amylase, which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the alpha-amylase in question. The term is also intended to indicate that the parent alpha-amylase may be a variant of a naturally occurring alpha-amylase, i.e., a variant, which is the result of a modification (insertion, substitution, deletion) of one or more amino acid residues of the naturally occurring alpha-amylase.
Parent Termamyl-Like Alpha-Amylases
[0039]According to the invention all Termamy-like alpha-amylases, as defined above, may be used as the parent (i.e., backbone) alpha-amylase. In a preferred embodiment of the invention the parent alpha-amylase is derived from B. licheniformis, e.g., one of those referred to above, such as the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8.
Parent Hybrid Termamyl-Like Alpha-Amylases
[0040]The parent alpha-amylase (i.e., backbone alpha-amylase) may also be a hybrid alpha-amylase, i.e., an alpha-amylase, which comprises a combination of partial amino acid sequences derived from at least two alpha-amylases.
[0041]The parent hybrid alpha-amylase may be one, which on the basis of amino acid homology (identity) and/or DNA hybridization (as defined above) can be determined to belong to the Termamyl-like alpha-amylase family. In this case, the hybrid alpha-amylase is typically composed of at least one part of a Termamyl-like alpha-amylase and part(s) of one or more other alpha-amylases selected from Termamyl-like alpha-amylases or non-Termamyl-like alpha-amylases of microbial (bacterial or fungal) and/or mammalian origin.
[0042]Thus, the parent hybrid alpha-amylase may comprise a combination of partial amino acid sequences deriving from at least two Termamyl-like alpha-amylases, or from at least one Termamyl-like and at least one non-Termamyl-like bacterial alpha-amylase, or from at least one Termamyl-like and at least one fungal alpha-amylase. The Termamyl-like alpha-amylase from which a partial amino acid sequence derives, may be any of the specific Termamyl-like alpha-amylase referred to herein.
[0043]For instance, the parent alpha-amylase may comprise a C-terminal part of an alpha-amylase derived from a strain of B. licheniformis, and a N-terminal part of an alpha-amylase derived from a strain of B. amyloliquefaciens or from a strain of B. stearothermophilus. For instance, the parent alpha-amylase may comprise at least 430 amino acid residues of the C-terminal part of the B. licheniformis alpha-amylase, and may, e.g., comprise a) an amino acid segment corresponding to the 37 N-terminal amino acid residues of the B. amyloliquefaciens alpha-amylase having the amino acid sequence shown in SEQ ID NO: 10 and an amino acid segment corresponding to the 445 C-terminal amino acid residues of the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8, or a hybrid Termamyl-like alpha-amylase being identical to the Termamyl sequence, i.e., the Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8, except that the N-terminal 35 amino acid residues (of the mature protein) has been replaced by the N-terminal 33 residues of BAN (mature protein), i.e., the Bacillus amyloliquefaciens alpha-amylase shown in SEQ ID NO: 10; or b) an amino acid segment corresponding to the 68 N-terminal amino acid residues of the B. stearothermophilus alpha-amylase having the amino acid sequence shown in SEQ ID NO: 6 and an amino acid segment corresponding to the 415 C-terminal amino acid residues of the B. licheniformis alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8.
[0044]Another suitable parent hybrid alpha-amylase is the one previously described in WO 96/23874 (from Novo Nordisk) constituting the N-terminus of BAN, Bacillus amyloliquefaciens alpha-amylase (amino acids 1-300 of the mature protein) and the C-terminus from Termamyl (amino acids 301-483 of the mature protein).
[0045]In a preferred embodiment of the invention the parent Termamyl-like alpha-amylase is a hybrid alpha-amylase of SEQ ID NO: 8 and SEQ ID NO: 10. Specifically, the parent hybrid Termamyl-like alpha-amylase may be a hybrid alpha-amylase comprising the 445 C-terminal amino acid residues of the B. licheniformis alpha-amylase shown in SEQ ID NO: 8 and the 37 N-terminal amino acid residues of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 10, which may suitably further have the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 8). The latter mentioned hybrid is used in the examples below and is referred to as LE174.
[0046]Other specifically contemplated parent alpha-amylase include LE174 with fewer mutations, i.e., the right above mentioned hydrid having the following mutations: A181T+N190F+A209V+Q264S; N190F+A209V+Q264S; A209V+Q264S; Q264S; H156Y+N190F+A209V+Q264S; H156Y+A209V+Q264S; H156Y+Q264S; H156Y+A181T+A209V+Q264S; H156Y+A181T+Q264S; H156Y+Q264S; H156Y+A181T+N190F+Q264S; H156Y+A181T+N190F; H156Y+A181T+N190F+A209V. These hybrids are also considered to be part of the invention.
[0047]In a preferred embodiment the parent Termamyl-like alpha amylase is LE174, SP722, or AA560 including any of
LE174+G48A+T49I+G107A+I201F;
LE174+M197L;
LE174+G48A+T49I+G107A+M197L+I201F;
SP722+D183*+G184*;
SP722+D183*+G184*+N195F;
SP722+D183*+G184*+M202L;
SP722+D183*+G184*+N195F+M202L;
BSG+I181*+G182*;
BSG+I181*+G182*+N193F;
BSG+I181*+G182*+M200L;
BSG+I181*+G182*+N193F+M200L;
AA560+D183*+G184*;
AA560+D183*+G184*+N195F; AA560+D183*+G184*+M202L;
AA560+D183*+G184*+N195F+M202L.
[0048]Other parent alpha-amylases contemplated include LE429, which is LE174 with an additional substitution in I201F. According to the invention LE335 is the alpha-amylase, which in comparison to LE429 has additional substitutions in T49I+G107A; LE399 is LE335+G48A, i.e., LE174, with G48A+T49I+G107A+I201F.
Altered Properties
[0049]The following section discusses the relationship between mutations, which are present in variants of the invention, and desirable alterations in properties (relative to those of a parent Termamyl-like alpha-amylase), which may result therefrom.
[0050]As mentioned above the invention relates to Termamyl-like alpha-amylases with altered properties (as mentioned above), in particular at high temperatures and/or at low pH, in particular at low calcium concentrations.
[0051]In the context of the present invention "high temperature" means temperatures from 70-120° C., preferably 80-100° C., especially 85-95° C.
[0052]In the context of the present invention the term "low pH" means from a pH in the range from 4-6, preferably 4.2-5.5, especially 4.5-5.
[0053]In the context of the present invention the term "high pH" means from a pH in the range from 8-11, especially 8.5-10.6.
[0054]In the context of the present invention the term "low calcium concentration" means free calcium levels lower than 60 ppm, preferably 40 ppm, more preferably 25 ppm, especially 5 ppm calcium.
[0055]Parent Termamyl-like alpha-amylase specifically contemplated in connection with going through the specifically contemplated altered properties are the above mentioned parent Termamyl-like alpha-amylase and parent hydrid Termamyl-like alpha-amylases.
[0056]The Termamyl® alpha-amylase is used as the starting point, but corresponding positions in, e.g., the SP722, BSG, BAN, AA560, SP690, KSM AP1378, and #707 should be understood as disclosed and specifically contemplated too.
[0057]In a preferred embodiment the variant of the invention has in particular at high temperatures and/or at low pH.
[0058]In an aspect the invention relates to variant with altered properties as mentioned above.
[0059]In the first aspect a variant of a parent Termamyl-like alpha-amylase, comprising an alteration at one or more positions (using SEQ ID NO: 8 for the amino acid numbering) selected from the group of:
49, 60, 104, 132, 161, 170, 176, 179, 180, 181, 183, 200, 203, 204, 207, 212, 237, 239, 250, 280, 298, 318, 374, 385, 393, 402, 406, 427, 430, 440, 444, 447, 482,wherein(a) the alteration(s) are independently
[0060](i) an insertion of an amino acid downstream of the amino acid which occupies the position,
[0061](ii) a deletion of the amino acid which occupies the position, or
[0062](iii) a substitution of the amino acid which occupies the position with a different amino acid,
(b) the variant has alpha-amylase activity and(c) each position corresponds to a position of the amino acid sequence of the parent Termamyl-like alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8.
[0063]In Termamyl® (SEQ ID NO: 8) such corresponding positions are:
T49; D60; N104; E132; D161; K170; K176; G179; K180; A181; D183; D200; Y203; D204; D207; 1212; K237; S239; E250; N280; Q298; L318; Q374; E385; Q393; Y402; H406; L427; D430; V440; N444; E447; Q482.
[0064]In SP722 (SEQ ID NO: 4) the corresponding positions are:
T51; D62; N106; D134; D163; Q172; K179; G184; K185; A186; D188; D205; M208; D209; X212; L217, K242, S244, N255, N285, S303, M323; D387, N395; Y404; H408; 1429; D432; V442; K446; Q449; K484.
[0065]Corresponding positions in other parent alpha-amylases can be found by alignment as described above and shown in the alignment in FIG. 1.
[0066]In a preferred embodiment the variant of the invention (using SEQ ID NO: 8 (Termamyl®) for the numbering) has one or more of the following substitutions:
T491; D60N; N104D; E132A,V,P; D161N; K170Q; K176R; G179N; K180T; A181N; D183N; D200N; X203Y; D2045; D207V,E,L,G; X2121; K237P; S239W; E250G,F; N280S; X298Q; L318M; Q374R; E385V; Q393R; Y402F; H406L,W; L427I; D430N; V440A; N444R,K; E447Q,K; Q482K.
[0067]In a preferred embodiment the variant of the invention (using SEQ ID NO: 4 (SP722) for the numbering) has one or more of the following substitutions:
T51I; D62N; N106D; D134A,V,P; D163N; X172Q; K179R; G184N; K185T; A186N; D188N; D205N; M208Y; D2095; X212V,E,L,G; L2171, K242P, S244W, N255G,F, N285S, S303Q, X323M; D387V, N395R; Y404F; H408L,W; X4291; D432N; V442A; X446R,K; X449Q,K; X484K, using SEQ ID NO: 4 (SP722) for numbering.
[0068]Preferred double, triple and multi-mutations--using SEQ ID NO: 8 as the basis for the numbering are selected from the group consisting of:
T491+D60N; T491+D60N+E132A; T491+D60N+E132V; T491+D60N+E132V+K170Q;
T491+D60N+E132A+K170Q; T491+D60N+E132V+K170Q+K176R;
T491+D60N+E132A+K170Q+K176R;
T491+D60N+E132V+K170Q+K176R+D207V; T491+D60N+E132A+K170Q+K176R+D207V;
T491+D60N+E132V+K170Q+K176R+D207E; T491+D60N+E132A+K170Q+K176R+D207E;
T491+D60N+E132V+K170Q+K176R+D207V+E250G;
T491+D60N+E132A+K170Q+K176R+D207V+E250G;
T491+D60N+E132V+K170Q+K176R+D207E+E250G;
T491+D60N+E132A+K170Q+K176R+D207E+E250G;
T491+D60N+E132V+K170Q+K176R+D207E+E250G+N280S;
T491+D60N+E132A+K170Q+K176R+D207E+E250G+N280S;
T491+D60N+E132V+K170Q+K176R+D207V+E250G+N280S;
T491+D60N+E132A+K170Q+K176R+D207V+E250G+N2805;
T491+D60N+E132V+K170Q+K176R+D207V+E250G+N2805+L318M;
T491+D60N+E132A+K170Q+K176R+D207V+E250G+N2805+L318M;
T491+D60N+E132V+K170Q+K176R+D207E+E250G+N2805+L318M;
T491+D60N+E132A+K170Q+K176R+D207E+E250G+N2805+L318M;
T491+D60N+E132V+K170Q+K176R+D207V+E250G+N2805+L318M+Q374R;
T491+D60N+E132A+K170Q+K176R+D207V+E250G+N2805+L318M+Q374R;
T491+D60N+E132V+K170Q+K176R+D207E+E250G+N2805+L318M+Q374R;
T491+D60N+E132A+K170Q+K176R+D207E+E250G+N2805+L318M+Q374R;
T491+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;
T491+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;
T491+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
T491+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
T491+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
T491+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
T491+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
T491+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
T491+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F;
T491+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F;
T491+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F;
T491+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F;
T491+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L;
T491+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L;
T491+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L;
T491+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L;
T491+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I;
T491+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I;
T491+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L4271;
T491+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I;
T491+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I+V440A;
T491+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I+V440A;
T491+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I+V440A;
T491+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I+V440A;
[0069]D60N+E132A; D60N+E132V; D60N+E132V+K170Q; D60N+E132A+K170Q;
D60N+E132V+K170Q+K176R; T491+D60N+E132A+K170Q+K176R;
D60N+E132V+K170Q+K176R+D207V; T491+D60N+E132A+K170Q+K176R+D207V;
D60N+E132V+K170Q+K176R+D207E; T491+D60N+E132A+K170Q+K176R+D207E;
D60N+E132V+K170Q+K176R+D207V+E250G;
D60N+E132A+K170Q+K176R+D207V+E250G;
D60N+E132V+K170Q+K176R+D207E+E250G;
D60N+E132A+K170Q+K176R+D207E+E250G;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40- 6L;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40- 6L;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40- 6L;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40- 6L;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40- 6L+L427I;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40- 6L+L427I;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40- 6L+L427I;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40- 6L+L4271;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40- 6L+L427I+V440A;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40- 6L+L427I+V440A;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40- 6L+L427I+V440A;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40- 6L+L427I+V440A;
E132V+K170Q; E132A+K170Q; E132V+K170Q+K176R; E132A+K170Q+K176R;
E132V+K170Q+K176R+D207V; E132A+K170Q+K176R+D207V;
E132V+K170Q+K176R+D207E; E132A+K170Q+K176R+D207E;
E132V+K170Q+K176R+D207V+E250G; E132A+K170Q+K176R+D207V+E250G;
E132V+K170Q+K176R+D207E+E250G; E132A+K170Q+K176R+D207E+E250G;
E132V+K170Q+K176R+D207E+E250G+N280S;
E132A+K170Q+K176R+D207E+E250G+N280S;
E132V+K170Q+K176R+D207V+E250G+N280S;
E132A+K170Q+K176R+D207V+E250G+N280S;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4- 27I;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4- 271;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4- 27I;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4- 27I;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4- 27I+V440A;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4- 27I+V440A;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4- 27I+V440A;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4- 27I+V440A;
K170Q+K176R; K170Q+K176R+D207V; K170Q+K176R+D207E;
K170Q+K176R+D207V+E250G; K170Q+K176R+D207E+E250G;
K170Q+K176R+D207V+E250G+N280S; K170Q+K176R+D207E+E250G+N280S;
K170Q+K176R+D207E+E250G+N280S+L318M;
K170Q+K176R+D207V+E250G+N280S+L318M;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V4- 40A;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V4- 40A;
K176R+D207V; K176R+D207E; K176R+D207V+E250G;
K176R+D207E+E250G; K176R+D207V+E250G+N280S;
K176R+D207E+E250G+N280S; K176R+D207E+E250G+N280S+L318M;
K176R+D207V+E250G+N280S+L318M;
K176R+D207E+E250G+N280S+L318M+Q374R;
K176R+D207V+E250G+N280S+L318M+Q374R;
K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
K176R+D207V+E250G+N280S+L318M+Q374R+E385V;
K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
D207V+E250G; D207E+E250G;
D207V+E250G+N280S; D207E+E250G+N280S+L318M;
D207V+E250G+N280S+L318M; D207E+E250G+N280S+L318M+Q374R;
D207V+E250G+N280S+L318M+Q374R;
D207E+E250G+N280S+L318M+Q374R+E385V;
D207V+E250G+N280S+L318M+Q374R+E385V;
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4271;
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
E250G+N280S; E250G+N280S+L318M; E250G+N280S+L318M+Q374R;
E250G+N280S+L318M+Q374R+E385V;
E250G+N280S+L318M+Q374R+E385V+Q393R;
E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
N280S+L318M; N280S+L318M+Q374R; N280S+L318M+Q374R+E385V;
N280S+L318M+Q374R+E385V+Q393R;
N280S+L318M+Q374R+E385V+Q393R+Y402F;
N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
N280S+L318 M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
L318M+Q374R; L318M+Q374R+E385V; L318M+Q374R+E385V+Q393R;
L318M+Q374R+E385V+Q393R+Y402F;
L318M+Q374R+E385V+Q393R+Y402F+H406L;
L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
Q374R+E385V; Q374R+E385V+Q393R; Q374R+E385V+Q393R+Y402F;
Q374R+E385V+Q393R+Y402F+H406L;
Q374R+E385V+Q393R+Y402F+H406L+L4271;
Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
E385V+Q393R; E385V+Q393R+Y402F; E385V+Q393R+Y402F+H406L;
E385V+Q393R+Y402F+H406L+L427I;
E385V+Q393R+Y402F+H406L+L427I+V440A;
Q393R+Y402F; Q393R+Y402F+H406L; Q393R+Y402F+H406L+L4271;
Q393R+Y402F+H406L+L427I+V440A; Y402F+H406L;
Y402F+H406L+L427I; Y402F+H406L+L427I+V440A; H406L+L427I;
H406L+L427I+V440A; L427I+V440A;
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N4- 44K+E447Q+Q482K;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E4- 47Q+Q482K;
D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K;
D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K;
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E4- 47Q+Q482K;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q4- 82K;
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N;
H406W+D430N; N444K+E447Q+Q482K; E447Q+Q482K;
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+
D430N+N444R+N444K+E447K+Q482K;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444R+N4- 44K+E447K+Q482K;
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W;
H406W+D430N; N444K+E447K+Q482K; E447K+Q482K;
N104D+D161N+A181N+D183N+D200N+D204S+K237P+S239W;
N104D+D161N+A181N+D183N+D200N+D204S+K237P;
N104D+D161N+A181N+D183N+D200N+D204S;
D161N+A181N+D183N+D200N+D204S+K237P+S239W;
D161N+A181N+D183N+D200N+D204S+K237P;
[0070]D161N+A181N+D183N+D200N+D204S; K237P+S239W, using SEQ ID NO: 8 for numbering.
[0071]In a preferred embodiment the variant has the following substitutions: K170Q+D207V+N280S; E132A+D207V; D207E+E250G+H406L+L427I; D207V+L318M; D60N+D207V+L318M; T491+E132V+V440A; T49I+K176R+D207V+Y402F; Q374R+E385V+Q393R; N190F+A209V+Q264S; G48A+T49I+G107A+I201F; T49I+G107A+I201F; G48A+T49I+I201F; G48A+T49I+G107A; T491+I201F; T49I+G107A; G48A+T49I; D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E- 447Q+Q482K using SEQ ID NO: 8 for numbering.
[0072]Specific variants include: LE399; LE174+G48A+T49I+G107A; LE174+G48A+T49I+I201F; LE174+G48A+G107A+I201F; LE174+T49I+G107A+I201F; LE174+G48A+T49I; LE174+G48A; LE174+G107A+I201F; and LE174+I201F.
Stability
[0073]In the context of the present invention, mutations (including amino acid substitutions and deletions) of importance with respect to achieving altered stability, in particular improved stability (i.e., higher or lower), at especially high temperatures (i.e., 70-120° C.) and/or extreme pH (i.e., low or high pH, i.e., pH 4-6 or pH 8-11, respectively), in particular at free (i.e., unbound, therefore in solution) calcium concentrations below 60 ppm, include any of the mutations listed in the "Altered properties" section. The stability may be determined as described in the "Materials & Methods" section below.
General Mutations in Variants of the Invention
[0074]A variant of the invention may in one embodiment comprise one or more modifications in addition to those outlined above. Thus, it may be advantageous that one or more Proline (Pro) residues present in the part of the alpha-amylase variant which is modified is/are replaced with a non-Proline residue which may be any of the possible, naturally occurring non-Proline residues, and which preferably is an Alanine, Glycine, Serine, Threonine, Valine or Leucine.
[0075]Analogously, in one embodiment one or more Cysteine residues present in the parent alpha-amylase may be replaced with a non-Cysteine residue such as Serine, Alanine, Threonine, Glycine, Valine or Leucine.
[0076]Furthermore, a variant of the invention may--either as the only modification or in combination with any of the above outlined modifications--be modified so that one or more Asp and/or Glu present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID NO: 10 is replaced by an Asn and/or Gln, respectively. Also of interest is the replacement, in the Termamyl-like alpha-amylase, of one or more of the Lys residues present in an amino acid fragment corresponding to the amino acid fragment 185-209 of SEQ ID NO: 10 by an Arg.
[0077]It is to be understood that the present invention encompasses variants incorporating two or more of the above outlined modifications.
[0078]Furthermore, it may be advantageous to introduce mutations in one or more of the following positions (using SEQ ID NO: 8 (Termamyl) for numbering):
M15, V128, A111, H133, W138, T149, M197, N188, A209, A210, H405, T412, in particular the following single, double or triple or multi mutations:M15X, in particular M15T,L;V128X, in particular V128E;H133X, in particular H133Y;N188X, in particular N188S,T,P;M197X, in particular M197T,L;A209X, in particular A209V;
M197T/W138F; M197T/W138Y; M15T/H133Y/N188S;
M15/V128E/H133Y/N188S; E119C/S130C; D124C/R127c; H133Y/T1491;
G475R, H133Y/S187D; H133Y/A209V.
Methods for Preparing Alpha-Amylase Variants of the Invention
[0079]Several methods for introducing mutations into genes are known in the art. After a brief description of cloning of alpha-amylase-encoding DNA sequences, methods for generating mutations at specific sites within the alpha-amylase-encoding sequence will be described.
Cloning a DNA Sequence Encoding an Alpha-Amylase
[0080]The DNA sequence encoding a parent alpha-amylase may be isolated from any cell or microorganism producing the alpha-amylase in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the alpha-amylase to be studied. Then, if the amino acid sequence of the alpha-amylase is known, homologous, labeled oligonucleotide probes may be synthesized and used to identify alpha-amylase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labeled oligonucleotide probe containing sequences homologous to a known alpha-amylase gene could be used as a probe to identify alpha-amylase-encoding clones, using hybridization and washing conditions of lower stringency.
[0081]Yet another method for identifying alpha-amylase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming alpha-amylase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for alpha-amylase, thereby allowing clones expressing the alpha-amylase to be identified.
[0082]Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g., the phosphoroamidite method described by Beaucage and Caruthers, 1981, Tetrahedron Letters 22: 1859-1869, or the method described by Matthes et al., 1984, The EMBO J. 3: 801-805. In the phosphoroamidite method, oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
[0083]Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the fragments corresponding to various parts of the entire DNA sequence), in accordance with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or Saiki et al., 1988, Science 239: 487-491.
Site-Directed Mutagenesis
[0084]Once an alpha-amylase-encoding DNA sequence has been isolated, and desirable sites for mutation identified, mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis. In a specific method, a single-stranded gap of DNA, bridging the alpha-amylase-encoding sequence, is created in a vector carrying the alpha-amylase gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase. A specific example of this method is described in Morinaga et al. (1984). U.S. Pat. No. 4,760,025 discloses the introduction of oligonucleotides encoding multiple mutations by performing minor alterations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the Morinaga method, because a multitude of oligonucleotides, of various lengths, can be introduced.
[0085]Another method for introducing mutations into alpha-amylase-encoding DNA sequences is described in Nelson and Long (1989). It involves the 3-step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.
[0086]Alternative methods for providing variants of the invention include gene shuffling, e.g., as described in WO 95/22625 (from Affymax Technologies N.V.) or in WO 96/00343 (from Novo Nordisk A/S), or other corresponding techniques resulting in a hybrid enzyme comprising the mutation(s), e.g., substitution(s) and/or deletion(s), in question. Examples of parent alpha-amylases, which suitably may be used for providing a hybrid with the desired mutations(s) according to the invention include the KSM-K36 and KSM-K38 alpha-amylases disclosed in EP 1,022,334 (hereby incorporated by reference).
Expression of Alpha-Amylase Variants
[0087]According to the invention, a DNA sequence encoding the variant produced by methods described above, or by any alternative methods known in the art, can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
[0088]The recombinant expression vector carrying the DNA sequence encoding an alpha-amylase variant of the invention may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
[0089]In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA sequence encoding an alpha-amylase variant of the invention, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis alpha-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens alpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
[0090]The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the alpha-amylase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
[0091]The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.
[0092]The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g., as described in WO 91/17243.
[0093]While intracellular expression may be advantageous in some respects, e.g., when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. In general, the Bacillus alpha-amylases mentioned herein comprise a preregion permitting secretion of the expressed protease into the culture medium. If desirable, this preregion may be replaced by a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions.
[0094]The procedures used to ligate the DNA construct of the invention encoding an alpha-amylase variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).
[0095]The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of an alpha-amylase variant of the invention. The cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
[0096]The cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g., a bacterial or a fungal (including yeast) cell.
[0097]Examples of suitable bacteria are gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomyces lividans or Streptomyces murinus, or gram-negative bacteria such as E. coli. The transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells in a manner known per se.
[0098]The yeast organism may favorably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g., Saccharomyces cerevisiae. The filamentous fungus may advantageously belong to a species of Aspergillus, e.g., Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.
[0099]In a yet further aspect, the present invention relates to a method of producing an alpha-amylase variant of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the variant and recovering the variant from the cells and/or culture medium.
[0100]The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the alpha-amylase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).
[0101]The alpha-amylase variant secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
INDUSTRIAL APPLICATIONS
[0102]The alpha-amylase variants of this invention possess valuable properties allowing for a variety of industrial applications. In particular, enzyme variants of the invention are applicable as a component in washing, dishwashing, and hard surface cleaning detergent compositions.
[0103]Variant of the invention with altered properties 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 patent publications Nos. 252730 and 63909, 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.
[0104]Further, variants of the invention are also particularly useful in the production of sweeteners and ethanol (see, e.g., U.S. Pat. No. 5,231,017 hereby incorporated by reference), such as fuel, drinking and industrial ethanol, from starch or whole grains.
[0105]A variant of the invention may also be used for textile desizing (see, e.g., WO 95/21247, U.S. Pat. No. 4,643,736, and EP 119,920, which are hereby incorporated by reference).
Detergent Compositions
[0106]As mentioned above, variants of the invention may suitably be incorporated in detergent compositions. Reference is made, for example, to WO 96/23874 and WO 97/07202 for further details concerning relevant ingredients of detergent compositions (such as laundry or dishwashing detergents), appropriate methods of formulating the variants in such detergent compositions, and for examples of relevant types of detergent compositions.
[0107]Detergent compositions comprising a variant of the invention may additionally comprise one or more other enzymes, such as a protease, a lipase, a peroxidase, another amylolytic enzyme, glucoamylase, maltogenic amylase, CGTase and/or a cellulase, mannanase (such as Mannaway® from Novozymes, Denmark)), pectinase, pectine lyase, cutinase, laccase, and/or another alpha-amylase.
[0108]Alpha-amylase variants of the invention may be incorporated in detergents at conventionally employed concentrations. It is at present contemplated that 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.
Compositions
[0109]The invention also relates to a composition comprising a variant of the invention, and in a preferred embodiment also a B. stearothermophilus alpha-amylase (BSG), in particular a variant thereof.
[0110]In another embodiment the composition comprises beside a variant of the invention a glucoamylase, in particular a glucoamylase originating from Aspergillus niger (e.g., the G1 or G2 A. niger AMG disclosed in Boel et al., 1984, "Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs", EMBO J. 3(5): 1097-1102, or a variant therefore, in particular a variant disclosed in WO 00/04136 or WO 01/04273 or the Talaromyces emersonii AMG disclosed in WO 99/28448.
[0111]A specific combination is LE399 and a variant disclosed in WO 00/04136 or WO 01/04273, in particular a variant with one or more of the following substitutions:
N9A, S56A, V59A, S119P, A246T, N313G, E342T, A393R, S394R, Y402F, E408R, in particular a variant with all mutation.
[0112]In an embodiment the composition of the invention also comprises a pullulanase, in particular a Bacillus pullulanase.
Materials and Methods
Enzymes:
[0113]Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8 and also available from Novozymes.AA560: SEQ ID NO: 12; disclosed in WO 00/60060; deposited on 25 Jan. 1999 at DSMZ and assigned the DSMZ no. 12649. AA560 was deposited by the inventors under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at Deutshe Sammmlung von Microorganismen and Zellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig DE.LB medium (In 1 liter H2O: 10 g bacto-tryptone, 5 g bacto-yeast extract, 10 g NaCl, pH adjusted to 7.0 w. NaOH, autoclaved).TY agar plates (In 1 liter H2O: 16 g bacto-tryptone, 10 g bacto-yeast extract, 5 g NaCl, pH adjusted to 7.0 w. NaOH, and 15 g bacto-agar is added prior to autoclaving).10% Lugol solution (Iodine/Potassium iodine solution; made by 10-fold dil. in H2O of stock: Sigma Cat. no. L 6146).Bacillus subtilis SHA273: see WO 95/10603
Plasmids
[0114]pDN1528 contains the complete gene encoding Termamyl, amyL, the expression of which is directed by its own promoter. Further, the plasmid contains the origin of replication, ori, from plasmid pUB110 and the cat gene from plasmid pC194 conferring resistance towards chloramphenicol. pDN1528 is shown in FIG. 9 of WO 96/23874.
Methods:
Low pH Filter Assay
[0115]Bacillus libraries are plated on a sandwich of cellulose acetate (OE 67, Schleicher & Schuell, Dassel, Germany)--and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates with 10 micrograms/ml chloramphenicol at 37° C. for at least 21 hours. The cellulose acetate layer is located on the TY agar plate.
[0116]Each filter sandwich is specifically marked with a needle after plating, but before incubation in order to be able to localize positive variants on the filter, and the nitrocellulose filter with bound variants is transferred to a container with citrate buffer, pH 4.5 and incubated at 80° C. for 20 minutes (when screening for variants in the wild type backbone) or 85° C. for 60 minutes (when screening for variants in the LE399 backbone). The cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on assay plates containing 1% agarose, 0.2% starch in citrate buffer, pH 6.0. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours at 50° C. After removal of the filters the assay plates are stained with 10% Lugol solution. Starch degrading variants are detected as white spots on dark blue background and then identified on the storage plates. Positive variants are re-screened twice under the same conditions as the first screen.
Secondary Screening
[0117]Positive transformants after rescreening are picked from the storage plate and tested in a secondary plate assay. Positive transformants are grown for 22 hours at 37° C. in 5 ml LB+chloramphenicol. The Bacillus culture of each positive transformant and as a control a clone expressing the corresponding backbone are incubated in citrate buffer, pH 4.5 at 90° C. and samples are taken at 0, 10, 20, 30, 40, 60 and 80 minutes. A 3 microliter sample is spotted on an assay plate. The assay plate is stained with 10% Lugol solution. Improved variants are seen as variants with higher residual activity (detected as halos on the assay plate) than the backbone. The improved variants are determined by nucleotide sequencing.
Stability Assay of Unpurified Variants:
[0118]Bacillus cultures expressing the variants to be analyzed are grown for 21 hours at 37° C. in 10 ml LB+chloramphenicol. 800 microliter culture is mixed with 200 microliters citrate buffer, pH 4.5. A number of 70 microliter aliquots corresponding to the number of sample time points are made in PCR tubes and incubated at 70° C. (for variants in the wt backbone) or 90° C. (for variants in LE399) for various time points (typically 5, 10, 15, 20, 25 and 30 minutes) in a PCR machine. The 0 min sample is not incubated at high temperature. Activity in the sample is measured by transferring 20 microliters to 200 microliters of the alpha-amylase PNP-G7 substrate MPR3 ((Boehringer Mannheim Cat. no. 1660730) as described below under "Assays for Alpha-Amylase Activity". Results are plotted as percentage activity (relative to the 0 time point) versus time, or stated as percentage residual activity after incubation for a certain period of time.
Fermentation and Purification of Alpha-Amylase Variants
[0119]A B. subtilis strain harboring the relevant expression plasmid is streaked on an LB-agar plate with 10 micrograms/ml kanamycin from -80° C. stock, and grown overnight at 37° C.
[0120]The colonies are transferred to 100 ml PS-1 media supplemented with 10 micrograms/ml chloamphinicol in a 500 ml shaking flask.
TABLE-US-00002 Composition of PS-1 medium: Pearl sugar 100 g/l Soy Bean Meal 40 g/l Na2HPO4, 12H2O 10 g/l PluronicTM PE 6100 0.1 g/l CaCO3 5 g/l The culture is shaken at 37° C. at 270 rpm for 5 days.
[0121]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 a UF-filter (10000 cut off membrane) and the buffer is changed to 20 mM Acetate pH 5.5. The UF-filtrate is applied on a S-sepharose F.F. and elution is carried out by step elution with 0.2 M NaCl in the same buffer. The eluate is dialysed 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 that contain the activity (measured by the Phadebas assay) are pooled, pH was adjusted to pH 7.5 and remaining color was removed by a treatment with 0.5% W/vol. active coal in 5 minutes.
Stability Determination of Purified Variants
[0122]All stability trials of purified variants are made using the same set up. The method is as follows:
[0123]The enzyme is incubated under the relevant conditions (1-4). Samples are taken at various time points, e.g., after 0, 5, 10, 15 and 30 minutes and diluted 25 times (same dilution for all taken samples) in assay buffer (0.1 M 50 mM Britton buffer pH 7.3) and the activity is measured using the Phadebas assay (Pharmacia) under standard conditions pH 7.3, 37° C.
[0124]The activity measured before incubation (0 minutes) is used as reference (100%). The decline in percent is calculated as a function of the incubation time. The table shows the residual activity after, e.g., 30 minutes of incubation.
Specific Activity Determination
[0125]The specific activity is determined using the Phadebas assay (Pharmacia) as activity/mg enzyme. The manufacturer's instructions are followed (see also below under "Assay for α-amylase activity").
Assays for Alpha-Amylase Activity
1. Phadebas Assay
[0126]Alpha-amylase activity is determined by a method employing Phadebas® tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-colored starch polymer, which has been mixed with bovine serum albumin and a buffer substance and tabletted.
[0127]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 CaCl2, pH adjusted to the value of interest with NaOH). The test is performed in a water bath at the temperature of interest. The alpha-amylase to be tested is diluted in x ml of 50 mM Britton-Robinson buffer. 1 ml of this alpha-amylase solution is added to the 5 ml 50 mM Britton-Robinson buffer. The starch 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.
[0128]It is important that the measured 620 nm absorbance after 10 or 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 must therefore be adjusted to fit this criterion. Under a specified set of conditions (temp., 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. The color intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure alpha-amylase protein) of the alpha-amylase in question under the given set of conditions.
2. Alternative Method
[0129]Alpha-amylase activity is determined by a method employing the PNP-G7 substrate. PNP-G7 which is a 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 digest the substrate to liberate a free PNP molecule which has a yellow colour and thus can be measured by visible spectophometry at λ=405 nm (400-420 nm). Kits containing PNP-G7 substrate and alpha-Glucosidase is manufactured by Boehringer-Mannheim (cat. No. 1054635).
[0130]To prepare the reagent solution 10 ml of substrate/buffer solution is added to 50 ml enzyme/buffer solution as recommended by the manufacturer. The assay is performed by transferring 20 microliter sample to a 96 well microtiter plate and incubating at 25° C. 200 microliters reagent solution pre-equilibrated to 25° C. is added. The solution is mixed and pre-incubated 1 minute and absorption is measured every 30 sec. over 4 minutes at OD 405 nm in an ELISA reader.
[0131]The slope of the time dependent absorption-curve is directly proportional to the activity of the alpha-amylase in question under the given set of conditions.
EXAMPLES
Example 1
[0132]Construction, by error-prone PCR mutagenesis, of Bacillus licheniformis alpha-amylase variants having an improved stability at low pH, high temperature and low calcium ion concentration compared to the parent enzyme.
Error-Prone PCR Mutagenesis and Library Construction
[0133]To improve the stability at low pH and low calcium concentration of the parent Bacillus licheniformis alpha-amylase, error-prone PCR mutagenesis was performed. The plasmid pDN1528 encoding the wild-type Bacillus licheniformis alpha-amylase gene was utilized as template to amplify this gene with primers: 22149: 5'-CGA TTG CTG ACG CTG TTA TTT GCG-3' (SEQ ID NO: 14) and 24814: 5'-GAT CAC CCG CGA TAC CGT C-3' (SEQ ID NO: 15) under PCR conditions where increased error rates leads to introduction of random point mutations. The PCR conditions utilized were: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, 0.3 mM MnCl2, 0.1 mM dGTP/dATP, 0.5 mM dTTP/dCTP, and 2.5 units Taq polymerase per 100 microliter reaction.
[0134]The resultant PCR fragment was purified on gel and used in a PCR-based multimerization step with a gel purified vector fragment created by PCR amplification of pDN1528 with primers #24: 5'-GAA TGT ATG TCG GCC GGC AAA ACG CCG GTG A-3' (SEQ ID NO: 16) and #27: 5''-GCC GCC GCT GCT GCA GAA TGA GGC AGC AAG-3' (SEQ ID NO: 17) forming an overlap to the insert fragment. The multimerization reaction was subsequently introduced into B. subtilis (Shafikhani et al., 1997, Biotechniques 23: 304-310).
Screening
[0135]The error-prone library described above was screened in the low pH filter assay (see "Materials & Methods"). Clones testing positive upon rescreening was submitted to secondary screening for stability in the liquid assay described in Materials and Methods.
Results:
Increased Stability at pH 4.5, 5 ppm Calcium Incubated at 90° C.
TABLE-US-00003 [0136] Name wt LE488 LE489 7.19.1 8.9.1 Mutations -- D207V K170Q E132A D207E D207V D207V E250G N280S H406L L427I Stability1) -- + + + + 1)A "+" indicates significant increase in stability relative to wild type.
Increased Stability at pH 4.5, 5 ppm Calcium Incubated at 90° C.
TABLE-US-00004 [0137]Name wt LE491 LE492 LE493 LE494 19.3.1 Mutations -- D60N T49I T49I Q374R N190F D207V E132V K176R E385V A209V L318M V440A D207V Q393R Q264S Y402F Stability1) -- + + + + + 1)A "+" indicates significant increase in stability relative to wt.
Increased Stability at pH 4.5, 5 ppm Calcium Incubated at 90° C.
TABLE-US-00005 [0138] Name wt E132-1 D207-7 D207-6 E250-8 Mutations -- E132P D207L D207G E250F Stability1) -- + + + + 1)A "+" indicates significant increase in stability relative to wt.
Example 2
[0139]Transfer, by site-directed mutagenesis, of a selection of mutations from Example 1 to a new (non-wild type) backbone to improve stability at low pH and low calcium ion concentration compared to the parent enzyme.
Site-Directed Mutagenesis
[0140]Mutations from LE493 (K176R+D207V+Y402F) were transferred to LE399 yielding LE495. This was performed by the overlap PCR method (Kirchhoff and Desrosiers, 1993, PCR Methods and Applications, 2: 301-304). 2 overlapping PCR fragments were generated by amplification of the LE399 template with the primers: Fragment A: #312 Mut176 5'-CCC GAA AGC TGA ACC GCA TCT ATA GGT TTC AAG GGA AGA CTT GGG ATT-3' (SEQ ID NO: 18) (mutated codon indicated in bold) and #290 D207overlap 5'-AGG ATG GTC ATA ATC AAA GTC GG-3'(SEQ ID NO: 19); Fragment B: #313 Mut207 5'-CCG ACT TTG ATT ATG ACC ATC CTG TTG TCG TAG CAG AGA TTA AGA GAT GGG G-3' (SEQ ID NO: 20) and #314 Mut402 5'-CGA CAA TGT CAT GGT GGT CGA AAA AAT CAT GCT GTG CTC CGT ACG-3' (SEQ ID NO: 21). Fragments A and B were mixed in equimolar ratios and subsequently the full-length fragment was amplified with the external primers: #312 Mut176 and #314 Mut402. This fragment was used in a multimerization reaction with the vector PCR fragment created with the primers #296 Y402multi 5'-TTT CGA CCA CCA TGA CAT TGT CG-3' (SEQ ID NO: 22) and #305 399Multi176 5'-TAT AGA TGC GGT TCA GCT TTC GGG-3' (SEQ ID NO: 23) on template LE399 as described above. The multimerization reaction was subsequently transformed into B. subtilis. Clones were screened for stability in the assay mentioned above. The presence of the mutations from LE493 in several clones with increased stability was confirmed by sequencing.
[0141]LE 497 was obtained in a similar manner by amplifying the LE399 encoding template with primers #312 Mut176 and #314 Mut402 and using the resulting PCR fragment in a multimerization reaction with a vector fragment obtained by PCR amplification of the LE399 template with the primers #296 Y402multi and #305 399Multi176.
Results:
Stabilization of LE399 Variant at pH 4.5, 5 ppm Calcium Incubated at 90° C.
TABLE-US-00006 [0142] Name LE399 LE495 LE497 Mutations -- K176R K176R (backbone) D207V Y402F Y402F Stability1) -- + + 1)A "+" indicates significant increase in stability relative to backbone.
Sequence CWU
1
3011455DNABacillus speciesCDS(1)..(1455) 1cat cat aat gga aca aat ggt act
atg atg caa tat ttc gaa tgg tat 48His His Asn Gly Thr Asn Gly Thr
Met Met Gln Tyr Phe Glu Trp Tyr1 5 10
15ttg cca aat gac ggg aat cat tgg aac agg ttg agg gat gac
gca gct 96Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp
Ala Ala 20 25 30aac tta aag
agt aaa ggg ata aca gct gta tgg atc cca cct gca tgg 144Asn Leu Lys
Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35
40 45aag ggg act tcc cag aat gat gta ggt tat gga
gcc tat gat tta tat 192Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly
Ala Tyr Asp Leu Tyr 50 55 60gat ctt
gga gag ttt aac cag aag ggg acg gtt cgt aca aaa tat gga 240Asp Leu
Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65
70 75 80aca cgc aac cag cta cag gct
gcg gtg acc tct tta aaa aat aac ggc 288Thr Arg Asn Gln Leu Gln Ala
Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90
95att cag gta tat ggt gat gtc gtc atg aat cat aaa ggt
gga gca gat 336Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly
Gly Ala Asp 100 105 110ggt acg
gaa att gta aat gcg gta gaa gtg aat cgg agc aac cga aac 384Gly Thr
Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115
120 125cag gaa acc tca gga gag tat gca ata gaa
gcg tgg aca aag ttt gat 432Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu
Ala Trp Thr Lys Phe Asp 130 135 140ttt
cct gga aga gga aat aac cat tcc agc ttt aag tgg cgc tgg tat 480Phe
Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr145
150 155 160cat ttt gat ggg aca gat
tgg gat cag tca cgc cag ctt caa aac aaa 528His Phe Asp Gly Thr Asp
Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys 165
170 175ata tat aaa ttc agg gga aca ggc aag gcc tgg gac
tgg gaa gtc gat 576Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp
Trp Glu Val Asp 180 185 190aca
gag aat ggc aac tat gac tat ctt atg tat gca gac gtg gat atg 624Thr
Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195
200 205gat cac cca gaa gta ata cat gaa ctt
aga aac tgg gga gtg tgg tat 672Asp His Pro Glu Val Ile His Glu Leu
Arg Asn Trp Gly Val Trp Tyr 210 215
220acg aat aca ctg aac ctt gat gga ttt aga ata gat gca gtg aaa cat
720Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225
230 235 240ata aaa tat agc
ttt acg aga gat tgg ctt aca cat gtg cgt aac acc 768Ile Lys Tyr Ser
Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr 245
250 255aca ggt aaa cca atg ttt gca gtg gct gag
ttt tgg aaa aat gac ctt 816Thr Gly Lys Pro Met Phe Ala Val Ala Glu
Phe Trp Lys Asn Asp Leu 260 265
270ggt gca att gaa aac tat ttg aat aaa aca agt tgg aat cac tcg gtg
864Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val
275 280 285ttt gat gtt cct ctc cac tat
aat ttg tac aat gca tct aat agc ggt 912Phe Asp Val Pro Leu His Tyr
Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295
300ggt tat tat gat atg aga aat att tta aat ggt tct gtg gtg caa aaa
960Gly Tyr Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys305
310 315 320cat cca aca cat
gcc gtt act ttt gtt gat aac cat gat tct cag ccc 1008His Pro Thr His
Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325
330 335ggg gaa gca ttg gaa tcc ttt gtt caa caa
tgg ttt aaa cca ctt gca 1056Gly Glu Ala Leu Glu Ser Phe Val Gln Gln
Trp Phe Lys Pro Leu Ala 340 345
350tat gca ttg gtt ctg aca agg gaa caa ggt tat cct tcc gta ttt tat
1104Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365ggg gat tac tac ggt atc cca
acc cat ggt gtt ccg gct atg aaa tct 1152Gly Asp Tyr Tyr Gly Ile Pro
Thr His Gly Val Pro Ala Met Lys Ser 370 375
380aaa ata gac cct ctt ctg cag gca cgt caa act ttt gcc tat ggt acg
1200Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr385
390 395 400cag cat gat tac
ttt gat cat cat gat att atc ggt tgg aca aga gag 1248Gln His Asp Tyr
Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu 405
410 415gga aat agc tcc cat cca aat tca ggc ctt
gcc acc att atg tca gat 1296Gly Asn Ser Ser His Pro Asn Ser Gly Leu
Ala Thr Ile Met Ser Asp 420 425
430ggt cca ggt ggt aac aaa tgg atg tat gtg ggg aaa aat aaa gcg gga
1344Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly
435 440 445caa gtt tgg aga gat att acc
gga aat agg aca ggc acc gtc aca att 1392Gln Val Trp Arg Asp Ile Thr
Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455
460aat gca gac gga tgg ggt aat ttc tct gtt aat gga ggg tcc gtt tcg
1440Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser465
470 475 480gtt tgg gtg aag
caa 1455Val Trp Val Lys
Gln 4852485PRTBacillus species 2His 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 Lys Gln
48531455DNABacillus speciesCDS(1)..(1455) 3cat cat aat ggg aca aat ggg
acg atg atg caa tac ttt gaa tgg cac 48His His Asn Gly Thr Asn Gly
Thr Met Met Gln Tyr Phe Glu Trp His1 5 10
15ttg cct aat gat ggg aat cac tgg aat aga tta aga gat
gat gct agt 96Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp
Asp Ala Ser 20 25 30aat cta
aga aat aga ggt ata acc gct att tgg att ccg cct gcc tgg 144Asn Leu
Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp 35
40 45aaa ggg act tcg caa aat gat gtg ggg tat
gga gcc tat gat ctt tat 192Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr
Gly Ala Tyr Asp Leu Tyr 50 55 60gat
tta ggg gaa ttt aat caa aag ggg acg gtt cgt act aag tat ggg 240Asp
Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65
70 75 80aca cgt agt caa ttg gag
tct gcc atc cat gct tta aag aat aat ggc 288Thr Arg Ser Gln Leu Glu
Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85
90 95gtt caa gtt tat ggg gat gta gtg atg aac cat aaa
gga gga gct gat 336Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys
Gly Gly Ala Asp 100 105 110gct
aca gaa aac gtt ctt gct gtc gag gtg aat cca aat aac cgg aat 384Ala
Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115
120 125caa gaa ata tct ggg gac tac aca att
gag gct tgg act aag ttt gat 432Gln Glu Ile Ser Gly Asp Tyr Thr Ile
Glu Ala Trp Thr Lys Phe Asp 130 135
140ttt cca ggg agg ggt aat aca tac tca gac ttt aaa tgg cgt tgg tat
480Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr145
150 155 160cat ttc gat ggt
gta gat tgg gat caa tca cga caa ttc caa aat cgt 528His Phe Asp Gly
Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165
170 175atc tac aaa ttc cga ggt gat ggt aag gca
tgg gat tgg gaa gta gat 576Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala
Trp Asp Trp Glu Val Asp 180 185
190tcg gaa aat gga aat tat gat tat tta atg tat gca gat gta gat atg
624Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met
195 200 205gat cat ccg gag gta gta aat
gag ctt aga aga tgg gga gaa tgg tat 672Asp His Pro Glu Val Val Asn
Glu Leu Arg Arg Trp Gly Glu Trp Tyr 210 215
220aca aat aca tta aat ctt gat gga ttt agg atc gat gcg gtg aag cat
720Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225
230 235 240att aaa tat agc
ttt aca cgt gat tgg ttg acc cat gta aga aac gca 768Ile Lys Tyr Ser
Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala 245
250 255acg gga aaa gaa atg ttt gct gtt gct gaa
ttt tgg aaa aat gat tta 816Thr Gly Lys Glu Met Phe Ala Val Ala Glu
Phe Trp Lys Asn Asp Leu 260 265
270ggt gcc ttg gag aac tat tta aat aaa aca aac tgg aat cat tct gtc
864Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val
275 280 285ttt gat gtc ccc ctt cat tat
aat ctt tat aac gcg tca aat agt gga 912Phe Asp Val Pro Leu His Tyr
Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295
300ggc aac tat gac atg gca aaa ctt ctt aat gga acg gtt gtt caa aag
960Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys305
310 315 320cat cca atg cat
gcc gta act ttt gtg gat aat cac gat tct caa cct 1008His Pro Met His
Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325
330 335ggg gaa tca tta gaa tca ttt gta caa gaa
tgg ttt aag cca ctt gct 1056Gly Glu Ser Leu Glu Ser Phe Val Gln Glu
Trp Phe Lys Pro Leu Ala 340 345
350tat gcg ctt att tta aca aga gaa caa ggc tat ccc tct gtc ttc tat
1104Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365ggt gac tac tat gga att cca
aca cat agt gtc cca gca atg aaa gcc 1152Gly Asp Tyr Tyr Gly Ile Pro
Thr His Ser Val Pro Ala Met Lys Ala 370 375
380aag att gat cca atc tta gag gcg cgt caa aat ttt gca tat gga aca
1200Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr385
390 395 400caa cat gat tat
ttt gac cat cat aat ata atc gga tgg aca cgt gaa 1248Gln His Asp Tyr
Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405
410 415gga aat acc acg cat ccc aat tca gga ctt
gcg act atc atg tcg gat 1296Gly Asn Thr Thr His Pro Asn Ser Gly Leu
Ala Thr Ile Met Ser Asp 420 425
430ggg cca ggg gga gag aaa tgg atg tac gta ggg caa aat aaa gca ggt
1344Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly
435 440 445caa gtt tgg cat gac ata act
gga aat aaa cca gga aca gtt acg atc 1392Gln Val Trp His Asp Ile Thr
Gly Asn Lys Pro Gly Thr Val Thr Ile 450 455
460aat gca gat gga tgg gct aat ttt tca gta aat gga gga tct gtt tcc
1440Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser465
470 475 480att tgg gtg aaa
cga 1455Ile Trp Val Lys
Arg 4854485PRTBacillus species 4His 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
48551548DNABacillus stearothermophilusCDS(1)..(1548) 5gcc gca ccg ttt aac
ggc acc atg atg cag tat ttt gaa tgg tac ttg 48Ala Ala Pro Phe Asn
Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu1 5
10 15ccg gat gat ggc acg tta tgg acc aaa gtg gcc
aat gaa gcc aac aac 96Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala
Asn Glu Ala Asn Asn 20 25
30tta tcc agc ctt ggc atc acc gct ctt tgg ctg ccg ccc gct tac aaa
144Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys
35 40 45gga aca agc cgc agc gac gta ggg
tac gga gta tac gac ttg tat gac 192Gly Thr Ser Arg Ser Asp Val Gly
Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55
60ctc ggc gaa ttc aat caa aaa ggg acc gtc cgc aca aaa tac gga aca
240Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr65
70 75 80aaa gct caa tat ctt
caa gcc att caa gcc gcc cac gcc gct gga atg 288Lys Ala Gln Tyr Leu
Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met 85
90 95caa gtg tac gcc gat gtc gtg ttc gac cat aaa
ggc ggc gct gac ggc 336Gln Val Tyr Ala Asp Val Val Phe Asp His Lys
Gly Gly Ala Asp Gly 100 105
110acg gaa tgg gtg gac gcc gtc gaa gtc aat ccg tcc gac cgc aac caa
384Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln
115 120 125gaa atc tcg ggc acc tat caa
atc caa gca tgg acg aaa ttt gat ttt 432Glu Ile Ser Gly Thr Tyr Gln
Ile Gln Ala Trp Thr Lys Phe Asp Phe 130 135
140ccc ggg cgg ggc aac acc tac tcc agc ttt aag tgg cgc tgg tac cat
480Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His145
150 155 160ttt gac ggc gtt
gat tgg gac gaa agc cga aaa ttg agc cgc att tac 528Phe Asp Gly Val
Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165
170 175aaa ttc cgc ggc atc ggc aaa gcg tgg gat
tgg gaa gta gac acg gaa 576Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp
Trp Glu Val Asp Thr Glu 180 185
190aac gga aac tat gac tac tta atg tat gcc gac ctt gat atg gat cat
624Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His
195 200 205ccc gaa gtc gtg acc gag ctg
aaa aac tgg ggg aaa tgg tat gtc aac 672Pro Glu Val Val Thr Glu Leu
Lys Asn Trp Gly Lys Trp Tyr Val Asn 210 215
220aca acg aac att gat ggg ttc cgg ctt gat gcc gtc aag cat att aag
720Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys225
230 235 240ttc agt ttt ttt
cct gat tgg ttg tcg tat gtg cgt tct cag act ggc 768Phe Ser Phe Phe
Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly 245
250 255aag ccg cta ttt acc gtc ggg gaa tat tgg
agc tat gac atc aac aag 816Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp
Ser Tyr Asp Ile Asn Lys 260 265
270ttg cac aat tac att acg aaa aca gac gga acg atg tct ttg ttt gat
864Leu His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp
275 280 285gcc ccg tta cac aac aaa ttt
tat acc gct tcc aaa tca ggg ggc gca 912Ala Pro Leu His Asn Lys Phe
Tyr Thr Ala Ser Lys Ser Gly Gly Ala 290 295
300ttt gat atg cgc acg tta atg acc aat act ctc atg aaa gat caa ccg
960Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro305
310 315 320aca ttg gcc gtc
acc ttc gtt gat aat cat gac acc gaa ccc ggc caa 1008Thr Leu Ala Val
Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln 325
330 335gcg ctg cag tca tgg gtc gac cca tgg ttc
aaa ccg ttg gct tac gcc 1056Ala Leu Gln Ser Trp Val Asp Pro Trp Phe
Lys Pro Leu Ala Tyr Ala 340 345
350ttt att cta act cgg cag gaa gga tac ccg tgc gtc ttt tat ggt gac
1104Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp
355 360 365tat tat ggc att cca caa tat
aac att cct tcg ctg aaa agc aaa atc 1152Tyr Tyr Gly Ile Pro Gln Tyr
Asn Ile Pro Ser Leu Lys Ser Lys Ile 370 375
380gat ccg ctc ctc atc gcg cgc agg gat tat gct tac gga acg caa cat
1200Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His385
390 395 400gat tat ctt gat
cac tcc gac atc atc ggg tgg aca agg gaa ggg ggc 1248Asp Tyr Leu Asp
His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly 405
410 415act gaa aaa cca gga tcc gga ctg gcc gca
ctg atc acc gat ggg ccg 1296Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala
Leu Ile Thr Asp Gly Pro 420 425
430gga gga agc aaa tgg atg tac gtt ggc aaa caa cac gct gga aaa gtg
1344Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val
435 440 445ttc tat gac ctt acc ggc aac
cgg agt gac acc gtc acc atc aac agt 1392Phe Tyr Asp Leu Thr Gly Asn
Arg Ser Asp Thr Val Thr Ile Asn Ser 450 455
460gat gga tgg ggg gaa ttc aaa gtc aat ggc ggt tcg gtt tcg gtt tgg
1440Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp465
470 475 480gtt cct aga aaa
acg acc gtt tct acc atc gct cgg ccg atc aca acc 1488Val Pro Arg Lys
Thr Thr Val Ser Thr Ile Ala Arg Pro Ile Thr Thr 485
490 495cga ccg tgg act ggt gaa ttc gtc cgt tgg
acc gaa cca cgg ttg gtg 1536Arg Pro Trp Thr Gly Glu Phe Val Arg Trp
Thr Glu Pro Arg Leu Val 500 505
510gca tgg cct tga
1548Ala Trp Pro 5156515PRTBacillus stearothermophilus 6Ala 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
51571920DNABacillus licheniformisCDS(421)..(1872) 7cggaagattg gaagtacaaa
aataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60gagacggaaa aatcgtctta
atgcacgata tttatgcaac gttcgcagat gctgctgaag 120agattattaa aaagctgaaa
gcaaaaggct atcaattggt aactgtatct cagcttgaag 180aagtgaagaa gcagagaggc
tattgaataa atgagtagaa gcgccatatc ggcgcttttc 240ttttggaaga aaatataggg
aaaatggtac ttgttaaaaa ttcggaatat ttatacaaca 300tcatatgttt cacattgaaa
ggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360cgattgctga cgctgttatt
tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420gca aat ctt aat ggg acg
ctg atg cag tat ttt gaa tgg tac atg ccc 468Ala Asn Leu Asn Gly Thr
Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro1 5
10 15aat gac ggc caa cat tgg agg cgt ttg caa aac gac
tcg gca tat ttg 516Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp
Ser Ala Tyr Leu 20 25 30gct
gaa cac ggt att act gcc gtc tgg att ccc ccg gca tat aag gga 564Ala
Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly 35
40 45acg agc caa gcg gat gtg ggc tac ggt
gct tac gac ctt tat gat tta 612Thr Ser Gln Ala Asp Val Gly Tyr Gly
Ala Tyr Asp Leu Tyr Asp Leu 50 55
60ggg gag ttt cat caa aaa ggg acg gtt cgg aca aag tac ggc aca aaa
660Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65
70 75 80gga gag ctg caa tct
gcg atc aaa agt ctt cat tcc cgc gac att aac 708Gly Glu Leu Gln Ser
Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn 85
90 95gtt tac ggg gat gtg gtc atc aac cac aaa ggc
ggc gct gat gcg acc 756Val Tyr Gly Asp Val Val Ile Asn His Lys Gly
Gly Ala Asp Ala Thr 100 105
110gaa gat gta acc gcg gtt gaa gtc gat ccc gct gac cgc aac cgc gta
804Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
115 120 125att tca gga gaa cac cta att
aaa gcc tgg aca cat ttt cat ttt ccg 852Ile Ser Gly Glu His Leu Ile
Lys Ala Trp Thr His Phe His Phe Pro 130 135
140ggg cgc ggc agc aca tac agc gat ttt aaa tgg cat tgg tac cat ttt
900Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe145
150 155 160gac gga acc gat
tgg gac gag tcc cga aag ctg aac cgc atc tat aag 948Asp Gly Thr Asp
Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165
170 175ttt caa gga aag gct tgg gat tgg gaa gtt
tcc aat gaa aac ggc aac 996Phe Gln Gly Lys Ala Trp Asp Trp Glu Val
Ser Asn Glu Asn Gly Asn 180 185
190tat gat tat ttg atg tat gcc gac atc gat tat gac cat cct gat gtc
1044Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val
195 200 205gca gca gaa att aag aga tgg
ggc act tgg tat gcc aat gaa ctg caa 1092Ala Ala Glu Ile Lys Arg Trp
Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215
220ttg gac ggt ttc cgt ctt gat gct gtc aaa cac att aaa ttt tct ttt
1140Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe225
230 235 240ttg cgg gat tgg
gtt aat cat gtc agg gaa aaa acg ggg aag gaa atg 1188Leu Arg Asp Trp
Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245
250 255ttt acg gta gct gaa tat tgg cag aat gac
ttg ggc gcg ctg gaa aac 1236Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp
Leu Gly Ala Leu Glu Asn 260 265
270tat ttg aac aaa aca aat ttt aat cat tca gtg ttt gac gtg ccg ctt
1284Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu
275 280 285cat tat cag ttc cat gct gca
tcg aca cag gga ggc ggc tat gat atg 1332His Tyr Gln Phe His Ala Ala
Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295
300agg aaa ttg ctg aac ggt acg gtc gtt tcc aag cat ccg ttg aaa tcg
1380Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser305
310 315 320gtt aca ttt gtc
gat aac cat gat aca cag ccg ggg caa tcg ctt gag 1428Val Thr Phe Val
Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325
330 335tcg act gtc caa aca tgg ttt aag ccg ctt
gct tac gct ttt att ctc 1476Ser Thr Val Gln Thr Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345
350aca agg gaa tct gga tac cct cag gtt ttc tac ggg gat atg tac ggg
1524Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365acg aaa gga gac tcc cag cgc
gaa att cct gcc ttg aaa cac aaa att 1572Thr Lys Gly Asp Ser Gln Arg
Glu Ile Pro Ala Leu Lys His Lys Ile 370 375
380gaa ccg atc tta aaa gcg aga aaa cag tat gcg tac gga gca cag cat
1620Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His385
390 395 400gat tat ttc gac
cac cat gac att gtc ggc tgg aca agg gaa ggc gac 1668Asp Tyr Phe Asp
His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405
410 415agc tcg gtt gca aat tca ggt ttg gcg gca
tta ata aca gac gga ccc 1716Ser Ser Val Ala Asn Ser Gly Leu Ala Ala
Leu Ile Thr Asp Gly Pro 420 425
430ggt ggg gca aag cga atg tat gtc ggc cgg caa aac gcc ggt gag aca
1764Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr
435 440 445tgg cat gac att acc gga aac
cgt tcg gag ccg gtt gtc atc aat tcg 1812Trp His Asp Ile Thr Gly Asn
Arg Ser Glu Pro Val Val Ile Asn Ser 450 455
460gaa ggc tgg gga gag ttt cac gta aac ggc ggg tcg gtt tca att tat
1860Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr465
470 475 480gtt caa aga tag
aagagcagag aggacggatt tcctgaagga aatccgtttt 1912Val Gln
Argtttatttt
19208483PRTBacillus licheniformis 8Ala 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 Arg92084DNABacillus amyloliquefaciensCDS(343)..(1794)
9gccccgcaca tacgaaaaga ctggctgaaa acattgagcc tttgatgact gatgatttgg
60ctgaagaagt ggatcgattg tttgagaaaa gaagaagacc ataaaaatac cttgtctgtc
120atcagacagg gtatttttta tgctgtccag actgtccgct gtgtaaaaat aaggaataaa
180ggggggttgt tattatttta ctgatatgta aaatataatt tgtataagaa aatgagaggg
240agaggaaaca tgattcaaaa acgaaagcgg acagtttcgt tcagacttgt gcttatgtgc
300acgctgttat ttgtcagttt gccgattaca aaaacatcag cc gta aat ggc acg
354 Val Asn Gly Thr
1ctg atg cag tat ttt gaa tgg tat
acg ccg aac gac ggc cag cat tgg 402Leu Met Gln Tyr Phe Glu Trp Tyr
Thr Pro Asn Asp Gly Gln His Trp5 10 15
20aaa cga ttg cag aat gat gcg gaa cat tta tcg gat atc
gga atc act 450Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp Ile
Gly Ile Thr 25 30 35gcc
gtc tgg att cct ccc gca tac aaa gga ttg agc caa tcc gat aac 498Ala
Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser Gln Ser Asp Asn 40
45 50gga tac gga cct tat gat ttg tat
gat tta gga gaa ttc cag caa aaa 546Gly Tyr Gly Pro Tyr Asp Leu Tyr
Asp Leu Gly Glu Phe Gln Gln Lys 55 60
65ggg acg gtc aga acg aaa tac ggc aca aaa tca gag ctt caa gat gcg
594Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu Leu Gln Asp Ala
70 75 80atc ggc tca ctg cat tcc cgg aac
gtc caa gta tac gga gat gtg gtt 642Ile Gly Ser Leu His Ser Arg Asn
Val Gln Val Tyr Gly Asp Val Val85 90 95
100ttg aat cat aag gct ggt gct gat gca aca gaa gat gta
act gcc gtc 690Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp Val
Thr Ala Val 105 110 115gaa
gtc aat ccg gcc aat aga aat cag gaa act tcg gag gaa tat caa 738Glu
Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser Glu Glu Tyr Gln
120 125 130atc aaa gcg tgg acg gat ttt
cgt ttt ccg ggc cgt gga aac acg tac 786Ile Lys Ala Trp Thr Asp Phe
Arg Phe Pro Gly Arg Gly Asn Thr Tyr 135 140
145agt gat ttt aaa tgg cat tgg tat cat ttc gac gga gcg gac tgg
gat 834Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Ala Asp Trp
Asp 150 155 160gaa tcc cgg aag atc agc
cgc atc ttt aag ttt cgt ggg gaa gga aaa 882Glu Ser Arg Lys Ile Ser
Arg Ile Phe Lys Phe Arg Gly Glu Gly Lys165 170
175 180gcg tgg gat tgg gaa gta tca agt gaa aac ggc
aac tat gac tat tta 930Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly
Asn Tyr Asp Tyr Leu 185 190
195atg tat gct gat gtt gac tac gac cac cct gat gtc gtg gca gag aca
978Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Thr
200 205 210aaa aaa tgg ggt atc tgg
tat gcg aat gaa ctg tca tta gac ggc ttc 1026Lys Lys Trp Gly Ile Trp
Tyr Ala Asn Glu Leu Ser Leu Asp Gly Phe 215 220
225cgt att gat gcc gcc aaa cat att aaa ttt tca ttt ctg cgt
gat tgg 1074Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe Leu Arg
Asp Trp 230 235 240gtt cag gcg gtc aga
cag gcg acg gga aaa gaa atg ttt acg gtt gcg 1122Val Gln Ala Val Arg
Gln Ala Thr Gly Lys Glu Met Phe Thr Val Ala245 250
255 260gag tat tgg cag aat aat gcc ggg aaa ctc
gaa aac tac ttg aat aaa 1170Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu
Glu Asn Tyr Leu Asn Lys 265 270
275aca agc ttt aat caa tcc gtg ttt gat gtt ccg ctt cat ttc aat tta
1218Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu His Phe Asn Leu
280 285 290cag gcg gct tcc tca caa
gga ggc gga tat gat atg agg cgt ttg ctg 1266Gln Ala Ala Ser Ser Gln
Gly Gly Gly Tyr Asp Met Arg Arg Leu Leu 295 300
305gac ggt acc gtt gtg tcc agg cat ccg gaa aag gcg gtt aca
ttt gtt 1314Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala Val Thr
Phe Val 310 315 320gaa aat cat gac aca
cag ccg gga cag tca ttg gaa tcg aca gtc caa 1362Glu Asn His Asp Thr
Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln325 330
335 340act tgg ttt aaa ccg ctt gca tac gcc ttt
att ttg aca aga gaa tcc 1410Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe
Ile Leu Thr Arg Glu Ser 345 350
355ggt tat cct cag gtg ttc tat ggg gat atg tac ggg aca aaa ggg aca
1458Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Thr
360 365 370tcg cca aag gaa att ccc
tca ctg aaa gat aat ata gag ccg att tta 1506Ser Pro Lys Glu Ile Pro
Ser Leu Lys Asp Asn Ile Glu Pro Ile Leu 375 380
385aaa gcg cgt aag gag tac gca tac ggg ccc cag cac gat tat
att gac 1554Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His Asp Tyr
Ile Asp 390 395 400cac ccg gat gtg atc
gga tgg acg agg gaa ggt gac agc tcc gcc gcc 1602His Pro Asp Val Ile
Gly Trp Thr Arg Glu Gly Asp Ser Ser Ala Ala405 410
415 420aaa tca ggt ttg gcc gct tta atc acg gac
gga ccc ggc gga tca aag 1650Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp
Gly Pro Gly Gly Ser Lys 425 430
435cgg atg tat gcc ggc ctg aaa aat gcc ggc gag aca tgg tat gac ata
1698Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr Trp Tyr Asp Ile
440 445 450acg ggc aac cgt tca gat
act gta aaa atc gga tct gac ggc tgg gga 1746Thr Gly Asn Arg Ser Asp
Thr Val Lys Ile Gly Ser Asp Gly Trp Gly 455 460
465gag ttt cat gta aac gat ggg tcc gtc tcc att tat gtt cag
aaa taa 1794Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr Val Gln
Lys 470 475 480ggtaataaaa aaacacctcc
aagctgagtg cgggtatcag cttggaggtg cgtttatttt 1854ttcagccgta tgacaaggtc
ggcatcaggt gtgacaaata cggtatgctg gctgtcatag 1914gtgacaaatc cgggttttgc
gccgtttggc tttttcacat gtctgatttt tgtataatca 1974acaggcacgg agccggaatc
tttcgccttg gaaaaataag cggcgatcgt agctgcttcc 2034aatatggatt gttcatcggg
atcgctgctt ttaatcacaa cgtgggatcc 208410483PRTBacillus
amyloliquefaciens 10Val 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
Lys111458DNABacillus speciesCDS(1)..(1458) 11cac cat aat ggt acg aac ggc
aca atg atg cag tac ttt gaa tgg tat 48His His Asn Gly Thr Asn Gly
Thr Met Met Gln Tyr Phe Glu Trp Tyr1 5 10
15cta cca aat gac gga aac cat tgg aat aga tta agg tct
gat gca agt 96Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser
Asp Ala Ser 20 25 30aac cta
aaa gat aaa ggg atc tca gcg gtt tgg att cct cct gca tgg 144Asn Leu
Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp 35
40 45aag ggt gcc tct caa aat gat gtg ggg tat
ggt gct tat gat ctg tat 192Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr
Gly Ala Tyr Asp Leu Tyr 50 55 60gat
tta gga gaa ttc aat caa aaa gga acc att cgt aca aaa tat gga 240Asp
Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly65
70 75 80acg cgc aat cag tta caa
gct gca gtt aac gcc ttg aaa agt aat gga 288Thr Arg Asn Gln Leu Gln
Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85
90 95att caa gtg tat ggc gat gtt gta atg aat cat aaa
ggg gga gca gac 336Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys
Gly Gly Ala Asp 100 105 110gct
acc gaa atg gtt agg gca gtt gaa gta aac ccg aat aat aga aat 384Ala
Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115
120 125caa gaa gtg tcc ggt gaa tat aca att
gag gct tgg aca aag ttt gac 432Gln Glu Val Ser Gly Glu Tyr Thr Ile
Glu Ala Trp Thr Lys Phe Asp 130 135
140ttt cca gga cga ggt aat act cat tca aac ttc aaa tgg aga tgg tat
480Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr145
150 155 160cac ttt gat gga
gta gat tgg gat cag tca cgt aag ctg aac aat cga 528His Phe Asp Gly
Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg 165
170 175att tat aaa ttt aga ggt gat gga aaa ggg
tgg gat tgg gaa gtc gat 576Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly
Trp Asp Trp Glu Val Asp 180 185
190aca gaa aac ggt aac tat gat tac cta atg tat gca gat att gac atg
624Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met
195 200 205gat cac cca gag gta gtg aat
gag cta aga aat tgg ggt gtt tgg tat 672Asp His Pro Glu Val Val Asn
Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215
220acg aat aca tta ggc ctt gat ggt ttt aga ata gat gca gta aaa cat
720Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225
230 235 240ata aaa tac agc
ttt act cgt gat tgg att aat cat gtt aga agt gca 768Ile Lys Tyr Ser
Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245
250 255act ggc aaa aat atg ttt gcg gtt gcg gaa
ttt tgg aaa aat gat tta 816Thr Gly Lys Asn Met Phe Ala Val Ala Glu
Phe Trp Lys Asn Asp Leu 260 265
270ggt gct att gaa aac tat tta aac aaa aca aac tgg aac cat tca gtc
864Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val
275 280 285ttt gat gtt ccg ctg cac tat
aac ctc tat aat gct tca aaa agc gga 912Phe Asp Val Pro Leu His Tyr
Asn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295
300ggg aat tat gat atg agg caa ata ttt aat ggt aca gtc gtg caa aga
960Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg305
310 315 320cat cca atg cat
gct gtt aca ttt gtt gat aat cat gat tcg caa cct 1008His Pro Met His
Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325
330 335gaa gaa gct tta gag tct ttt gtt gaa gaa
tgg ttc aaa cca tta gcg 1056Glu Glu Ala Leu Glu Ser Phe Val Glu Glu
Trp Phe Lys Pro Leu Ala 340 345
350tat gct ttg aca tta aca cgt gaa caa ggc tac cct tct gta ttt tat
1104Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365gga gat tat tat ggc att cca
acg cat ggt gta cca gcg atg aaa tcg 1152Gly Asp Tyr Tyr Gly Ile Pro
Thr His Gly Val Pro Ala Met Lys Ser 370 375
380aaa att gac ccg att cta gaa gcg cgt caa aag tat gca tat gga aga
1200Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg385
390 395 400caa aat gac tac
tta gac cat cat aat atc atc ggt tgg aca cgt gaa 1248Gln Asn Asp Tyr
Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405
410 415ggg aat aca gca cac ccc aac tcc ggt tta
gct act atc atg tcc gat 1296Gly Asn Thr Ala His Pro Asn Ser Gly Leu
Ala Thr Ile Met Ser Asp 420 425
430ggg gca gga gga aat aag tgg atg ttt gtt ggg cgt aat aaa gct ggt
1344Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly
435 440 445caa gtt tgg acc gat atc act
gga aat cgt gca ggt act gtt acg att 1392Gln Val Trp Thr Asp Ile Thr
Gly Asn Arg Ala Gly Thr Val Thr Ile 450 455
460aat gct gat gga tgg ggt aat ttt tct gta aat gga gga tca gtt tct
1440Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser465
470 475 480att tgg gta aac
aaa taa 1458Ile Trp Val Asn
Lys 48512485PRTBacillus species 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 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
48513197PRTBacillus species 13Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn
Ala Ser Lys Ser Gly1 5 10
15Gly Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg
20 25 30His Pro Ser His Ala Val Thr
Phe Val Asp Asn His Asp Ser Gln Pro 35 40
45Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu
Ala 50 55 60Tyr Ala Leu Thr Leu Thr
Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr65 70
75 80Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val
Pro Ala Met Arg Ser 85 90
95Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys
100 105 110Gln Asn Asp Tyr Leu Asp
His His Asn Ile Ile Gly Trp Thr Arg Glu 115 120
125Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met
Ser Asp 130 135 140Gly Ala Gly Gly Ser
Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly145 150
155 160Gln Val Trp Ser Asp Ile Thr Gly Asn Arg
Thr Gly Thr Val Thr Ile 165 170
175Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser
180 185 190Ile Trp Val Asn Lys
1951424DNAArtificial SequenceSynthetic construct 14cgattgctga
cgctgttatt tgcg
241519DNAArtificial SequenceSynthetic construct 15gatcacccgc gataccgtc
191631DNAArtificial
SequenceSynthetic construct 16gaatgtatgt cggccggcaa aacgccggtg a
311730DNAArtificial SequenceSynthetic construct
17gccgccgctg ctgcagaatg aggcagcaag
301848DNAArtificial SequenceSynthetic construct 18cccgaaagct gaaccgcatc
tataggtttc aagggaagac ttgggatt 481923DNAArtificial
SequenceSynthetic construct 19aggatggtca taatcaaagt cgg
232052DNAArtificial SequenceSynthetic construct
20ccgactttga ttatgaccat cctgttgtcg tagcagagat taagagatgg gg
522145DNAArtificial SequenceSynthetic construct 21cgacaatgtc atggtggtcg
aaaaaatcat gctgtgctcc gtacg 452223DNAArtificial
SequenceSynthetic construct 22tttcgaccac catgacattg tcg
232324DNAArtificial SequenceSynthetic construct
23tatagatgcg gttcagcttt cggg
24241650DNABacillus species 24cttgaatcat tatttaaagc tggttatgat atatgtaagc
gttatcatta aaaggaggta 60tttgatgaaa agatgggtag tagcaatgct ggcagtgtta
tttttatttc cttcggtagt 120agttgcagat ggcttgaatg gaacgatgat gcagtattat
gagtggcatc tagagaatga 180tgggcaacac tggaatcggt tgcatgatga tgccgaagct
ttaagtaatg cgggtattac 240agctatttgg atacccccag cctacaaagg aaatagtcag
gctgatgttg ggtatggtgc 300atacgacctt tatgatttag gggagtttaa tcaaaaaggt
accgttcgaa cgaaatacgg 360gacaaaggct cagcttgagc gagctatagg gtccctaaag
tcgaatgata tcaatgttta 420tggggatgtc gtaatgaatc ataaattagg agctgatttc
acggaggcag tgcaagctgt 480tcaagtaaat ccttcgaacc gttggcagga tatttcaggt
gtctacacga ttgatgcatg 540gacgggattt gactttccag ggcgcaacaa tgcctattcc
gattttaaat ggagatggtt 600ccattttaat ggcgttgact gggatcaacg ctatcaagaa
aaccatcttt ttcgctttgc 660aaatacgaac tggaactggc gagtggatga agagaatggt
aattatgact atttattagg 720atcgaacatt gactttagcc acccagaggt tcaagaggaa
ttaaaggatt gggggagctg 780gtttacggat gagctagatt tagatgggta tcgattggat
gctattaagc atattccatt 840ctggtatacg tcagattggg ttaggcatca gcgaagtgaa
gcagaccaag atttatttgt 900cgtaggggag tattggaagg atgacgtagg tgctctcgaa
ttttatttag atgaaatgaa 960ttgggagatg tctctattcg atgttccgct caattataat
ttttaccggg cttcaaagca 1020aggcggaagc tatgatatgc gtaatatttt acgaggatct
ttagtagaag cacatccgat 1080tcatgcagtt acgtttgttg ataatcatga tactcagcca
ggagagtcat tagaatcatg 1140ggtcgctgat tggtttaagc cacttgctta tgcgacaatc
ttgacgcgtg aaggtggtta 1200tccaaatgta ttttacggtg actactatgg gattcctaac
gataacattt cagctaagaa 1260ggatatgatt gatgagttgc ttgatgcacg tcaaaattac
gcatatggca cacaacatga 1320ctattttgat cattgggata tcgttggatg gacaagagaa
ggtacatcct cacgtcctaa 1380ttcgggtctt gctactatta tgtccaatgg tcctggagga
tcaaaatgga tgtacgtagg 1440acagcaacat gcaggacaaa cgtggacaga tttaactggc
aatcacgcgg cgtcggttac 1500gattaatggt gatggctggg gcgaattctt tacaaatgga
ggatctgtat ccgtgtatgt 1560gaaccaataa taaaaagcct tgagaaggga ttcctcccta
actcaaggct ttctttatgt 1620cgtttagctc aacgcttcta cgaagcttta
165025501PRTBacillus species 25Met Lys Arg Trp Val
Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro1 5
10 15Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr
Met Met Gln Tyr Tyr 20 25
30Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His Asp
35 40 45Asp Ala Glu Ala Leu Ser Asn Ala
Gly Ile Thr Ala Ile Trp Ile Pro 50 55
60Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr65
70 75 80Asp Leu Tyr Asp Leu
Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr 85
90 95Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala
Ile Gly Ser Leu Lys 100 105
110Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys Leu
115 120 125Gly Ala Asp Phe Thr Glu Ala
Val Gln Ala Val Gln Val Asn Pro Ser 130 135
140Asn Arg Trp Gln Asp Ile Ser Gly Val Tyr Thr Ile Asp Ala Trp
Thr145 150 155 160Gly Phe
Asp Phe Pro Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp
165 170 175Arg Trp Phe His Phe Asn Gly
Val Asp Trp Asp Gln Arg Tyr Gln Glu 180 185
190Asn His Leu Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg
Val Asp 195 200 205Glu Glu Asn Gly
Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe 210
215 220Ser His Pro Glu Val Gln Glu Glu Leu Lys Asp Trp
Gly Ser Trp Phe225 230 235
240Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His
245 250 255Ile Pro Phe Trp Tyr
Thr Ser Asp Trp Val Arg His Gln Arg Ser Glu 260
265 270Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp
Lys Asp Asp Val 275 280 285Gly Ala
Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu 290
295 300Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg
Ala Ser Lys Gln Gly305 310 315
320Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala
325 330 335His Pro Ile His
Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro 340
345 350Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp
Phe Lys Pro Leu Ala 355 360 365Tyr
Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr 370
375 380Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn
Ile Ser Ala Lys Lys Asp385 390 395
400Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly
Thr 405 410 415Gln His Asp
Tyr Phe Asp His Trp Asp Ile Val Gly Trp Thr Arg Glu 420
425 430Gly Thr Ser Ser Arg Pro Asn Ser Gly Leu
Ala Thr Ile Met Ser Asn 435 440
445Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Gln Gln His Ala Gly 450
455 460Gln Thr Trp Thr Asp Leu Thr Gly
Asn His Ala Ala Ser Val Thr Ile465 470
475 480Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly
Gly Ser Val Ser 485 490
495Val Tyr Val Asn Gln 500261745DNABacillus
speciesCDS(190)..(1692)sig_peptide(190)..(253)mat_peptide(253)..()
26aactaagtaa catcgattca ggataaaagt atgcgaaacg atgcgcaaaa ctgcgcaact
60actagcactc ttcagggact aaaccacctt ttttccaaaa atgacatcat ataaacaaat
120ttgtctacca atcactattt aaagctgttt atgatatatg taagcgttat cattaaaagg
180aggtatttg atg aga aga tgg gta gta gca atg ttg gca gtg tta ttt tta
231 Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu
-20 -15 -10ttt cct tcg gta gta
gtt gca gat gga ttg aac ggt acg atg atg cag 279Phe Pro Ser Val Val
Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln -5 -1 1
5tat tat gag tgg cat ttg gaa aac gac ggg cag cat tgg aat cgg
ttg 327Tyr Tyr Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg
Leu10 15 20 25cac gat
gat gcc gca gct ttg agt gat gct ggt att aca gct att tgg 375His Asp
Asp Ala Ala Ala Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp 30
35 40att ccg cca gcc tac aaa ggt aat
agt cag gcg gat gtt ggg tac ggt 423Ile Pro Pro Ala Tyr Lys Gly Asn
Ser Gln Ala Asp Val Gly Tyr Gly 45 50
55gca tac gat ctt tat gat tta gga gag ttc aat caa aag ggt act
gtt 471Ala Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr
Val 60 65 70cga acg aaa tac gga
act aag gca cag ctt gaa cga gct att ggg tcc 519Arg Thr Lys Tyr Gly
Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser 75 80
85ctt aaa tct aat gat atc aat gta tac gga gat gtc gtg atg
aat cat 567Leu Lys Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met
Asn His90 95 100 105aaa
atg gga gct gat ttt acg gag gca gtg caa gct gtt caa gta aat 615Lys
Met Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn
110 115 120cca acg aat cgt tgg cag gat
att tca ggt gcc tac acg att gat gcg 663Pro Thr Asn Arg Trp Gln Asp
Ile Ser Gly Ala Tyr Thr Ile Asp Ala 125 130
135tgg acg ggt ttc gac ttt tca ggg cgt aac aac gcc tat tca
gat ttt 711Trp Thr Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser
Asp Phe 140 145 150aag tgg aga tgg
ttc cat ttt aat ggt gtt gac tgg gat cag cgc tat 759Lys Trp Arg Trp
Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr 155
160 165caa gaa aat cat att ttc cgc ttt gca aat acg aac
tgg aac tgg cga 807Gln Glu Asn His Ile Phe Arg Phe Ala Asn Thr Asn
Trp Asn Trp Arg170 175 180
185gtg gat gaa gag aac ggt aat tat gat tac ctg tta gga tcg aat atc
855Val Asp Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile
190 195 200gac ttt agt cat cca
gaa gta caa gat gag ttg aag gat tgg ggt agc 903Asp Phe Ser His Pro
Glu Val Gln Asp Glu Leu Lys Asp Trp Gly Ser 205
210 215tgg ttt acc gat gag tta gat ttg gat ggt tat cgt
tta gat gct att 951Trp Phe Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg
Leu Asp Ala Ile 220 225 230aaa cat
att cca ttc tgg tat aca tct gat tgg gtt cgg cat cag cgc 999Lys His
Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg 235
240 245aac gaa gca gat caa gat tta ttt gtc gta ggg
gaa tat tgg aag gat 1047Asn Glu Ala Asp Gln Asp Leu Phe Val Val Gly
Glu Tyr Trp Lys Asp250 255 260
265gac gta ggt gct ctc gaa ttt tat tta gat gaa atg aat tgg gag atg
1095Asp Val Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met
270 275 280tct cta ttc gat gtt
cca ctt aat tat aat ttt tac cgg gct tca caa 1143Ser Leu Phe Asp Val
Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Gln 285
290 295caa ggt gga agc tat gat atg cgt aat att tta cga
gga tct tta gta 1191Gln Gly Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg
Gly Ser Leu Val 300 305 310gaa gcg
cat ccg atg cat gca gtt acg ttt gtt gat aat cat gat act 1239Glu Ala
His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Thr 315
320 325cag cca ggg gag tca tta gag tca tgg gtt gct
gat tgg ttt aag cca 1287Gln Pro Gly Glu Ser Leu Glu Ser Trp Val Ala
Asp Trp Phe Lys Pro330 335 340
345ctt gct tat gcg aca att ttg acg cgt gaa ggt ggt tat cca aat gta
1335Leu Ala Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val
350 355 360ttt tac ggt gat tac
tat ggg att cct aac gat aac att tca gct aaa 1383Phe Tyr Gly Asp Tyr
Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys 365
370 375aaa gat atg att gat gag ctg ctt gat gca cgt caa
aat tac gca tat 1431Lys Asp Met Ile Asp Glu Leu Leu Asp Ala Arg Gln
Asn Tyr Ala Tyr 380 385 390ggc acg
cag cat gac tat ttt gat cat tgg gat gtt gta gga tgg act 1479Gly Thr
Gln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp Thr 395
400 405agg gaa gga tct tcc tcc aga cct aat tca ggc
ctt gcg act att atg 1527Arg Glu Gly Ser Ser Ser Arg Pro Asn Ser Gly
Leu Ala Thr Ile Met410 415 420
425tcg aat gga cct ggt ggt tcc aag tgg atg tat gta gga cgt cag aat
1575Ser Asn Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Asn
430 435 440gca gga caa aca tgg
aca gat tta act ggt aat aac gga gcg tcc gtt 1623Ala Gly Gln Thr Trp
Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val 445
450 455aca att aat ggc gat gga tgg ggc gaa ttc ttt acg
aat gga gga tct 1671Thr Ile Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr
Asn Gly Gly Ser 460 465 470gta tcc
gtg tac gtg aac caa taacaaaaag ccttgagaag ggattcctcc 1722Val Ser
Val Tyr Val Asn Gln 475 480ctaactcaag gctttcttta tgt
174527501PRTBacillus species
27Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro -20
-15 -10Ser Val Val Val Ala Asp Gly Leu Asn
Gly Thr Met Met Gln Tyr Tyr-5 -1 1 5
10Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His
Asp 15 20 25Asp Ala Ala Ala
Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro 30
35 40Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly
Tyr Gly Ala Tyr 45 50 55Asp Leu Tyr
Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr60 65
70 75Lys Tyr Gly Thr Lys Ala Gln Leu
Glu Arg Ala Ile Gly Ser Leu Lys 80 85
90Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His
Lys Met 95 100 105Gly Ala Asp
Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr 110
115 120Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr
Ile Asp Ala Trp Thr 125 130 135Gly Phe
Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp140
145 150 155Arg Trp Phe His Phe Asn Gly
Val Asp Trp Asp Gln Arg Tyr Gln Glu 160
165 170Asn His Ile Phe Arg Phe Ala Asn Thr Asn Trp Asn
Trp Arg Val Asp 175 180 185Glu
Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe 190
195 200Ser His Pro Glu Val Gln Asp Glu Leu
Lys Asp Trp Gly Ser Trp Phe 205 210
215Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His220
225 230 235Ile Pro Phe Trp
Tyr Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu 240
245 250Ala Asp Gln Asp Leu Phe Val Val Gly Glu
Tyr Trp Lys Asp Asp Val 255 260
265Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu
270 275 280Phe Asp Val Pro Leu Asn Tyr
Asn Phe Tyr Arg Ala Ser Gln Gln Gly 285 290
295Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val Glu
Ala300 305 310 315His Pro
Met His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro
320 325 330Gly Glu Ser Leu Glu Ser Trp
Val Ala Asp Trp Phe Lys Pro Leu Ala 335 340
345Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val
Phe Tyr 350 355 360Gly Asp Tyr Tyr
Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp 365
370 375Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr
Ala Tyr Gly Thr380 385 390
395Gln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp Thr Arg Glu
400 405 410Gly Ser Ser Ser Arg
Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn 415
420 425Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg
Gln Asn Ala Gly 430 435 440Gln Thr
Trp Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile 445
450 455Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn
Gly Gly Ser Val Ser460 465 470
475Val Tyr Val Asn Gln 48028501PRTBacillus species
28Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro1
5 10 15Ser Val Val Val Ala Asp
Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr 20 25
30Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg
Leu His Asp 35 40 45Asp Ala Ala
Ala Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro 50
55 60Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly
Tyr Gly Ala Tyr65 70 75
80Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr
85 90 95Lys Tyr Gly Thr Lys Ala
Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys 100
105 110Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met
Asn His Lys Met 115 120 125Gly Ala
Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr 130
135 140Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr
Ile Asp Ala Trp Thr145 150 155
160Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp
165 170 175Arg Trp Phe His
Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu 180
185 190Asn His Ile Phe Arg Phe Ala Asn Thr Asn Trp
Asn Trp Arg Val Asp 195 200 205Glu
Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe 210
215 220Ser His Pro Glu Val Gln Asp Glu Leu Lys
Asp Trp Gly Ser Trp Phe225 230 235
240Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys
His 245 250 255Ile Pro Phe
Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu 260
265 270Ala Asp Gln Asp Leu Phe Val Val Gly Glu
Tyr Trp Lys Asp Asp Val 275 280
285Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu 290
295 300Phe Asp Val Pro Leu Asn Tyr Asn
Phe Tyr Arg Ala Ser Gln Gln Gly305 310
315 320Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser
Leu Val Glu Ala 325 330
335His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro
340 345 350Gly Glu Ser Leu Glu Ser
Trp Val Ala Asp Trp Phe Lys Pro Leu Ala 355 360
365Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val
Phe Tyr 370 375 380Gly Asp Tyr Tyr Gly
Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp385 390
395 400Met Ile Asp Glu Leu Leu Asp Ala Arg Gln
Asn Tyr Ala Tyr Gly Thr 405 410
415Gln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp Thr Arg Glu
420 425 430Gly Ser Ser Ser Arg
Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn 435
440 445Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg
Gln Asn Ala Gly 450 455 460Gln Thr Trp
Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile465
470 475 480Asn Gly Asp Gly Trp Gly Glu
Phe Phe Thr Asn Gly Gly Ser Val Ser 485
490 495Val Tyr Val Asn Gln
500291920DNABacillus licheniformisCDS(421)..(1872) 29cggaagattg
gaagtacaaa aataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60gagacggaaa
aatcgtctta atgcacgata tttatgcaac gttcgcagat gctgctgaag 120agattattaa
aaagctgaaa gcaaaaggct atcaattggt aactgtatct cagcttgaag 180aagtgaagaa
gcagagaggc tattgaataa atgagtagaa gcgccatatc ggcgcttttc 240ttttggaaga
aaatataggg aaaatggtac ttgttaaaaa ttcggaatat ttatacaaca 300tcatatgttt
cacattgaaa ggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360cgattgctga
cgctgttatt tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420gca aat ctt
aat ggg acg ctg atg cag tat ttt gaa tgg tac atg ccc 468Ala Asn Leu
Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro1 5
10 15aat gac ggc caa cat tgg agg cgt ttg
caa aac gac tcg gca tat ttg 516Asn Asp Gly Gln His Trp Arg Arg Leu
Gln Asn Asp Ser Ala Tyr Leu 20 25
30gct gaa cac ggt att act gcc gtc tgg att ccc ccg gca tat aag gga
564Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45acg agc caa gcg gat gtg ggc
tac ggt gct tac gac ctt tat gat tta 612Thr Ser Gln Ala Asp Val Gly
Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55
60ggg gag ttt cat caa aaa ggg acg gtt cgg aca aag tac ggc aca aaa
660Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65
70 75 80gga gag ctg caa
tct gcg atc aaa agt ctt cat tcc cgc gac att aac 708Gly Glu Leu Gln
Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn 85
90 95gtt tac ggg gat gtg gtc atc aac cac aaa
ggc ggc gct gat gcg acc 756Val Tyr Gly Asp Val Val Ile Asn His Lys
Gly Gly Ala Asp Ala Thr 100 105
110gaa gat gta acc gcg gtt gaa gtc gat ccc gct gac cgc aac cgc gta
804Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val
115 120 125att tca gga gaa cac cta att
aaa gcc tgg aca cat ttt cat ttt ccg 852Ile Ser Gly Glu His Leu Ile
Lys Ala Trp Thr His Phe His Phe Pro 130 135
140ggg cgc ggc agc aca tac agc gat ttt aaa tgg cat tgg tac cat ttt
900Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe145
150 155 160gac gga acc gat
tgg gac gag tcc cga aag ctg aac cgc atc tat aag 948Asp Gly Thr Asp
Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165
170 175ttt caa gga aag gct tgg gat tgg gaa gtt
tcc aat gaa aac ggc aac 996Phe Gln Gly Lys Ala Trp Asp Trp Glu Val
Ser Asn Glu Asn Gly Asn 180 185
190tat gat tat ttg atg tat gcc gac atc gat tat gac cat cct gat gtc
1044Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val
195 200 205gca gca gaa att aag aga tgg
ggc act tgg tat gcc aat gaa ctg caa 1092Ala Ala Glu Ile Lys Arg Trp
Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215
220ttg gac ggt ttc cgt ctt gat gct gtc aaa cac att aaa ttt tct ttt
1140Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe225
230 235 240ttg cgg gat tgg
gtt aat cat gtc agg gaa aaa acg ggg aag gaa atg 1188Leu Arg Asp Trp
Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245
250 255ttt acg gta gct gaa tat tgg cag aat gac
ttg ggc gcg ctg gaa aac 1236Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp
Leu Gly Ala Leu Glu Asn 260 265
270tat ttg aac aaa aca aat ttt aat cat tca gtg ttt gac gtg ccg ctt
1284Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu
275 280 285cat tat cag ttc cat gct gca
tcg aca cag gga ggc ggc tat gat atg 1332His Tyr Gln Phe His Ala Ala
Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295
300agg aaa ttg ctg aac ggt acg gtc gtt tcc aag cat ccg ttg aaa tcg
1380Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser305
310 315 320gtt aca ttt gtc
gat aac cat gat aca cag ccg ggg caa tcg ctt gag 1428Val Thr Phe Val
Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325
330 335tcg act gtc caa aca tgg ttt aag ccg ctt
gct tac gct ttt att ctc 1476Ser Thr Val Gln Thr Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345
350aca agg gaa tct gga tac cct cag gtt ttc tac ggg gat atg tac ggg
1524Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly
355 360 365acg aaa gga gac tcc cag cgc
gaa att cct gcc ttg aaa cac aaa att 1572Thr Lys Gly Asp Ser Gln Arg
Glu Ile Pro Ala Leu Lys His Lys Ile 370 375
380gaa ccg atc tta aaa gcg aga aaa cag tat gcg tac gga gca cag cat
1620Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His385
390 395 400gat tat ttc gac
cac cat gac att gtc ggc tgg aca agg gaa ggc gac 1668Asp Tyr Phe Asp
His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405
410 415agc tcg gtt gca aat tca ggt ttg gcg gca
tta ata aca gac gga ccc 1716Ser Ser Val Ala Asn Ser Gly Leu Ala Ala
Leu Ile Thr Asp Gly Pro 420 425
430ggt ggg gca aag cga atg tat gtc ggc cgg caa aac gcc ggt gag aca
1764Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr
435 440 445tgg cat gac att acc gga aac
cgt tcg gag ccg gtt gtc atc aat tcg 1812Trp His Asp Ile Thr Gly Asn
Arg Ser Glu Pro Val Val Ile Asn Ser 450 455
460gaa ggc tgg gga gag ttt cac gta aac ggc ggg tcg gtt tca att tat
1860Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr465
470 475 480gtt caa aga tag
aagagcagag aggacggatt tcctgaagga aatccgtttt 1912Val Gln
Argtttatttt
192030483PRTBacillus licheniformis 30Ala 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 Arg
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