Alpha-Amylase Mutants with Altered Properties

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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No. 13/052,373 filed on Mar. 21, 2011, now abandoned, which is a divisional of U.S. application Ser. No. 12/758,346 filed on Apr. 12, 2010, now pending, which is a continuation of U.S. application Ser. No. 12/645,116 filed on Dec. 22, 2009, now U.S. Pat. No. 7,713,723, which is a continuation of U.S. application Ser. No. 12/566,238 filed Sep. 24, 2009, now abandoned, which is a continuation of U.S. application Ser. No. 10/630,203 filed Jul. 29, 2003, now abandoned, 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 variants have alpha-amylase activity and exhibit 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 variants of the invention are suitable for starch conversion, ethanol production, laundry wash, dish wash, hard surface cleaning, textile desizing, and/or sweetner 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, have alpha-amylase activity and exhibit 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 A30N a 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] Ala30A1aLys 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 *36D for an insertion of an aspartic acid in position 36.

[0015] Multiple mutations are separated by plus signs, i.e.:

[0016] Ala30Asp+Glu34Ser or A30N+E34S representing 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, A300, 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] FIGS. 1A, 1B and 1C provide 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 252666 (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 Io, 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.

[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, are considered to be Termamyl-like alpha-amylases. 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, 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, SxDenhardt'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: H156Y+A181T+N190F; H156Y+A181T+N190F+A209V; H156Y+A181T+N190F+Q264S; H156Y+A181T+A209V+Q264S; H156Y+A181T+Q264S; H156Y+N190F+A209V+Q264S; H156Y+A209V+Q264S; H156Y+Q264S; A181T+N190F+A209V+Q264S; N190F+A209V+Q264S; A209V+Q264S; and Q264S. 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+M197L+I201F;

LE174+G48A+T49I+G107A+I201F;

LE174+M197L;

SP722+D183*+G184*;

SP722+D183*+G184*+N195F;

SP722+D183*+G184*+N195F+M202L;

SP722+D183*+G184*+M202L;

BSG+1181*+G182*;

BSG+1181*+G182*+N193F;

BSG+1181*+G182*+N193F+M200L;

BSG+1181*+G182*+M200L;

AA560+D183*+G184*;

AA560+D183*+G184*+N195F;

AA560+D183*+G184*+N195F+M202L; and

AA560+D183*+G184*+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., SP722, BSG, BAN, AA560, SP690, KSM AP1378, and #707 should be understood as disclosed and specifically comtemplated 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

[0060] (a) the alteration(s) are independently

[0061] (i) an insertion of an amino acid downstream of the amino acid which occupies the position,

[0062] (ii) a deletion of the amino acid which occupies the position, or

[0063] (iii) a substitution of the amino acid which occupies the position with a different amino acid,

[0064] (b) the variant has alpha-amylase activity and

[0065] (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.

[0066] 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.

[0067] 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.

[0068] Corresponding positions in other parent alpha-amylases can be found by alignment as described above and shown in the alignment in FIG. 1.

[0069] 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:

T49I; 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.

[0070] 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:

T511; 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, 5303Q, X323M; D387V, N395R; Y404F; H408L,W; X4291; D432N; V442A; X446R,K; X449Q,K; X484K, using SEQ ID NO: 4 (SP722) for numbering.

[0071] Preferred double, triple and multi-mutations--using SEQ ID NO: 8 as the basis for the numbering--are selected from the group consisting of:

T49I+D60N; T49I+D60N+E132A; T49I+D60N+E132V; T49I+D60N+E132V+K170Q;

T49I+D60N+E132A+K170Q; T49I+D60N+E132V+K170Q+K176R;

T49I+D60N+E132A+K170Q+K176R;

T49I+D60N+E132V+K170Q+K176R+D207V; T49I+D60N+E132A+K170Q+K176R+D207V;

T49I+D60N+E132V+K170Q+K176R+D207E; T49I+D60N+E132A+K170Q+K176R+D207E;

T49I+D60N+E132V+K170Q+K176R+D207V+E250G;

T49I+D60N+E132A+K170Q+K176R+D207V+E250G;

T49I+D60N+E132V+K170Q+K176R+D207E+E250G;

T49I+D60N+E132A+K170Q+K176R+D207E+E250G;

T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S;

T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S;

T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S;

T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S;

T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M;

T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M;

T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M;

T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M;

T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;

T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;

T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;

T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;

T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;

T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;

T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;

T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;

T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;

T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;

T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;

T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;

T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F;

T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F;

T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F;

T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F;

T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L;

T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L;

T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L;

T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L;

T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I;

T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I;

T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I;

T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I;

T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I+V440A;

T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I+V440A;

T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I+V440A;

T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402- F+H406L+L427I+V440A;

D60N+E132A;

D60N+E132A+K170Q;

D60N+E132V;

D60N+E132V+K170Q;

D60N+E132V+K170Q+K176R; T49I+D60N+E132A+K170Q+K176R;

D60N+E132V+K170Q+K176R+D207V; T49I+D60N+E132A+K170Q+K176R+D207V;

D60N+E132V+K170Q+K176R+D207E; T49I+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+L427I;

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;

E132A+K170Q;

E132A+K170Q+K176R;

E132A+K170Q+K176R+D207E;

E132A+K170Q+K176R+D207E+E250G;

E132A+K170Q+K176R+D207V;

E132A+K170Q+K176R+D207V+E250G;

E132A+K170Q+K176R+D207E+E250G+N280S;

E132V+K170Q;

E132V+K170Q+K176R;

E132V+K170Q+K176R+D207E;

E132V+K170Q+K176R+D207E+E250G;

E132V+K170Q+K176R+D207E+E250G+N280S;

E132V+K170Q+K176R+D207V;

E132V+K170Q+K176R+D207V+E250G;

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- 27I;

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+L427I;

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+L318M+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+L427I;

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+L427I;

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+N4- 44R+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;

[0072] D161N+A181N+D183N+D200N+D204S; K237P+S239W, using SEQ ID NO: 8 for numbering.

[0073] In a preferred embodiment the variant has the following substitutions: K170Q+D207V+N280S; E132A+D207V; D207E+E250G+H406L+L427I; D207V+L318M; D60N+D207V+L318M; T49I+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; T49I+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.

[0074] 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

[0075] 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

[0076] 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.

[0077] 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.

[0078] 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.

[0079] It is to be understood that the present invention encompasses variants incorporating two or more of the above outlined modifications.

[0080] 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/T149I;

G475R, H133Y/S187D; H133Y/A209V.

Methods for Preparing Alpha-Amylase Variants of the Invention

[0081] 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

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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

[0086] 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.

[0087] 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.

[0088] 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 NS), 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 1022334 (hereby incorporated by reference).

Expression of Alpha-Amylase Variants

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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).

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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 238023.

[0101] 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.

[0102] 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).

[0103] 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

[0104] 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.

[0105] 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 252730 and EP 063909, WO 99/19467, and WO 96/28567, which are all hereby incorporated by reference). Also contemplated are compositions for starch conversion purposes, which may beside the variant of the invention also comprise an AMG, pullulanase, and other alpha-amylases.

[0106] 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.

[0107] 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 119920, which are hereby incorporated by reference).

Detergent Compositions

[0108] 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.

[0109] 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.

[0110] 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

[0111] 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.

[0112] In another embodiment the composition comprises besides 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.

[0113] 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.

[0114] In an embodiment the composition of the invention also comprises a pullulanase, in particular a Bacillus pullulanase.

Materials and Methods

Enzymes:

[0115] 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 1 b, D-38124 Braunschweig DE.

[0116] 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).

[0117] 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).

[0118] 10% Lugol solution (Iodine/Potassium iodine solution; made by 10-fold dil. in H₂O of stock: Sigma Cat. no. L 6146).

[0119] Bacillus subtilis SHA273: see WO 95/10603

Plasmids

[0120] 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, oh, 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

[0121] 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.

[0122] 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

[0123] 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:

[0124] 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 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

[0125] 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.

[0126] The colonies are transferred to 100 ml PS-1 media supplemented with 10 micrograms/ml chloamphinicol in a 500 ml shaking flask.

Composition of PS-1 Medium:

TABLE-US-00002

[0127] Pearl sugar 100 g/l Soy Bean Meal 40 g/l Na₂HPO₄, 12 H₂O 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.

[0128] 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 dialyzed against 10 mM Tris, pH 9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradient from 0-0.3 M NaCl over 6 column volumes. The fractions 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

[0129] All stability trials of purified variants are made using the same set up. The method is as follows:

[0130] 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.

[0131] 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

[0132] 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 Alpha-Amylase Activity").

Assays for Alpha-Amylase Activity

1. Phadebas Assay

[0133] 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.

[0134] 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.

[0135] 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

[0136] 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 2=405 nm (400-420 nm). Kits containing PNP-G7 substrate and alpha-Glucosidase is manufactured by Boehringer-Mannheim (cat. no. 1054635).

[0137] 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.

[0138] 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

[0139] 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

[0140] 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.

[0141] 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

[0142] 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:

[0143] Increased stability at pH 4.5, 5 ppm calcium incubated at 90° C.

TABLE-US-00003 Name wt LE488 LE489 7.19.1 8.9.1 Mutations -- D207V K170Q E132A D207E D207V D207V E250G N280S H406L L427I Stability 1) - + + + + 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 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 Stability 1) - + + + + + 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 Name wt E132-1 D207-7 D207-6 E250-8 Mutations -- E132P D207L D207G E250F Stability 1) - + + + + 1) A "+" indicates significant increase in stability relative to wt.

Example 2

[0144] 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

[0145] 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.

[0146] 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:

[0147] Stabilization of LE399 variant at pH 4.5, 5 ppm calcium incubated at 90° C.

TABLE-US-00006 Name LE399 LE495 LE497 Mutations -- K176R K176R (backbone) D207V Y402F Y402F Stability 1) - + + 1) A "+" indicates significant increase in stability relative to backbone.

Sequence CWU 1

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 Tyr 1 5 10 15 ttg 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 30 aac 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 45 aag 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 60 gat 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 Gly 65 70 75 80 aca 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 95 att 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 100 105 110 ggt 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 115 120 125 cag 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 130 135 140 ttt 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 Tyr 145 150 155 160 cat 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 165 170 175 ata 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 180 185 190 aca 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 195 200 205 gat 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 210 215 220 acg 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 His 225 230 235 240 ata 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 245 250 255 aca 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 260 265 270 ggt 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 275 280 285 ttt 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 290 295 300 ggt 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 Lys 305 310 315 320 cat 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 325 330 335 ggg 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 340 345 350 tat 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 355 360 365 ggg 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 370 375 380 aaa 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 Thr 385 390 395 400 cag 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 405 410 415 gga 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 420 425 430 ggt 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 435 440 445 caa 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 450 455 460 aat 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 Ser 465 470 475 480 gtt tgg gtg aag caa 1455Val Trp Val Lys Gln 485 2485PRTBacillus species 2His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120 125 Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys 165 170 175 Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205 Asp His Pro Glu Val Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr 245 250 255 Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Tyr Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys 305 310 315 320 His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr 385 390 395 400 Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445 Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Val Trp Val Lys Gln 485 31455DNABacillus 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 His 1 5 10 15 ttg 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 30 aat 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 45 aaa 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 60 gat 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 Gly 65 70 75 80 aca 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 95 gtt 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 100 105 110 gct 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 115 120 125 caa 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 130 135 140 ttt 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 Tyr 145 150 155 160 cat 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 165 170 175 atc 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 180 185 190 tcg 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 195 200 205 gat 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 210 215 220 aca 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 His 225 230 235 240 att 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 245 250 255 acg 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 260 265 270 ggt 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 275 280 285 ttt 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 290 295 300 ggc 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 Lys 305 310 315 320 cat 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 325 330 335 ggg 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 340 345 350 tat 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 355 360 365 ggt 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 370 375 380 aag 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 Thr 385 390 395 400 caa 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 405 410 415 gga 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 420 425 430 ggg 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 435 440 445 caa 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 450 455 460 aat 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 Ser 465 470 475 480 att tgg gtg aaa cga 1455Ile Trp Val Lys Arg 485 4485PRTBacillus species 4His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30 Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90

95 Val Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala 245 250 255 Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys 305 310 315 320 His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr 385 390 395 400 Gln His Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala Gly 435 440 445 Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Lys Arg 485 51548DNABacillus 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 Leu 1 5 10 15 ccg 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 30 tta 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 45 gga 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 60 ctc 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 Thr 65 70 75 80 aaa 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 95 caa 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 100 105 110 acg 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 115 120 125 gaa 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 130 135 140 ccc 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 His 145 150 155 160 ttt 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 165 170 175 aaa 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 180 185 190 aac 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 195 200 205 ccc 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 210 215 220 aca 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 Lys 225 230 235 240 ttc 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 245 250 255 aag 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 260 265 270 ttg 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 275 280 285 gcc 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 290 295 300 ttt 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 Pro 305 310 315 320 aca 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 325 330 335 gcg 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 340 345 350 ttt 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 355 360 365 tat 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 370 375 380 gat 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 His 385 390 395 400 gat 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 405 410 415 act 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 420 425 430 gga 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 435 440 445 ttc 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 450 455 460 gat 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 Trp 465 470 475 480 gtt 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 485 490 495 cga 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 500 505 510 gca tgg cct tga 1548Ala Trp Pro 515 6515PRTBacillus stearothermophilus 6Ala Ala Pro Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu 1 5 10 15 Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30 Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45 Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60 Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr 65 70 75 80 Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala Ala Gly Met 85 90 95 Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly 100 105 110 Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gln 115 120 125 Glu Ile Ser Gly Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140 Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His 145 150 155 160 Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His 195 200 205 Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr Val Asn 210 215 220 Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys 225 230 235 240 Phe Ser Phe Phe Pro Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly 245 250 255 Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 260 265 270 Leu His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp 275 280 285 Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala 290 295 300 Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro 305 310 315 320 Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gln 325 330 335 Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile 370 375 380 Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His 385 390 395 400 Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly 405 410 415 Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val 435 440 445 Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser 450 455 460 Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp 465 470 475 480 Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala Arg Pro Ile Thr Thr 485 490 495 Arg Pro Trp Thr Gly Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val 500 505 510 Ala Trp Pro 515 71920DNABacillus 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 Pro 1 5 10 15 aat 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 30 gct 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 45 acg 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 60 ggg 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 Lys 65 70 75 80 gga 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 95 gtt 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 100 105 110 gaa 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 115 120 125 att 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 130 135 140 ggg 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 Phe 145 150 155 160 gac 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 165 170 175 ttt 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 180 185 190 tat 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 195 200 205 gca 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 210 215 220 ttg 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 Phe 225 230

235 240 ttg 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 245 250 255 ttt 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 260 265 270 tat 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 275 280 285 cat 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 290 295 300 agg 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 Ser 305 310 315 320 gtt 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 325 330 335 tcg 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 340 345 350 aca 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 355 360 365 acg 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 370 375 380 gaa 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 His 385 390 395 400 gat 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 405 410 415 agc 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 420 425 430 ggt 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 435 440 445 tgg 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 450 455 460 gaa 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 Tyr 465 470 475 480 gtt caa aga tag aagagcagag aggacggatt tcctgaagga aatccgtttt 1912Val Gln Arg tttatttt 19208483PRTBacillus licheniformis 8Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn 85 90 95 Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160 Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175 Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200 205 Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215 220 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu 275 280 285 His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380 Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445 Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455 460 Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475 480 Val Gln Arg 92084DNABacillus 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 1 ctg 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 Trp 5 10 15 20 aaa 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 35 gcc 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 50 gga 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 65 ggg 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 80 atc 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 Val 85 90 95 100 ttg 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 105 110 115 gaa 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 120 125 130 atc 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 135 140 145 agt 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 150 155 160 gaa 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 Lys 165 170 175 180 gcg 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 185 190 195 atg 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 200 205 210 aaa 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 215 220 225 cgt 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 230 235 240 gtt 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 Ala 245 250 255 260 gag 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 265 270 275 aca 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 280 285 290 cag 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 295 300 305 gac 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 310 315 320 gaa 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 Gln 325 330 335 340 act 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 345 350 355 ggt 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 360 365 370 tcg 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 375 380 385 aaa 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 390 395 400 cac 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 Ala 405 410 415 420 aaa 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 425 430 435 cgg 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 440 445 450 acg 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 455 460 465 gag 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 470 475 480 ggtaataaaa 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 Asp 1 5 10 15 Gly Gln His Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30 Ile Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser 35 40 45 Gln Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu 50 55 60 Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu 65 70 75 80 Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr 85 90 95 Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp 100 105 110 Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser 115 120 125 Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly Arg 130 135 140 Gly Asn Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly 145 150 155 160 Ala Asp Trp Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg 165 170 175 Gly Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val 195 200 205 Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser 210 215 220 Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Gln Ala Val Arg Gln Ala Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asn Ala Gly Lys Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu 275 280 285 His Phe Asn Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala 305 310 315 320 Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile 370 375 380 Glu Pro Ile Leu Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His 385 390 395 400 Asp Tyr Ile Asp His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405

410 415 Ser Ser Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr 435 440 445 Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser 450 455 460 Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr 465 470 475 480 Val Gln Lys 111458DNABacillus 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 Tyr 1 5 10 15 cta 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 30 aac 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 45 aag 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 60 gat 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 Gly 65 70 75 80 acg 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 95 att 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 100 105 110 gct 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 115 120 125 caa 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 130 135 140 ttt 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 Tyr 145 150 155 160 cac 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 165 170 175 att 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 180 185 190 aca 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 195 200 205 gat 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 210 215 220 acg 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 His 225 230 235 240 ata 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 245 250 255 act 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 260 265 270 ggt 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 275 280 285 ttt 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 290 295 300 ggg 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 Arg 305 310 315 320 cat 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 325 330 335 gaa 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 340 345 350 tat 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 355 360 365 gga 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 370 375 380 aaa 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 Arg 385 390 395 400 caa 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 405 410 415 ggg 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 420 425 430 ggg 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 435 440 445 caa 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 450 455 460 aat 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 Ser 465 470 475 480 att tgg gta aac aaa taa 1458Ile Trp Val Asn Lys 485 12485PRTBacillus species 12His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30 Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95 Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Lys Leu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Gly Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245 250 255 Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 290 295 300 Gly Asn Tyr Asp Met Arg Gln Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315 320 His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg 385 390 395 400 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440 445 Gln Val Trp Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Asn Lys 485 13197PRTBacillus species 13Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 1 5 10 15 Gly Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg 20 25 30 His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 35 40 45 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 50 55 60 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 65 70 75 80 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Arg Ser 85 90 95 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Lys 100 105 110 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 115 120 125 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 130 135 140 Gly Ala Gly Gly Ser Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 145 150 155 160 Gln Val Trp Ser Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 165 170 175 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 180 185 190 Ile Trp Val Asn Lys 195 1424DNAArtificial 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 Pro 1 5 10 15 Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr 20 25 30 Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His Asp 35 40 45 Asp Ala Glu Ala Leu Ser Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro 50 55 60 Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr 65 70 75 80 Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr 85 90 95 Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys 100 105 110 Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys Leu 115 120 125 Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Ser 130 135 140 Asn Arg Trp Gln Asp Ile Ser Gly Val Tyr Thr Ile Asp Ala Trp Thr 145 150 155 160 Gly Phe Asp Phe Pro Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp 165 170 175 Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu 180 185 190 Asn His Leu Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp 195 200 205 Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe 210 215 220 Ser His Pro Glu Val Gln Glu Glu Leu Lys Asp Trp Gly Ser Trp Phe 225 230 235 240 Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His 245 250 255 Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Ser Glu 260 265

270 Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val 275 280 285 Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu 290 295 300 Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Lys Gln Gly 305 310 315 320 Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala 325 330 335 His Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro 340 345 350 Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala 355 360 365 Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr 370 375 380 Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp 385 390 395 400 Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr 405 410 415 Gln His Asp Tyr Phe Asp His Trp Asp Ile Val Gly Trp Thr Arg Glu 420 425 430 Gly Thr Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn 435 440 445 Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Gln Gln His Ala Gly 450 455 460 Gln Thr Trp Thr Asp Leu Thr Gly Asn His Ala Ala Ser Val Thr Ile 465 470 475 480 Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser 485 490 495 Val Tyr Val Asn Gln 500 261745DNABacillus 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 -10 ttt 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 5 tat 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 Leu 10 15 20 25 cac 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 40 att 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 55 gca 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 70 cga 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 85 ctt 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 His 90 95 100 105 aaa 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 110 115 120 cca 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 125 130 135 tgg 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 140 145 150 aag 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 155 160 165 caa 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 Arg 170 175 180 185 gtg 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 190 195 200 gac 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 205 210 215 tgg 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 220 225 230 aaa 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 235 240 245 aac 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 Asp 250 255 260 265 gac 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 270 275 280 tct 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 285 290 295 caa 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 300 305 310 gaa 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 315 320 325 cag 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 Pro 330 335 340 345 ctt 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 350 355 360 ttt 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 365 370 375 aaa 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 380 385 390 ggc 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 395 400 405 agg 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 Met 410 415 420 425 tcg 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 430 435 440 gca 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 445 450 455 aca 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 460 465 470 gta tcc gtg tac gtg aac caa taacaaaaag ccttgagaag ggattcctcc 1722Val Ser Val Tyr Val Asn Gln 475 480 ctaactcaag gctttcttta tgt 174527501PRTBacillus species 27Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro -20 -15 -10 Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr -5 -1 1 5 10 Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His Asp 15 20 25 Asp Ala Ala Ala Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro 30 35 40 Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr 45 50 55 Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr 60 65 70 75 Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys 80 85 90 Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys Met 95 100 105 Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr 110 115 120 Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr 125 130 135 Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp 140 145 150 155 Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu 160 165 170 Asn His Ile Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp 175 180 185 Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe 190 195 200 Ser His Pro Glu Val Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe 205 210 215 Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His 220 225 230 235 Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu 240 245 250 Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val 255 260 265 Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu 270 275 280 Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly 285 290 295 Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala 300 305 310 315 His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro 320 325 330 Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala 335 340 345 Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr 350 355 360 Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp 365 370 375 Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr 380 385 390 395 Gln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp Thr Arg Glu 400 405 410 Gly Ser Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn 415 420 425 Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly 430 435 440 Gln Thr Trp Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile 445 450 455 Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser 460 465 470 475 Val Tyr Val Asn Gln 480 28501PRTBacillus species 28Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro 1 5 10 15 Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr 20 25 30 Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His Asp 35 40 45 Asp Ala Ala Ala Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro 50 55 60 Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr 65 70 75 80 Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr 85 90 95 Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys 100 105 110 Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys Met 115 120 125 Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr 130 135 140 Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr 145 150 155 160 Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp 165 170 175 Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu 180 185 190 Asn His Ile Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp 195 200 205 Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe 210 215 220 Ser His Pro Glu Val Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe 225 230 235 240 Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His 245 250 255 Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu 260 265 270 Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val 275 280 285 Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu 290 295 300 Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly 305 310 315 320 Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala 325 330 335 His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro 340 345 350 Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala 355 360 365 Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr 370 375 380 Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp 385 390 395 400 Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr 405 410 415 Gln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp Thr Arg Glu 420 425 430 Gly Ser Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn 435 440 445 Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly 450 455 460 Gln Thr Trp Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile 465 470 475 480 Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser 485 490 495 Val Tyr Val Asn Gln 500 291920DNABacillus 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 Pro 1 5 10 15 aat 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 30 gct 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 45 acg 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 60 ggg 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 Lys 65 70 75 80 gga 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 95 gtt 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 100 105 110 gaa 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 115 120 125 att 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 130 135 140 ggg 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 Phe 145 150 155 160 gac 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 165 170 175 ttt 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 180 185 190 tat 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 195 200 205 gca 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 210 215 220 ttg 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 Phe 225 230 235 240 ttg 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 245 250 255 ttt 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 260 265 270 tat 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 275 280 285 cat 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 290 295 300 agg 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 Ser 305 310 315 320 gtt 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 325 330 335 tcg 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 340 345 350 aca 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 355 360 365 acg 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 370 375 380 gaa 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 His 385 390 395 400 gat 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 405 410 415 agc 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 420 425 430 ggt 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 435 440 445 tgg 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 450 455 460 gaa 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 Tyr 465 470 475 480 gtt caa aga tag aagagcagag aggacggatt tcctgaagga aatccgtttt 1912Val Gln Arg tttatttt 192030483PRTBacillus licheniformis 30Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30 Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn 85 90 95 Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160 Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175 Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200 205 Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215 220 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu 275 280 285 His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser 305 310 315 320 Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380 Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445 Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455 460 Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475 480 Val Gln Arg

Claims

1. A variant of an alpha-amylase having at least 60% homology to SEQ ID NO. 8, comprising 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, 239, 250, 280, 298, 318, 374, 385, 393, 402, 406, 427, 430, 440, 444, 447, and 482, wherein (a) the alteration(s) are independently (i) an insertion of an amino acid downstream of the amino acid which occupies the position, (ii) a deletion of the amino acid which occupies the position, or (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 alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8.

2. The variant of claim 1, which variant has one or more of the following mutations: T49I; 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 using SEQ ID NO: 8 for the numbering.

3. The variant of claim 1, wherein the variant has the following mutations: G48A+T49I; G48A+T49I+G107A; G48A+T49I+G107A+I201F; G48A+T49I+I201F; T49I+G107A; T49I+G107A+I201F; T49I+E132V+V440A; T49I+K176R+D207V+Y402F; T49I+I201F; D60N+D207V+L318M; N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W; N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N- 444K+E447Q+Q482K; E132A+D207V; N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N; N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N- 444R+N444K+E447K+Q482K; N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E- 447Q+Q482K; N104D+D161N+A181N+D183N+D200N+D204S; N104D+D161N+A181N+D183N+D200N+D204S+K237P; N104D+D161N+A181N+D183N+D200N+D204S+K237P+S239W; D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W; D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N; D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E- 447Q+Q482K; D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444R+N- 444K+E447K+Q482K; D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q- 482K; D161N+A181N+D183N+D200N+D204S; D161N+A181N+D183N+D200N+D204S+K237P; D161N+A181N+D183N+D200N+D204S+K237P+S239W; D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K; D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K; K170Q+D207V+N280S; N190F+A209V+Q264S; D207E+E250G+H406L+L427I; D207V+L318M; K237P+S239W, Q374R+E385V+Q393R; H406W+D430N; N444K+E447K+Q482K; N444K+E447Q+Q482K; E447K+Q482K; E447Q+Q482K; using SEQ ID NO: 8 for numbering.

4. The variant of claim 1, wherein the parent alpha-amylase is derived from a strain of B. licheniformis (SEQ ID NO: 8), B. amyloliquefaciens (SEQ ID NO: 10), or B. stearothermophilus (SEQ ID NO: 6).

5. The variant of claim 1, wherein the parent alpha-amylase is any of: LE174; LE174+G48A+T49I+G107A+M197L+I201F; LE174+G48A+T49I+G107A+I201F; or LE174+M197L.

6. The variant of claim 1, wherein the variant is mutated in one or more of the following positions: T511; D62N; N106D; D134A,V,P; D163N; X172Q; K179R; G184N; K185T; A186N; D188N; D205N; M208Y; D209S; 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 for numbering.

7. The variant of claim 1, wherein the variant has the following mutations: T51I+D134V+V442A; T511+K179R+E212V+Y404F; D62N+E212V+X323M; D134A+E212V; N195F+X212V+K269S, E212V+N285S; E212V+X323M; N255G+H408L+X4291; D387V+N395R; using SEQ ID NO: 4 for numbering.

8. The variant of claim 1, wherein the parent alpha-amylase is selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 12; SEQ ID NO: 13; or KSM-AP1378.

9. The variant of claim 1, wherein the parent alpha amylase is any of: SEQ ID NO: 4+D183*+G184*; SEQ ID NO: 4+D183*+G184*+N195F; SEQ ID NO: 4+D183*+G184*+N195F+M202L; SP722+D183*+G184*+M202L; SEQ ID NO: 6+1181*+G182*; SEQ ID NO: 6+1181*+G182*+N193F; SEQ ID NO: 6+1181*+G182*+N193F+M200L; SEQ ID NO: 6+1181*+G182*+M200L; SEQ ID NO: 12+D183*+G184* SEQ ID NO: 12+D183*+G184*+N195F; SEQ ID NO: 12+D183*+G184*+N195F+M202L; and SEQ ID NO: 12+D183*+G184*+M202L.

10. The variant of claim 1, wherein the parent alpha-amylase has an amino acid sequence which has a degree of identity to SEQ ID NO: 8 of at least 70%, more preferably at least 80%, even more preferably at least about 90%, even more preferably at least 95%, even more preferably at least 97%, and even more preferably at least 99%.

11. The variant of claim 1, wherein the parent alpha-amylase is encoded by a nucleic acid sequence, which hybridizes under low, preferably medium, preferred high stringency conditions, with the nucleic acid sequence of SEQ ID NO: 7.

12. The variant of claim 1, which variant has altered stability, in particular at high temperatures from 70-120.degree. C. and/or low pH in the range from pH 4-6.

13. A composition comprising an alpha-amylase variant of claim 1.

14. The composition of claim 13, further comprising a B. stearothermophilus alpha-amylase, particular in a ratio of 1:10 to 10:1, preferably 1:2.

15. The composition of claim 13, wherein the composition further comprises a glucoamylase, pullulanase and/or a phytase.

16. A detergent composition comprising an alpha-amylase variant of claim 1 and a surfactant.

17. A detergent composition of claim 16, which additionally comprises one or more other enzymes selected from the group consisting of lipase, peroxidase, protease, another amylolytic enzyme, glucoamylase, maltogenic amylase, CGTase, mannanase, cutinase, laccase and/or a cellulase.

18. A method for liquefying starch, comprising treating the starch with an alpha-amylase variant of claim 1.

19. A method for producing ethanol, comprising liquefying starch with an alpha-amylase variant of claim 1, hydrolyzing the liquefied starch with a glucoamylase, and fermenting the hydrolyzed starch.

20. A method for desizing a textile, comprising treating a sized textile with an alpha-amylase variant of claim 1.

21. A DNA construct comprising a DNA sequence encoding an alpha-amylase variant of claim 1.

22. A recombinant expression vector which carries a DNA construct according to claim 21.

23. A cell which is transformed with a DNA construct according to claim 21.

24. The cell of claim 23, which is a microorganism, preferably a bacterium or a fungus.

25. The cell of claim 24, which cell is a gram-positive bacterium, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis.

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