WO2000060060A2 - Polypeptides having alkaline alpha-amylase activity and nucleic acids encoding same - Google Patents

Abstract

The present invention relates to isolated polypeptides having alpha-amylase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.

Description

POLYPEPTIDES HAVING ALKALINE ALPHA-AMYLASE ACTIVITY AND NUCLEIC ACIDS ENCODING SAME

Background of the Invention

Field of the Invention

The present invention relates to isolated polypeptides having alpha-amylase activity and isolated nucleic acid sequences encoding the polypeptides. Further, the invention relates to variants of the alpha-amylase of the invention. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides. Further, the invention also relates to compositions for laundry, dish wash and/or hard surface cleaning .

Description of the Related Art

For a number of years alpha-amylase enzymes have been used for a variety of different purposes, the most important of which are starch liquefaction, textile desizmg, starch modification m the paper and pulp industry, and for brewing and baking. A further use of alpha-amylases , which is becoming increasingly important, is the removal of starchy stains during washing with a detergent at alkaline pH .

Examples of commercial alpha-amylase products are Termamyl™, Duramyl™, Natalase , BAN™ and Fungamyl™, all available from Novo Nordisk A/S, Denmark. These and similar products from other commercial sources have an acidic to a neutral pH optimum, typically m the range of from pH 5 to pH 7.5, and they do not display opmmal activity m detergent solutions at alkaline pH .

WO 95/26397 discloses an alpha-amylase from a Bacillus strain. WO 96/23873 describes variants of Bacillus amylases with improved performance under washing conditions. US 5,147,796 describe an alkaline pullulanase having alpha-amylase activity. Fig. 2b of the document shows optimum amylase activity at pH 8-8.5. . Takagi et al . , J. Ferment. Bioeng., vol 81, No. 6, 557-

559 (1996) describes an alkaliphilic alpha-amylase-pullulanase from Bacillus s . The enzyme has optimum amylase activity at pH

9, but the activity drops rapidly at higher pH, and the activity at pH 10 is lower than at pH 7.

WO 97/00324 (KAO) discloses a gene encoding an alkaline liquefying alpha-amylase derived from Bacillus sp . strain KSM- AP1378 with the deposited no. FERM BP-3048 suitable for detergents .

Tsukamoto et . al . , (1988), Biochem. Biophys . Res Commun. 151, p. 25-33) disclose an alkaline alpha-amylase from Bacillus sp. #707. It is an object of the present invention to provide novel alpha-amylases with altered performance, m particular with improved performance m alkaline solutions, especially m alkaline detergent solutions at pH around 9-11.

Summary of the Invention

The present invention relates to isolated polypeptides having alpha-amylase activity and one or more characteristics or properties selected from the group consisting of:

(a) a polypeptide having an ammo acid sequence which has at least 96% identity with ammo acids 1 to 485 of SEQ ID NO : 2 or SEQ ID NO: 4; (b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (1) the nucleic acid sequence of SEQ ID NO : 1 or SEQ ID NO: 3, (n) the cDNA sequence of SEQ ID NO : 1 or SEQ ID NO: 3, (in) a subsequence of (I) or (n) of at least 100 nucleotides, or (IV) a complementary strand of (l) , (n) , or (in) ;

(c) an allelic variant of (a) or (b) ;

(d) a fragment of (a) , (b) or (c) that has alpha-amylase activity; (e) a polypeptide with a pH optimum determined using the Phadebas method (37°C) m the range between pH 8 and 9 ;

(f) a polypeptide with a temperature optimum determined using the Phasebas method (pH 9.0) m the range between 55 and 65°C; (g) a polypeptide with a pi between 7-8 determined by isoelectric focusing (Pharmacia, Ampholme, pH 3.5-9.3); and

(l) a polypeptide with improved wash and/or dishwash performance between pH 9-11.

It is to be understood that the alpha-amylase of the invention may have one or more of the above characteristics.

Further, the invention relates to variants of the alpha- amylase of the invention. The present invention also relates to isolated nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.

Brief Description of the Figures

Figure 1 shows an alignment of a number of Bacillus alpha- amylases .

Figure 2 shows the pH Profile of the AA560 alpha-amylase compared to the SP722 and SP690 alpha-amylases . The pH profile was measured at 37°C. The activity is shown m absolute values as Abs650/mg enzyme.

Figure 3 shows the Temperature Profile of the AA560 alpha- amylase compared to the SP722 and SP690 alpha-amylases. The temperature profile is shown as Abs650/mg enzyme.

Figure 4 shows the wash performance of AA560 alpha-amylase m the AP Model Detergent 97 m comparison to SP722, SP690 and Termamyl^®, respectively. Figure 5 shows the wash performance of AA560 m the Omo Multi Acao m comparison to SP722, SP690 and Termamyl^®, repsectively .

Figure 6 shows the wash performance of AA560 the Omo Concentrated m comparison to SP722, SP690 and Termamyl^®, respectively.

Figure 7 shows the wash performance of AA560 the Ariel Futur liquid m comparison to SP722, SP690 and Termamyl^®, repsectively.

Detailed Description of the Invention Microbial source

The alkaline alpha-amylase of the invention may be derived from a strain of Bacillus . Preferred strains are of Bacillus sp . DSM 12649 (the AA560 alpha-amylase) or Bacillus sp . DSM 12648 (the AA349 alpha-amylase) . These strains were deposited on 25^th January 1999 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 und Zellkulturen GmbH (DSMZ) , Mascheroder Weg lb, D-38124 Braunschweig DE .

Two Escherichia coll strains termed NN049467 and NN049470 containing the alpha-amylase genes cloned m plasmids pLιH1274 and pTVB299, respectively, have also been deposited on 7^th April 1999 under the terms of the Budapest Treaty with the Deutshe Sammmlung von Microorganismen und Zellkulturen GmbH (DSMZ) , Mascheroder Weg lb, D-38124 Braunschweig DE, and given the accession numbers DSM12761 and DSM12764, respectively.

Polypeptides Having Alpha-amylase Activity

Alpha-amylases (alpha- 1 , 4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes, which catalyze hydrolysis of starch and other linear and branched 1 , 4-glucosιdιc oligo- and polysaccharides . For purposes of the present invention, alpha-amylase activity is determined using the Phadebas assay or the pNPG7 assay described below m the "Materials and Methods" section.

Homology of Enzyme

In a first embodiment, the present invention relates to isolated polypeptides having an ammo acid sequence which has a degree of homology to ammo acids 1 to 485 of SEQ ID NO : 2 or SEQ ID NO: 4 (i.e., the mature polypeptide) of at least about 96%, preferably at least about 97%, more preferably at least about 98%, even more preferably at least about 99%, which have alpha-amylase activity (hereinafter "homologous polypeptides"). In a preferred embodiment, the homologous polypeptides have an ammo acid sequence which differs by five ammo acids, preferably by four ammo acids, more preferably by three ammo acids, even more preferably by two ammo acids, and most preferably by one ammo acid from ammo acids 1 to 485 of SEQ ID NO: 2 or SEQ ID NO : 4. It is to be noted that SEQ ID NO : 2 and SEQ ID NO: 4 are identical. However, the DNA sequences, i . e . , SEQ ID NO: 1 and SEQ ID NO: 3, respectively, encoding the alpha-amylase of the invention shown m SEQ ID NO: 2 and SEQ ID NO: 4 are not identical.

The ammo acid sequence homology may be determined as the degree of homology 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. Thus, GAP provided m GCG version 8 (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453) may be used for a pairwise alignment of the sequences and calculation of the degree of identity or degree of homology using the default settings. The gap settings may be the following parameters: gap creation penalty of 5.0 and gap extension penalty of 0.3.

Homology to known Bacillus sp . Alpha-Amylases

A homology search of known sequences showed homologies for the sequences of the invention with a number of Bacillus amylases m the range 65-95 % on ammo acid basis determined as described above.

Specifically, the most homologous alpha-amylases found are SP690 (SEQ ID NO: 1 of US patent no. 5,856,164 which is about 87% homologous), SP722 (SEQ ID NO: 2 of US patent no. 5,856,164 which is about 87% homologous), the mature part (i.e., ammo acids no. 31-516) of the alpha-amylase obtained from Bacillus sp. KSM-AP1378 disclosed as SEQ ID NO : 2 of WO 97/00324 which is about 86% homologous, and the alpha-amylase disclosed m Tsukamoto et . al . , (1988), Biochem. Biophys . Res Commun . 151, p. 25-33) (shown as sequence "707 amy" the alignment m fig. 1) which is about 95% homologous to SEQ ID NO: 2 and SEQ ID NO: 4 determined as describe above . Preferably, the polypeptides of the present invention comprise the ammo acid sequence of SEQ ID NO : 2 or SEQ ID NO:

4 or allelic variants thereof; or fragments thereof that has alpha-amylase activity. SEQ ID NO: 2 and SEQ ID NO: 4 show the mature part of the alkaline alpha-amylases of the invention.

A fragment of SEQ ID NO : 2 or SEQ ID NO: 4 are polypeptides having one or more ammo acids deleted from the ammo and/or carboxyl terminus of this ammo acid sequence.

An allelic variant denotes any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result m polymorphism within populations. Gene mutations can be silent

(no change the encoded polypeptide) or may encode polypeptides having altered ammo acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene .

The ammo acid sequences of the homologous polypeptides may differ from the ammo acid sequence of SEQ ID NO : 2 or SEQ

ID NO: 4 by an insertion or deletion of one or more ammo acid residues and/or the substitution of one or more ammo acid residues by different ammo acid residues. Preferably, ammo acid changes are of a minor nature, that is conservative ammo acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 ammo acids; small ammo- or carboxyl -terminal extensions, such as an ammo-terminal methion e residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidme tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine) , acidic amino acids (glutamic acid and aspartic acid) , polar amino acids (glutamine and asparagine) , hydrophobic amino acids (leucine, isoleucine and valine) , aromatic amino acids (phenylalanine, tryptophan and tyrosine) , and small amino acids (glycine, alanine, serine, threonine and methionine) . Amino acid substitutions, which do not generally alter the specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins , Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val , Ala/Glu, and Asp/Gly as well as these in reverse.

In a second embodiment, the present invention relates to isolated polypeptides having alpha-amylase activity which are encoded by nucleic acid sequences which hybridize under medium stringency conditions, preferably medium-high stringency conditions, more preferably high stringency conditions, and most preferably very high stringency conditions with a nucleic acid probe which hybridizes under the same conditions with (i) the nucleic acid sequence of SEQ ID NO : 1 or SEQ ID NO : 3, (ii) the cDNA sequence of SEQ ID NO : 1 or SEQ ID NO: 3, (iii) a subsequence of (i) or (ii) , or (iv) a complementary strand of (i) , (ii) , or (iii) (J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Labora tory Manual , 2d edition, Cold Spring Harbor, New York) . The subsequence of SEQ ID NO:l may be at least 100 nucleotides or preferably at least 200 nucleotides. Moreover, the subsequence may encode a polypeptide fragment, which has alpha-amylase activity. The polypeptides may also be allelic variants or fragments of the polypeptides that have alpha-amylase activity. The nucleic acid sequence of SEQ ID NO : 1 or SEQ ID NO: 3 or a subsequence thereof, as well as the ammo acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having alpha-amylase activity from strains of different genera or species according to methods well known m the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, m order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, and more preferably at least 35 nucleotides m length. Longer probes can also be used. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ^3SS, biotm, or avidm) . Such probes are encompassed by the present invention.

Thus, a genomic DNA or cDNA library prepared from such other organisms may be screened for DNA, which hybridizes with the probes described above and which encodes a polypeptide having alpha-amylase activity. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis , or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material . In order to identify a clone or DNA which is homologous with SEQ ID NO : 1 or SEQ ID NO : 3 or subsequences thereof, the carrier material is used m a Southern blot.

For purposes of the present invention, hybridization indicates that the nucleic acid sequence hybridizes to a nucleic acid probe corresponding to the nucleic acid sequence shown in SEQ ID NO : 1 or SEQ ID NO: 3, its complementary strand, or subsequences thereof, under medium to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions are detected using X-ray film.

In another preferred embodiment, the nucleic acid probe is the nucleic acid sequence contained in plasmids pLiH1274 (AA349) or pTVB299 (AA560) which are contained in Escheri chia coli DSM12761 or Escherichia coli DSM12764, respectively, or, wherein the nucleic acid sequence encodes a polypeptide having acid alpha-amylase activity of the invention and shown in SEQ ID NO: 2 and SEQ ID NO : 4, respectively.

For long probes of at least 100 nucleotides in length, medium to very high stringency conditions are defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micro g/ml sheared and denatured salmon sperm DNA, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures. For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2 x SSC, 0.2% SDS preferably at least at 55°C

(medium stringency), preferably at least at 60°C (medium-high stringency) , more preferably at least at 65 °C (high stringency) , and most preferably at least at 70°C (very high stringency) .

For short probes which are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at 5°C to 10°C below the calculated T,. using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, IX Denhardt ' s solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures.

For short probes, which are about 15 nucleotides to about 70 nucleotides m length, the carrier material is washed once m 6X SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6X SSC at 5°C to 10°C below the calculated T_m. In a third embodiment, the present invention relates to isolated polypeptides, i.e., the polypeptides shown m SEQ ID NO: 2 or SEQ ID NO: 4, having the following physicochemical properties :

A pH optimum (see Fig. 2) determined using the Phadebas method (37°C) was found to be m the range between pH 8 and 9, more precisely at about 8.5.

A temperature optimum (See Fig. 3) determined using the Phasebas method (pH 9.0) was found to be the range between 55 and 65°C, more precisely about 60°C. A pi between 7-8 (See Table 1 m Example 6) was determined by isoelectric focusing (Pharmacia, Amphol e, pH 3.5-9.3).

A specific activity (see Table 1 of Example 6) of 35,000 NU/mg was determined using the Phadebas method and 6,000 NU/mg using the pNPG7 method. The polypeptides of the present invention have at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, even more preferably at least 90%, and most preferably at least 100% of the alpha- amylase activity of the mature polypeptide shown m SEQ ID NO: 2 and SEQ ID NO: 4.

A polypeptide of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by the nucleic acid sequence is produced by the source or by a cell in which the nucleic acid sequence from the source has been inserted.

A polypeptide of the present invention is a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus polypeptide, e . g . , a Bacillus alkalophilus, Bacillus amyloliquefaci ens , Bacillus brevis , Bacillus circulans , Bacillus coagulans , Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus , Bacillus subtilis, or Bacillus thuringi ensis polypeptide; or a Strep tomyces polypeptide, e . g. , a Streptomyces lividans or

Strepto yces murinus polypeptide; or a gram negative bacterial polypeptide, e . g. , an E. coli or a Pseudomonas s . polypeptide.

In another preferred embodiment, the polypeptide is a

Bacillus sp . polypeptide, more preferred embodiment, the polypeptide is a Bacillus s . DSM 12648 or Bacillus sp . DSM 12649 polypeptide, e . g. , the polypeptides with the amino acid sequence of SEQ ID NO : 2 and SEQ ID NO : 4, respectively.

It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC) , Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) , Centraalbureau Voor Schimmelcultures (CBS) , and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL) . Furthermore, such polypeptides may be identified and obtained from other sources including microorganisms isolated from nature { e . g. , soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known m the art. The nucleic acid sequence may then be derived by m a similar manner screening a genomic or cDNA library of another microorganism. Once a nucleic acid sequence encoding a polypeptide has been detected with the probe (s) , the sequence may be isolated or cloned by utilizing techniques which are known to those of ordinary skill m the art (see, e.g., Sambrook et al . , 1989, supra) .

As defined herein, an "isolated" polypeptide is a polypeptide which is essentially free of other non-alpha- amylase polypeptides, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably about 60% pure, even more preferably about 80% pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS- PAGE.

Polypeptides encoded by nucleic acid sequences of the present invention also include fused polypeptides or cleavable fusion polypeptides m which another polypeptide is fused at the N-termmus or the C-termmus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleic acid sequence (or a portion thereof) encoding another polypeptide to a nucleic acid sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known m the art, and include ligatmg the coding sequences encoding the polypeptides so that they are m frame and that expression of the fused polypeptide is under control of the same promoter (s) and terminator. The term "improved wash performance" means m the context of the present invention a performance determined under the washing conditions described Example 8, which is higher than other alpha-amylase used for washing, e.g., SP690, SP722 and Termamyl^®, or m the case of a mutant/variant of the invention m comparison to the parent alpha-amylase, i.e., the un- mutated, such as un-substituted alpha-amylase backbone.

Mutant Alpha-Amylases

Altered properties of AA560 variants

The following discusses the relationship between mutations, especially substitutions and deletions, which may be introduced into the AA560 or A349 alpha-amylases of the invention, and desirable alterations m properties relative to those of a parent alpha-amylase.

Invention also relates to a mutant of the alpha- amylases (i.e., alpha-amylase variants) shown m SEQ ID NO: 2 or SEQ ID NO: 4. The mutant alpha-amylase of to the invention is characterized by the fact that one or more of the Methionme ammo acid residues is exchanged with any ammo acid residue except for Cys and Met. Thus, according to the invention the ammo acid residues to replace the methionme ammo acid residue are the following: Ala, Arg, Asn, Asp, Gin, Glu, Gly, His, lie, Leu, Lys , Phe , Pro, Ser, Thr, Trp, Tyr, and Val . A preferred embodiment of the mutant alpha-amylase of the invention is characterized by the fact that one or more of the Methionme ammo acid residues is (are) exchanged with a Leu, Thr, Ala, Gly, Ser, lie, or Val ammo acid residue, preferably a Leu, Thr, Ala, or Gly ammo acid residue. In this embodiment a very satisfactory activity level and stability m the presence of oxidizing agents is obtained. Specifically this means that one or more of the Methionmes m the following position may be replaced or deleted using any suitable technique known m the art, including especially site directed mutagenesis and gene shuffling. Contemplated position, using the SEQ ID NO: 2 numbering, are: 9, 10, 105, 116, 202, 208, 261, 309, 323, 382, 430, 440.

In a preferred embodiment of the mutant alpha-amylase of the invention is characterized by the fact that the Methionme ammo acid residue at position 202 is exchanged with any of ammo acid residue expect for Cys and Met, preferably with a Leu, Thr, Ala, Gly, Ser, lie, or Asp.

Other contemplated preferred mutations include deletion of one, two or more residues of ammo acids R181, G182, D183 or G184, K185, G186 or substitution of one or more of these residues. A preferred mutation is the deletion of D183-G184. Particularly relevant mutations are substitutions of G186 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. A particularly preferred substitution is G186R. Also contemplated is substitution of N195 with Ala,

Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. A particularly interesting substitution is N195F.

The following combinations of the above mentioned mutations include: deletion of (D183-G184 ) +N195F; deletion of (D183-G184) +G186R; deletion of (D183 -G184 ) +G186R+N195F, and G186R+N195F.

Increased thermostability

Mutations resulting m variants of the invention, e.g., having increased thermostability, m particular at acidic pH and/or at low Ca²⁺ concentration include mutations at the following positions (using the AA560 alpha-amylase numbering, i.e. , SEQ ID NO: 2) :

R158, N174, G186, N195, N197, H210, E212, V214, V215, E216, K269, N270.

In the context of the invention the term "acidic pH" means a pH below 7.0, especially below the pH range, which industrial starch liquefaction processes are normally performed, which is between pH 5.5 and 6.2. In the context of the present invention the term "low Calcium concentration" means concentrations below the normal level used m industrial starch liquefaction. Normal concentrations vary depending of the concentration of free Ca²" m the corn. Normally a dosage corresponding to 1 mM (40ppm) is added which together with the level m corn gives between 40 and 60ppm free Ca²⁺ .

In the context of the invention the term "high temperatures" means temperatures between 95°C and 160°C, especially the temperature range which industrial starch liquefaction processes are normally performed, which is between 95°C and

105°C.

The inventors have now found that the thermostability, m particular at acidic pH and/or at low Ca²⁺ concentration can be increased even more by combining other mutations including the above-mentioned mutations and/or 1206 with each other.

Said "other" mutations are the following (relative to the AA560 alpha-amylase, SEQ ID NO : 2) : N195, E212, E216, K269 and 1206.

Said mutation may further be combined with deletions in one, preferably two or even three positions as described in WO 96/23873 (i.e., in positions R181, G182, D183, G184 in SEQ ID NO: 2 herein) .

According to the present invention variants of a parent AA560 alpha-amylase with alpha-amylase activity comprise mutations in two, three, four, five, six or seven of the above positions are contemplated.

It should be emphasized that not only the AA560 alpha- amylases mentioned are contemplated. Also alpha-amylases having a degree of homologous (identical) as defined below are contemplated to be within the scope of the present invention. It may be mentioned here that amino acid residues, respectively, at positions corresponding to N195, 1206, E212 and E216, respectively, in SEQ ID NO: 2 constitute amino acid residues, which are conserved in numerous Termamyl-like alpha- amylases, i.e., Termamyl^® (B . licheniformis alpha-amylase). Thus, for example, the corresponding positions of residues in AA560 and Termamyl can be see in the alignment in Fig. 1 and in Table 1 and Table 2, below.

Table 1

Termamyl-like alpha-amylase

B . licheniformis (Termamyl) N190 1201 H205 E211 N265

AA560 (SEQ ID NO: 5) N195 1206 H210 E211 N270 Table 2

Termamyl-like alpha-amylase

SP690 R181, G182, T183, G184, K185, A186 SP722 R181, G182, D183, G184, K185, A186 AA560 (SEQ ID NO : 2) R181, G182, D183, G184, K185, G186

Mutations of these conserved amino acid residues are very important in relation to alter properties.

When using SEQ ID NO: 2 for numbering two, three, four, five, six or seven mutations may according to the invention be made in the following positions to alter the properties, in particular to increase the thermostability at acidic pH and/or at low Ca²⁺ concentrations (relative to SEQ ID NO: 2 herein) :

1: R181*, G182*, D183*, G184*;

2: N195A,R,D,C,E,Q,G,H, I,L,K,M,F,P,S,T,W,Y,V;

3: I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V;

4: E212A,R,D,N,C,Q,G,H, I,L,K,M,F,P,S,T,W,Y,V; 5: E216A,R,D,N,C,Q,G,H, I , L, K, M, F, P, S , T, W, Y, V;

6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

7: R181A,N,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V.

Contemplated according to the present invention is combining three, four, five, six or seven mutations. Specific double mutations are according to the invention

(using SEQ ID NO : 2 for the numbering) :

-R181*/G182*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

-G182*/T183*/N195A,R,D,C,E,Q,G,H, I,L,K,M,F,P,S,T,W,Y,V;

-T183*/G184*/N195A,R,D,C,E,Q,G,H, I,L,K,M,F,P,S,T,W,Y,V; -T183*/G184*/R181A,N,D,C,E,Q,G,H, I , L, K, M, F, P, S , T, W, Y, V;

-R181*/G182*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W, Y,V;

-G182*/T183*/I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V; -T183*/G184*/I206A,R,D K,M,F,P,S,T,W,Y,V -R181*/G182*/E212A,R,D K,M,F,P,S,T,W,Y,V -G182*/T183*/E212A,R,D K,M,F,P,S,T,W,Y,V -T183*/G184*/E212A,R,D K,M,F,P,S,T,W,Y,V -R181*/G182*/E216A,R,D K,M,F,P,S,T,W,Y,V -G182*/T183*/E216A,R,D K,M,F,P,S,T,W,Y,V -T183*/G184*/E216A,R,D K,M,F,P,S,T,W,Y,V -R181*/G182*/K269A,R,D L,M,F,P,S,T,W,Y,V -G182*/T183*/K269A,R,D L,M,F,P,S,T,W,Y,V -T183*/G184*/K269A,R,D L,M,F,P,S,T,W,Y,V -N195A,R,D,C,E,Q,G,H,I W,Y,V /I206A,R,D,N,C,E,Q,G,H W,Y,V; -N195A,R,D,C,E,Q,G,H, I W,Y,V /E212A,R,D,N,C,Q,G,H,I W,Y,V; -N195A,R,D,C,E,Q,G,H,I W,Y,V /E216A,R,D,N,C,Q,G,H,I W,Y,V; -N195A,R,D,C,E,Q,G,H, I W,Y,V /K269A,R,D,N,C,E,Q,G,H W,Y,V; -I206A,R,D,N,C,E,Q,G,H W,Y,V /E212A,R,D,N,C,Q,G,H,I W,Y,V; -I206A,R,D,N,C,E,Q,G,H W,Y,V /E216A,R,D,N,C,Q,G,H,I W,Y,V; -I206A,R,D,N,C,E,Q,G,H W,Y,V /K269A,R,D,N,C,E,Q,G,H W,Y,V; -E212A,R,D,N,C,Q,G,H,I W,Y,V /E216A,R,D,N,C,Q,G,H, I W,Y,V; E212A,R,D,N,C,Q,G,H, I,: ,Y,V /K269A,R,D,N,C,E,Q,G,H W,Y,V; -E216A,R,D,N,C,Q,G,H,I W,Y,V /K269A,R,D,N,C,E,Q,G,H

W,Y,V.

In a preferred embodiment the variant comprises the following mutations: N195F/K264S SEQ ID NO: 2 or corresponding positions m 96% homologous alpha-amylases as defined herein. In another embodiment the variant of the invention comprises the following mutations: R181*/G182*/N195F SEQ ID NO: 2 or m corresponding positions m another homologous alpha-amylases. Said variant may further comprise a substitution m position E216Q.

Improved Ca²⁺ stability of AA560 variant at pH 8-10.5 Improved Ca²⁺ stability means the stability of the enzyme under Ca²⁺ depletion has been improved. In the context of the present invention, mutations (including ammo acid substitutions and deletions) of importance, with respect to achieving improved Ca²⁺ stability at high pH, include mutations and/or deletions disclosed above the section "increased thermostability".

General mutations of the invention

It may be preferred that a variant of the invention comprises one or more modifications addition to those outlined above. Thus, it may be advantageous that one or more prolme residues present m the part of the alpha-amylase variant which is modified is/are replaced with a non-prolme residue which may be any of the possible, naturally occurring non-prolme residues, and which preferably is an alanine, glyc e, serme, threonme, val e or leucme.

Analogously, it may be preferred that one or more cysteme residues present among the ammo acid residues with which the parent alpha-amylase is modified is/are replaced with a non- cysteme residue such as ser e, alanine, threonme, glycme, val e or leucme.

Furthermore, a variant of the invention may - either as the only modification or m combination with any of the above outlined modifications - be modified so that one or more Asp and/or Glu present m an ammo acid fragment corresponding to the ammo acid fragment 190-214 of SEQ ID NO: 2 is replaced by an Asn and/or Gin, respectively. Also of interest is the replacement, m the alpha-amylase, of one or more of the Lys residues present m an am o acid fragment corresponding to the ammo acid fragment 190-214 of SEQ ID NO : 2 by an Arg.

It will be understood that the present invention encompasses variants incorporating two or more of the above outlined modifications .

Furthermore, it may be advantageous to introduce point mutations m any of the variants described herein.

Mutations may suitably include mutations m the following positions: Y133, L17, M202, V214.

Cloning a DNA sequence encoding an alpha-amylase

The DNA sequence encoding a parent alpha-amylase as defined above may be isolated from any cell or microorganism producing the alpha-amylase m question, using various methods well known m 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 am o acid sequence of the alpha-amylase is known, homologous, labelled oligonucleotide probes may be synthesized and used to identify alpha-amylase-encodmg clones from a genomic library prepared from the organism m question. Alternatively, a labelled oligonucleotide probe containing sequences homologous to a known alpna-amylase gene could be used as a probe to identify alpha-amylase-encodmg clones, using hybridization and washing conditions of lower stringency. Yet another method for identifying alpha-amylase-encodmg 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.

Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoroamidite method described by S.L. Beaucage and M.H. Caruthers (1981) or the method described by Matthes et al . (1984) . In the phosphoroamidite method, oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors. 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 US 4,683,202 or R.K. Saiki et al. (1988) .

Site-directed mutagenesis

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 smgle-stranded gap of DNA, bridging the alpha-amylase-encodmg 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 re- maming gap is then filled m with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase. A specific example of this method is described m Mormaga et al . (1984). US 4,760,025 discloses the introduction of oligonucleotides encoding multiple mutations by performing minor alt- erations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the Mormaga method, because a multitude of oligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into alpha-amylase- encoding DNA sequences is described m 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 m 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.

Random Mutagenesis

Random mutagenesis is suitably performed either as localised or region-specific random mutagenesis m at least three parts of the gene translating to the ammo acid sequence shown question, or within the whole gene.

The random mutagenesis of a DNA sequence encoding a parent alpha-amylase may be conveniently performed by use of any method known the art.

In relation to the above, a further aspect of the present invention relates to a method for generating a variant of a parent alpha-amylase, e.g. wherein the variant exhibits altered or increased thermal stability relative to the parent, the method comprising: (a) subjecting a DNA sequence encoding the parent alpha- amylase to random mutagenesis,

(b) expressing the mutated DNA sequence obtained in step (a) in a host cell, and

(c) screening for host cells expressing an alpha-amylase variant which has an altered property (e.g., thermal stability) relative to the parent alpha-amylase.

Step (a) of the above method of the invention is preferably performed using doped primers.

For instance, the random mutagenesis may be performed by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the random mutagenesis may be performed by use of any combination of these mutagenizing agents. The mutagenizing agent may, e.g., be one that induces transitions, transversions, inversions, scrambling, deletions, and/or insertions.

Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N¹ -nitro-N-nitrosoguanidine (MNNG) , O- methyl hydroxylamine, nitrous acid, ethyl methane sulphonate

(EMS), sodium bisulphite, formic acid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed by incubating the DNA sequence encoding the parent enzyme to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions for the mutagenesis to take place, and selecting for mutated DNA having the desired properties .

When the mutagenesis is performed by the use of an oligonucleotide, the oligonucleotide may be doped or spiked with the three non-parent nucleotides during the synthesis of the oligonucleotide at the positions which are to be changed. The doping or spiking may be done so that codons for unwanted am o acids are avoided. The doped or spiked oligonucleotide can be incorporated into the DNA encoding the alpha-amylase enzyme by any published technique, using e.g. PCR, LCR or any DNA polymerase and ligase as deemed appropriate.

Preferably, the doping is carried out using "constant random doping", m which the percentage of wild-type and mutation each position is predefined. Furthermore, the doping may be directed toward a preference for the introduction of certain nucleotides, and thereby a preference for the introduction of one or more specific ammo acid residues. The doping may be made, e.g., so as to allow for the introduction of 90% wild type and 10% mutations m each position. An additional consideration m the choice of a doping scheme is based on genetic as well as protein-structural constraints. The doping scheme may be made using the DOPE program (see "Material and Methods" section) , which, inter alia, ensures that introduction of stop codons is avoided.

When PCR-generated mutagenesis is used, either a chemically treated or non-treated gene encoding a parent alpha-amylase is subjected to PCR under conditions that increase the mis- mcorporation of nucleotides (Deshler 1992; Leung et al . , Technique, Vol .1 , 1989, pp. 11-15).

A mutator strain of E. coli (Fowler et al . , Molec . Gen. Genet., 133, 1974, pp. 179-191), S . cereviseae or any other microbial organism may be used for the random mutagenesis of the DNA encoding the alpha-amylase by, e.g., transforming a plasmid containing the parent glycosylase into the mutator strain, growing the mutator strain with the plasmid and isolating the mutated plasmid from the mutator strain. The mutated plasmid may be subsequently transformed into the expression organism.

The DNA sequence to be mutagenized may be conveniently present m a genomic or cDNA library prepared from an organism expressing the parent alpha-amylase. Alternatively, the DNA sequence may be present on a suitable vector such as a plasmid or a bacteπophage, which as such may be incubated with or otherwise exposed to the mutagemsmg agent. The DNA to be mutagenized may also be present a host cell either by being integrated m the genome of said cell or by being present on a vector harboured m the cell. Finally, the DNA to be mutagenized may be m isolated form. It will be understood that the DNA sequence to be subjected to random mutagenesis is preferably a cDNA or a genomic DNA sequence.

In some cases it may be convenient to amplify the mutated DNA sequence prior to performing the expression step b) or the screening step c) . Such amplification may be performed accordance with methods known m the art, the presently preferred method being PCR-generated amplification using oligonucleotide primers prepared on the basis of the DNA or ammo acid sequence of the parent enzyme. Subsequent to the incubation with or exposure to the mutagemsmg agent, the mutated DNA is expressed by culturmg a suitable host cell carrying the DNA sequence under conditions allowing expression to take place. The host cell used for this purpose may be one which has been transformed with the mutated DNA sequence, optionally present on a vector, or one which was carried the DNA sequence encoding the parent enzyme during the mutagenesis treatment. Examples of suitable host cells are the following: 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 , Streptomyces lividans or Streptomyces murinus; and gram-negative bacteria such as E. coli .

The mutated DNA sequence may further comprise a DNA sequence encoding functions permitting expression of the mutated DNA sequence .

Localized random mutagenesis

The random mutagenesis may be advantageously localized to a part of the parent alpha-amylase in question. This may, e . g. , be advantageous when certain regions of the enzyme have been identified to be of particular importance for a given property of the enzyme, and when modified are expected to result in a variant having improved properties. Such regions may normally be identified when the tertiary structure of the parent enzyme has been elucidated and related to the function of the enzyme.

The localized, or region-specific, random mutagenesis is conveniently performed by use of PCR generated mutagenesis techniques as described above or any other suitable technique known in the art. Alternatively, the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e.g., by insertion into a suitable vector, and said part may be subsequently subjected to mutagenesis by use of any of the mutagenesis methods discussed above. Alternative methods of providing alpha-amylase variants

Alternative methods for providing variants of the invention include gene-shuffling method known in the art including the methods, e . g. , described in WO 95/22625 (from Affymax Technologies N.V.) and WO 96/00343 (from Novo Nordisk A/S) ._

Expression of alpha-amylase variants

According to the invention, a DNA sequence encoding the variant produced by methods described above, or by any alterna- tive 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. 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.

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 protemase, A . niger neutral alpha- amylase, A . niger acid stable alpha-amylase, A . niger glu- coamylase, Rhizomucor miehei lipase, A . oryzae alkaline protease, A . oryzae triose phosphate isomerase or A . nidulans acetamidase .

The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, poly- adenylation 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.

The vector may further comprise a DNA sequence enabling the vector to replicate the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl and pIJ702.

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, kanamyc , chloramphenicol or tetracyclm resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, maD and sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, e.g., as described m WO 91/17243.

While mtracellular expression may be advantageous m 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 . 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 m the art (cf., for instance, Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).

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 m 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 (m one or more copies) m the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained m 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 m connection with the different types of host cells. 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.

Examples of suitable bacteria are grampositive bacteria such as Bacillus subtilis , Bacillus licheniformis , Bacillus lentus , Bacillus brevis, Bacillus stearothermophilus , Bacillus alkalo- philus, Bacillus amyloliquefaciens , Bacillus coagulans , Bacillus circulans , Bacillus lautus , Bacillus megaterium, Bacillus thuringiensis , or Streptomyces lividans or Streptomyces murinus, or gramnegative bacteria such as E. coli . The transformation of the bacteria may, for instance, be effected by protoplast transformation or by using competent cells m a manner known per se .

The yeast organism may favourably 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 Aspergil lus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall m a manner known per se . A suitable procedure for transformation of Aspergillus host cells is described m EP 238 023.

In yet a 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. The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell m 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 m catalogues of the American Type Culture Collection) .

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 centπfugation or filtration, and precipitating protemaceous 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.

Nucleic Acid Sequences

The present invention also relates to isolated nucleic acid sequences, which encode a polypeptide of the present invention. In a preferred embodiment, the nucleic acid sequence is set forth m SEQ ID NO: 1 or SEQ ID NO: 3. In another more preferred embodiment, the nucleic acid sequence is the sequence contained m plasmid pLιH1274 or plasmid pTVB299 that is contained m Escheri chia coli DSM12761 and Escheri chia coli DSM12764, respectively In another preferred embodiment, the nucleic acid sequence is tne mature polypeptide coding region of SEQ ID NO:l or SEQ ID NO : 3 The present invention also encompasses nucleic acid sequences which encode a polypeptide having the ammo acid sequence of SEQ ID NO : 2 which differ from SEQ ID NO:l or SEQ ID NO: 3 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO : 1 or SEQ ID NO: 3 which encode fragments of SEQ ID NO : 2 or SEQ ID NO: 4, respectively, that have alpha-amylase activity.

Subsequences of SEQ ID NO : 1 or SEQ ID NO : 3 are nucleic acid sequences encompassed by SEQ ID NO : 1 or SEQ ID NO: 3 except that one or more nucleotides from the 5 ' and/or 3 ' end have been deleted.

The present invention also relates to mutant nucleic acid sequences comprising at least one mutation m the mature polypeptide coding sequence of SEQ ID NO : 1 or SEQ ID NO: 3, m which the mutant nucleic acid sequence encodes a polypeptide which consists of ammo acids 1 to 485 of SEQ ID NO : 2 or SEQ ID NO: 4.

The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are known m the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al . , 1990, PCR : A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR) , ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used. The nucleic acid sequence may be cloned from a strain of Bacillus, or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleic acid sequence.

The term "isolated nucleic acid sequence" as used herein refers to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, and most preferably at least about 90% pure as determined by agarose electrophoresis . For example, an isolated nucleic acid sequence can be obtained by standard cloning procedures used m genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

Homology of DNA sequence encoding the enzyme The present invention also relates to nucleic acid sequences which have a degree of homology to the mature polypeptide coding sequence of SEQ ID NO : 1 ( i . e . , nucleotides 1 to 1458) or SEQ ID NO: 3 (i.e., nucleotide 1 to 1458) of at least about 96% homology on DNA level, preferably aat least about 97%, preferably at least about 98%, more preferably at least about 99% homology, which encode an active polypeptide.

The DNA sequence 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 m the art such as GAP provided m the GCG program package (described above) . Thus, Gap GCGv8 may be used with the following default parameters: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, default scoring matrix. GAP uses the method of Needleman/Wunsch/Sellers to make alignments. Modification of a nucleic acid sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ m some engineered way from the polypeptide isolated from its native source, e.g., variants that differ m specific activity, thermostability, pH optimum, or the like. The variant sequence may be constructed on the basis of the nucleic acid sequence presented as the polypeptide encoding part of SEQ ID NO : 1 or SEQ ID NO: 3, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another ammo acid sequence of the polypeptide encoded by the nucleic acid sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different ammo acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al . , 1991, Protein Expression and Purification 2: 95-107. It will be apparent to those skilled m the art that such substitutions can be made outside the regions critical to the function of the molecule and still result m an active polypeptide. Ammo acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known m the art, such as site-directed mutagenesis or alanme- scannmg mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue m the molecule, and the resultant mutant molecules are tested for [enzyme] activity to identify ammo acid residues that are critical to the activity of the molecule. Sites of substrate- enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffmity labelling (see, e.g., de Vos et al . , 1992, Sci ence 255: 306-312; Smith et al . , 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al . , 1992, FEBS Letters 309: 59-64) . The present invention also relates to isolated nucleic acid sequences encoding a polypeptide of the present invention, which hybridize under medium stringency conditions, preferably medium-high stringency conditions, more preferably high stringency conditions, and most preferably very high stringency conditions with a nucleic acid probe which hybridizes under the same conditions with the nucleic acid sequence of SEQ ID NO : 1 or SEQ ID NO: 3 or its complementary strand; or allelic variants and subsequences thereof (Sambrook et al . , 1989, supra) , as defined herein. The present invention also relates to isolated nucleic acid sequences produced by (a) hybridizing a DNA under medium, medium-high, high, or very high stringency conditions with the sequence of SEQ ID NO : 1 or SEQ ID NO: 3, or their complementary strands, or a subsequence thereof; and (b) isolating the nucleic acid sequence. The subsequence is preferably a sequence of at least 100 nucleotides such as a sequence, which encodes a polypeptide fragment, which has alpha-amylase activity.

Methods for Producing Mutant Nucleic Acid Sequences The present invention further relates to methods for producing a mutant nucleic acid sequence, comprising introducing at least one mutation into the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3 or a subsequence thereof, wherein the mutant nucleic acid sequence encodes a polypeptide which consists of 1 to 485 of SEQ ID NO: 2 or SEQ ID NO: 4 or a fragment thereof which has alpha-amylase activity.

The introduction of a mutation into the nucleic acid sequence to exchange one nucleotide for another nucleotide may be accomplished by site-directed mutagenesis using any of the methods known m the art. Particularly useful is the procedure, which utilizes a supercoiled, double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, extend during temperature cycling by means of Pfu DNA polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicks is generated. Following temperature cycling, the product is treated with Dpnl , which is specific for methylated and hemimethylated DNA to digest the parental DNA template and to select for mutation-containing synthesized DNA.

Other procedures known in the art may also be used. These other procedures include gene shuffling, e.g., as described WO 95/22625 (from Affy ax Technologies N.V.) and WO 96/00343 (from Novo Nordisk A/S) . Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprising a nucleic acid sequence of the present invention operably linked to one or more control sequences, which direct the expression of the coding sequence a suitable host cell under conditions compatible with the control sequences. Expression will be understood to include any step involved m the production of the polypeptide including, but not limited to, transcription, post-transcπptional modification, translation, post-translational modification, and secretion.

"Nucleic acid construct" is defined herein as a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid which are combined and juxtaposed m a manner which would not otherwise exist m nature. The term nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. The term "coding sequence" is defined herein as a portion of a nucleic acid sequence, which directly specifies the ammo acid sequence of its protein product . The boundaries of the coding sequence are generally determined by a ribosome binding site (prokaryotes) or by the ATG start codon (eukaryotes) located just upstream of the open reading frame at the 5' end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3' end of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

An isolated nucleic acid sequence encoding a polypeptide of the present invention may be manipulated m a variety of ways to provide for expression of the polypeptide. Manipulation of the nucleic acid sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing recombinant DNA methods are well known m the art .

The term "control sequences" is defined herein to include all components, which are necessary or advantageous for the expression of a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide. The term "operably linked" is defined herein as a configuration m which a control sequence is appropriately placed at a position relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of a polypeptide.

Promoter Sequence

The control sequence may be an appropriate promoter sequence, a nucleic acid sequence, which is recognized by a host cell for expression of the nucleic acid sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence which shows transcriptional activity m the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or mtracellular polypeptides either homologous or heterologous to the host cell .

Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially m a bacterial host cell, are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene ( dagA) , Bacillus subtilis levansucrase gene ( sacB) , Bacillus licheniformis alpha-amylase gene ( amyL) , Bacillus stearothermophilus maltogenic amylase gene ( amyM) , Bacillus amyloliquefaciens alpha-amylase gene ( amyQ) , Bacillus licheniformis penicillmase gene (penP) , Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa- Kamaroff et al . , 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoer et al . , 1983, Proceedings of the Na tional Academy of Sciences USA 80: 21-25). Further promoters are described m "Useful proteins from recombinant bacteria" m Scientific American, 1980, 242: 74-94; and m Sambrook et al . , 1989, supra .

Terminator sequence

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator, which is functional m the host cell of choice may be used m the present invention. Signal Peptide

The control sequence may also be a signal peptide-codmg region that codes for an ammo acid sequence linked to the ammo terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide-codmg region naturally linked in translation reading frame with the segment of the coding region, which encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide- codmg region, which is foreign to the coding sequence. The foreign signal peptide-codmg region may be required where the coding sequence does not naturally contain a signal peptide- coding region. Alternatively, the foreign signal peptide- codmg region may simply replace the natural signal peptide- codmg region m order to enhance secretion of the polypeptide. However, any signal peptide-codmg region, which directs the expressed polypeptide into the secretory pathway of a host cell of choice, may be used m the present invention.

Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilism, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS , nprM) , and Bacillus subtilis prsA . Further signal peptides are described by Simonen and Palva, 1993, Mi crobiologi cal Reviews 57: 109- 137. Regulatory system

It may also be desirable to add regulatory sequences, which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off m response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems m prokaryotic systems include the lac, tac , and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those, which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene, which is amplified m the presence of methotrexate , and the metallothionem genes which are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the polypeptide would be operably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites. Alternatively, the nucleic acid sequence of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. 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, an extrachromosomal element, a mmichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication . Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome (s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.

The vectors of the present invention preferably contain one or more selectable markers, which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillm, kanamycm, chloramphenicol or tetracyclme resistance. Suitable markers for yeast host cells are ADE2 , HIS3, LEU2 , LYS2 , MET3 , TRP1, and URA3. A selectable marker for use in a filamentous fungal host cell may be selected from the group including, but not limited to, a dS (acetamidase) , argB (ornith e carbamoyltransferase) , bar (phosphmothπcm acetyltransferase) , hygB (hygromycm phosphotransferase) , niaD (nitrate reductase), pyrG (orotιdme-5 ' -phosphate decarboxylase) , sC (sulfate adenyltransferase) , trpC

(anthranilate synthase) , as well as equivalents thereof. Preferred for use an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus .

The vectors of the present invention preferably contain an element (s) that permits stable integration of the vector into the host cell genome or autonomous replication of the vector m the cell independent of the genome of the cell .

For integration into the host cell genome, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell . The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location (s) m the chromosome (s) . To increase the likelihood of integration at a precise location, the mtegrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The mtegrational elements may be any sequence that is homologous with the target sequence the genome of the host cell . Furthermore, the mtegrational elements may be non-encoding or encoding nucleic acid sequences. On the other hand, the vector may be integrated into the genome of the host cell by non- homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously m the host cell question. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication m E. coli , and pUBHO, pE194, pTA1060, and pAMβl permitting replication m Bacillus . Examples of origins of replication for use m a yeast host cell are the 2 micron origin of replication, ARSl, ARS4 , the combination of ARSl and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive m the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sci ences USA 75: 1433).

More than one copy of a nucleic acid sequence of the present invention may be inserted into the host cell to increase production of the gene product . An increase m the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al . , 1989, supra) .

Host Cells

The present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a nucleic acid sequence of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self -replicating extra-chromosomal vector as described earlier.

The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote .

Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus , Bacillus amyloliquefaci ens, Bacillus brevis, Bacillus circulans , Bacillus clausii , Bacillus coagulans , Bacillus lautus , Bacillus lentus , Bacillus licheniformis , Bacillus megaterium, Baci llus stearothermophilus , Bacillus subtilis, and Bacillus thuringiensis ; or a Streptomyces cell, e.g., Streptomyces lividans or Streptomyces muπnus , or gram negative bacteria such as E. coli and Pseudomonas sp . In a preferred embodiment, the bacterial host cell is a Bacillus lentus , Bacillus licheniformis , Bacillus stearothermophilus or Bacillus subtilis cell. In another preferred embodiment, the Bacillus cell is an alkalophilic Bacillus . The introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation

(see, e.g., Chang and Cohen, 1979, Molecular General Genetics

168: 111-115), using competent cells (see, e.g., Young and

Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771- 5278) .

Methods of Production

The present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a strain, which m its wild-type form is capable of producing the polypeptide, to produce a supernatant comprising the polypeptide; and (b) recovering the polypeptide. Preferably, the strain is of the genus Bacillus sp

The present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide .

The present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide, wherein the host cell comprises a mutant nucleic acid sequence having at least one mutation the mature polypeptide coding region of SEQ ID NO : 1 or SEQ ID NO: 3, wherein the mutant nucleic acid sequence encodes a polypeptide which consists of ammo acids 1 to 485 of SEQ ID NO: 2 or SEQ ID NO : 4, and (b) recovering the polypeptide.

Compositions

In a still further aspect, the present invention relates to compositions comprising an alpha-amylase or a variant thereof of the present invention. Preferably, the compositions are enriched m an alpha-amylase or variant thereof of the present invention. In the present context, the term "enriched" indicates that the alpha-amylase activity of the composition has been increased, e.g., with an enrichment factor of 1.1. The composition may comprise a polypeptide of the invention as the major enzymatic component, e.g., a mono- component composition. Alternatively, the composition may comprise multiple enzymatic activities, such as an ammopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitmase, cutmase, cyclodextrm glycosyltransferase, deoxyribonuclease, esterase, alpha- galactosidase, beta-galactosidase, glucoamylase, alpha- glucosidase, beta-glucosidase , haloperoxidase, mvertase, laccase, lipase, mannosidase, oxidase, pectmolytic enzyme, peptidoglutammase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutammase, or xylanase. The additional enzyme (s) may be producible by means of a microorganism belonging to the genus Aspergillus , preferably Aspergillus aculeatus , Aspergillus awamori , Aspergillus niger, or Aspergillus oryzae, or Trichoderma , Humicola , preferably Humicola msolens , or Fusarium, preferably Fusarium bactridioides , Fusarium cerealis, Fusarium crookwellense , Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporu , Fusarium negundi , Fusarium oxysporum, Fusarium reticulatum, Fusarium roseu , Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium tri chothecioides , or Fusarium venenatu .

The polypeptide compositions may be prepared m accordance with methods known in the art and may be m the form of a liquid or a dry composition. For instance, the polypeptide composition may be m the form of granulate or a micro- granulate. The polypeptide to be included m the composition may be stabilized m accordance with methods known m the art. Examples are given below of preferred uses of the polypeptide compositions of the invention. The dosage of the polypeptide composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known m the art.

Industrial Applications

Owing to their activity at alkaline pH values, the alpha- amylases of the invention are well suited for use m a variety of industrial processes, m particular the enzyme finds potential applications as a component m detergents, e.g., laundry, dishwashing and hard surface cleaning detergent compositions, but it may also be useful for desizmg of textiles, fabrics and garments, beer making or brewing, m pulp and paper production, and further m the production of sweeteners and ethanol , such as fuel, drinking and industrial ethanol , from starch or whole grams.

Starch Conversion

Conventional starch-conversion processes, such as liquefaction and sacchaπfication processes, are described, e.g., US Patent No. 3,912,590 and EP patent publications Nos . 252,730 and 63,909, hereby incorporated by reference.

Pulp and Paper Production

The alkaline alpha-amylase of the invention may also be used m the production of lignocellulosic materials, such as pulp, paper and cardboard, from starch reinforced waste paper and cardboard, especially where re-pulpmg occurs at pH above 7 and where amylases facilitate the disintegration of the waste material through degradation of the reinforcing starch. The alpha-amylase of the invention is especially useful m a process for producing a papermakmg pulp from starch-coated prmted-paper. The process may be performed as described m WO 95/14807, comprising the following steps: a) disintegrating the paper to produce a pulp, b) treating with a starch-degrading enzyme before, during or after step a) , and c) separating ink particles from the pulp after steps a) and b) .

The alpha-amylases of the invention may also be very useful m modifying starch where enzymatically modified starch is used papermakmg together with alkaline fillers such as calcium carbonate, kaolin and clays. With the alkaline alpha- amylases of the invention t becomes possible to modify the starch m the presence of the filler thus allowing for a simpler integrated process .

Desizmg of Textiles, Fabrics and Garments

An alpha-amylase of the invention may also be very useful m textile, fabric or garment desizmg. In the textile processing industry, alpha-amylases are traditionally used as auxiliaries m the desizmg process to facilitate the removal of starch-contammg size, which has served as a protective coating on weft yarns during weaving. Complete removal of the size coating after weaving is important to ensure optimum results m the subsequent processes, m which the fabric is scoured, bleached and dyed. Enzymatic starch breakdown is preferred because it does not involve any harmful effect on the fiber material . In order to reduce processing cost and increase mill throughput, the desizmg processing is sometimes combined with the scouring and bleaching steps. In such cases, non- enzymatic auxiliaries such as alkali or oxidation agents are typically used to break down the starch, because traditional alpha-amylases are not very compatible with high pH levels and bleaching agents. The non-enzymatic breakdown of the starch size does lead to some fiber damage because of the rather aggressive chemicals used. Accordingly, it would be desirable to use the alpha-amylases of the invention as they have an improved performance m alkaline solutions. The alpha-amylases may be used alone or m combination with a cellulase when desizmg cellulose-containing fabric or textile.

Desizmg and bleaching processes are well known m the art. For instance, such processes are described m WO 95/21247, US patent 4,643,736, EP 119,920 hereby m corporate by reference .

Commercially available products for desizmg include Aquazyme^® and Aquazyme^® Ultra from Novo Nordisk A/S.

Beer making

The alpha-amylases of the invention may also be very useful m a beer-mak g process; the alpha-amylases will typically be added during the mashing process.

Detergent Compositions

The enzyme of the invention may be added to and thus become a component of a detergent composition.

The detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rmse added fabric softener composition, or be formulated as a detergent composition for use m general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additive comprising the enzyme of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutmase, an amylase, a carbohydrase, a cellulase, a pectmase, a mannanase, an arabmase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme (s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme (s) should be present effective amounts.

Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serme protease or a metallo protease, preferably an alkaline microbial protease or a trypsm-like protease. Examples of alkaline proteases are subtilisms, especially those derived from Bacillus, e.g., subtilism Novo, subtilism Carlsberg, subtilism 309, subtilism 147 and subtilism 168 (described m WO 89/06279) . Examples of trypsm- like pro-teases are trypsm (e.g., of porcine or bovine origin) and the Fusarium protease described m WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described m WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions m one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and 274.

Preferred commercially available protease enzymes include Alcalase^®, Savmase^®, Primase^®, Duralase^®, Esperase^®, and Kannase^® (Novo Nordisk A/S) , Maxatase^®, Maxacal , Maxapem^®, Properase^®, Purafect^®, Purafect OxP^®, FN2^®, and FN3^® (Genencor International Inc.) .

Lipases : Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces) , e.g., from H. lanugmosa ( T. lanugmosus) as described m EP 258 068 and EP 305 216 or from H. msolens as described m WO 96/13580, a Pseudomonas lipase, e.g., from P . alcaligenes or P. pseudoalcaligenes (EP 218 272) , P. cepacia (EP 331 376) , P. stutzen (GB 1,372,034), P . fluorescens, Pseudomonas sp . strain SD 705 (WO 95/06720 and WO 96/27002) , P. wisconsmensi s (WO 96/12012), a Bacillus lipase, e.g., from B . subtili s (Dartois et al . (1993), Biochemica et Biophysica Acta, 1131, 253-360), B . stearothermophilus (JP 64/744992) or B . pumilus (WO 91/16422) .

Other examples are lipase variants such as those described m WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Preferred commercially available lipase enzymes include Lipolase™ and Lipolase Ultra™ (Novo Nordisk A/S) .

Amylases: Suitable amylases (alpha and/or beta) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus , e.g., a special strain of B . licheniformis , described m more detail m GB 1,296,839. Examples of useful alpha-amylases are the variants described WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions m one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ and BAN™ (Novo Nordisk A/S) , Rapidase™ and Puras ar™ (from Genencor International Inc.) .

Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus , Pseudomonas , Humicola, Fusari um, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola msolens , Myceliophthora thermophila and Fusarium oxysporum disclosed m US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described m EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluzyme^®, and Carezyme^® (Novo Nordisk A/S) , Clazmase^®, and Puradax HA^® (Genencor International Inc.), and KAC-500(B)^® (Kao Corporation) . Peroxidases/Oxidases : Suitable peroxidases/oxidases include those of plant, bac-terial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprmus, e.g., from C. cinereus , and variants thereof as those described m WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme^® (Novo Nordisk A/S) .

The detergent enzyme (s) may be included m a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive of the invention, i.e., a separate additive or a combined additive, can be formulated, e.g., granulate, a liquid, a slurry, etc. Preferred detergent additive formulations are granulates, m particular non-dustmg granulates, liquids, m particular stabilized liquids, or slurries . Non-dustmg granulates may be produced, e.g., as disclosed US 4,106,991 and 4,661,452 and may optionally be coated by methods known m the art. Examples of waxy coating materials are poly (ethylene oxide) products (polyethyleneglycol , PEG) with mean molar weights of 1000 to 20000; ethoxylated nonyl- phenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols m which the alcohol contains from 12 to 20 carbon atoms and m which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglyceπdes of fatty acids. Examples of film- forming coating materials suitable for application by fluid bed techniques are given m GB 1483591. Liquid enzyme pre-parations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed m EP 238,216.

The detergent composition of the invention may be m any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 70 % water and 0-30 % organic solvent, or non- aqueous .

The detergent composition comprises one or more surfactants, which may be non- ionic including semi -polar and/or anionic and/or catiomc and/or zwitterionic . The surfactants are typically present at a level of from 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate) , alcohol ethoxysulfate, secondary alkanesulfonate , alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccmic acid or soap. When included therein the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonyl -phenol ethoxylate, alkylpolyglycoside, alkyldimethylamme-oxide, ethoxylated fatty acid monoethanol-amide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamme ( "glucamides " ) .

The detergent may contain 0-65 % of a detergent builder or complexmg agent such as zeolite, diphosphate, tπpho-sphate, phosphonate, carbonate, citrate, nitrilotπacetic acid, ethylenediammetetraacetic acid, diethylenetπ-ammepen- taacetic acid, alkyl- or alkenylsuccmic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst) .

The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly (vmyl-pyrrolidone) , poly

(ethylene glycol), poly(vmyl alcohol), poly (vmylpyridme-N- oxide) , poly (vmylimidazole) , polycarboxylates such as polyacrylates , maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid co-polymers. The detergent may contain a bleaching system, which may comprise a H₂0₂ source such as perborate or percarbonate which may be combined with a peracid- forming bleach activator such as tetraacetylethylenediamme or nonanoyloxyben-zenesul-fonate . Alternatively, the bleaching system may comprise peroxyacids of, e.g., the amide, lmide, or sulfone type.

The enzyme (s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol , a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the corn-position may be formulated as described m, e.g., WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti- corrosion agents, soil -suspending agents, anti-soil re- deposition agents, dyes, bactericides, optical bπghteners, hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that m the detergent compositions any enzyme, m particular the enzyme of the invention, may be added m an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liquor, preferably 0.05- 5 mg of enzyme protein per liter of wash liquor, m particular 0.1-1 mg of enzyme protein per liter of wash liquor.

The enzyme of the invention may additionally be incorporated m the detergent formulations disclosed m WO 97/07202, which is hereby incorporated as reference.

Dishwash Deterget Compositions The enzyme of the invention mat also be used m dish wash detergent compositions, including the following: 1) POWDER AUTOMATIC DISHWASHING COMPOSITION

Nonionic surfactant 0.4 - 2.5%

Sodium metasilicate 0 - 20%

Sodium disilicate 3 - 20%

Sodium tπphosphate 20 - 40%

Sodium carbonate 0 - 20%

Sodium perborate 2 - 9%

Tetraacetyl ethylene diamme (TAED) 1 - 4%

Sodium sulphate 5 - 33%

POWDER AUTOMATIC DISHWASHING COMPOSITION

Nonionic surfactant 1 - 2% (e.g. alcohol ethoxylate)

Sodium disilicate 2 - 30%

Sodium carbonate 10 - 50%

Sodium phosphonate 0 - 5%

Trisodium citrate dihydrate 9 - 30%

Nitrilotrisodium acetate (NTA) 0 - 20%

Sodium perborate monohydrate 5 - 10%

Tetraacetyl ethylene diamine (TAED) 1 - 2%

Polyacrylate polymer (e.g. maleic acid/acrylic acid co- 6 - 25% polymer)

Enzymes 0.0001 - 0.1%

Perfume 0.1 - 0.5%

Water 5 - 10

3) POWDER AUTOMATIC DISHWASHING COMPOSITION

Nonionic surfactant 0.5 - 2.0%

Sodium disilicate 25 - 40%

Sodium citrate 30 - 55%

Sodium carbonate 0 - 29%

Sodium bicarbonate 0 - 20%

Sodium perborate monohydrate 0 - 15%

Tetraacetyl ethylene diamine (TAED) 0 - 6%

Maleic acid/acrylic 0 - 5% acid copolymer

Clay 1 - 3%

Polyamino acids 0 - 20%

POWDER AUTOMATIC DISHWASHING COMPOSITION

Nonionic surfactant 1 2%

Zeolite MAP 15 42%

Sodium disilicate 30 34%

Sodium citrate 0 12%

Sodium carbonate 0 20%

Sodium perborate monohydrate 7 15%

Tetraacetyl ethylene diam e (TAED) 0 3%

Polymer 0 4%

Maleic acid/acrylic acid copolymer 0 5%

Organic phosphonate 0 4%

Clay 1 2%

Enzymes 0.0001 - 0.1%

Sodium sulphate Balance

5) POWDER AUTOMATIC DISHWASHING COMPOSITION

Nonionic surfactant 1 - 7%

Sodium disilicate 18 - 30%

Trisodium citrate 10 - 24%

Sodium carbonate 12 - 20%

Monopersulphate (2 KHS0₅. KHS0₄. K₂S0₄) 15 - 21%

Bleach stabilizer 0.1 - 2%

Maleic acid/acrylic acid copolymer 0 - 6%

Diethylene tπamme pentaacetate, pentasodium salt 0 - 2.5%

Enzymes 0.0001 - 0.1%

6) POWDER AND LIQUID DISHWASHING COMPOSITION WITH CLEANING SURFACTANT SYSTEM

Nonionic surfactant 0 - 1.5%

Octadecyl dimethylamme N-oxide dihydrate 0 - 5%

80:20 wt.C18/C16 blend of octadecyl dimethylamme N-oxide dihydrate and hexadecyldimethyl amine N-oxide 0 - 4% dihydrate

70:30 wt.C18/C16 blend of octadecyl bis (hydroxyethyl) amme N-oxide anhydrous and hexadecyl bis 0 - 5% (hydroxyethyl) amme N-oxide anhydrous ^ci₃~^ci₅ alkyl ethoxysulfate with an average degree of ethoxylation of 3 0 - 10%

C₁₂-C₁₅ alkyl ethoxysulfate with an average degree of ethoxylation of 3 0 ^ci₃ ^_ci₅ ethoxylated alcohol with an average degree of ethoxylation of 12 0 - 5%

A blend of C₁₂-C₁₅ ethoxylated alcohols with an average degree of 0 - 6.5% ethoxylation of 9

A blend of C₁₃-C₁₅ ethoxylated alcohols with an average degree of 0 - 4% ethoxylation of 30

Sodium disilicate 0 - 33%

Sodium tπpolyphosphate 0 - 46%

Sodium citrate 0 - 28%

Citric acid 0 - 29%

Sodium carbonate 0 - 20%

Sodium perborate monohydrate 0 - 11.5%

Tetraacetyl ethylene diam e (TAED) 0 - 4% Maleic acid/acrylic acid copolymer 0 - 7.5%

Sodium sulphate 0 - 12.5%

Enzymes 0.0001 - 0.1%

7) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING COMPOSITION

Liquid nonionic surfactant (e.g. alcohol ethoxylates) 2.0 - 10.0%

Alkali metal silicate 3.0 - 15.0%

Alkali metal phosphate 20.0 - 40.0%

Liquid carrier selected from higher glycols, polyglycols, polyoxides, 25.0 - 45.0% glycolethers

Stabilizer (e.g. a partial ester of phosphoric acid and a C₁₆-C_ιa alkanol) 0.5 - 7.0%

Foam suppressor (e.g. silicone) 0 - 1.5%

Enzymes 0.0001 - 0.1%

8) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION

Liquid nonionic surfactant (e.g. alcohol ethoxylates) 2.0 - 10.0%

Sodium silicate 3.0 - 15.0%

Alkali metal carbonate 7.0 - 20.0%

Sodium citrate 0.0 - 1.5%

Stabilizing system (e.g. mixtures of finely divided silicone and low molecular weight dialkyl poiyglycol 0.5 - 7.0% ethers)

Low molecule weight polyacrylate polymer 5.0 - 15.0%

Clay gel thickener (e.g. bentonite) 0.0 - 10.0%

Hydroxypropyl cellulose polymer 0.0 - 0.6%

Enzymes 0.0001 - 0.1%

Liquid carrier selected from higher lycols, polyglycols, polyoxides and Balance glycol ethers

9) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING COMPOSITION ^ci₂ ^_ci₄ fatty acid 0 - 0.5%

Block co-polymer surfactant 1.5 - 15.0%

Sodium citrate 0 - 12%

Sodium tripolyphosphate 0 - 15%

Sodium carbonate 0 - 8%

Aluminium tπstearate 0 - 0.1%

Sodium cumene sulphonate 0 - 1.7%

Polyacrylate thickener 1.32 - 2.5%

Sodium polyacrylate 2.4 - 6.0%

Boric acid 0 - 4.0%

Sodium formate 0 - 0.45%

Calcium formate 0 - 0.2%

Sodium n-decydiphenyl oxide disulphonate 0 - 4.0%

Monoethanol amme (MEA) 0 - 1.86%

Sodium hydroxide (50%) 1.9 - 9.3%

1,2- Propanediol 0 - 9.4%

Enzymes 0.0001 - 0.1%

Suds suppressor, dye, perfumes, water Balance

10) LIQUID AUTOMATIC DISHWASHING COMPOSITION

Alcohol ethoxylate 0 - 20%

Fatty acid ester sulphonate 0 - 30%

Sodium dodecyl sulphate 0 - 20%

Alkyl polyglycoside 0 - 21%

Oleic acid 0 - 10%

Sodium disilicate monohydrate 18 - 33% Sodium citrate dihydrate 18 - 33%

Sodium stearate 0 - 2.5%

Sodium perborate monohydrate 0 - 13%

Tetraacetyl ethylene diam e (TAED) 0 - 8%

Maleic acid/acrylic acid copolymer 4 - 8%

Enzymes 0.0001 - 0.1%

11) LIQUID AUTOMATIC DISHWASHING COMPOSITION CONTAINING PROTECTED BLEACH PARTICLES

Sodium silicate 5 - 10%

Tetrapotassium pyrophosphate 15 - 25%

Sodium tπphosphate 0 - 2%

Potassium carbonate 4 - 8%

Protected bleach particles, e.g. chlorine 5 - 10%

Polymeric thickener 0.7 - 1.5%

Potassium hydroxide 0 - 2%

Enzymes 0.0001 ^■ - 0.1%

Water Balance

11) Automatic dishwashing compositions as described m 1), 2), 3) , 4) , 6) and 10) , wherein perborate is replaced by percarbonate .

12) Automatic dishwashing compositions as described m 1) - 6) which additionally contain a manganese catalyst. The manganese catalyst may, e.g., be one of the compounds described m "Efficient manganese catalysts for low-temperature bleaching", Nature 369, 1994, pp. 637-639.

Uses

The present invention is also directed to methods for using the polypeptides having alpha-amylase activity of the invention in detergents, in particular laundry detergent compositions and dishwashing detergent compositions, hard surface cleaning compositions, and in composition for desizing of textiles, fabrics or garments, for production of pulp and paper, beer making, and starch conversion processes as described above.

The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

Materials and Methods

Chemicals used as buffers and substrates were commercial products of at least reagent grade.

Materials :

Enzymes :

SP690: alpha-amylase disclosed in SEQ ID NO: 1 of US patent no.

5, 856, 164. SP722 : alpha-amylase disclosed in SEQ ID NO: 2 of US patent no.

US patent no. 5,856,164.

Termamyl^®: alpha-amylase from Bacillus licheniformis disclosed in SEQ ID NO: 1 of US patent no. 5,830,837.

AA560: alpha-amylase of the invention shown m SEQ ID NO: 2. BAN: alpha-amylase derived from Bacillus amyloliquefaci ens and available from Novo Nordisk A/S

BSG: alpha-amylase derived from Bacillus stearothermophilus and available from Novo Noridsk A/S

Model detergent :

A/P (Asia/Pacific) Model Detergent has the following composition: 20% STPP (sodium tπpolyphosphate) , 25% Na₂S0₄, 15% Na₂C0₃, 20% LAS (linear alkylbenzene sulfonate, Nansa 80S) , 5% C₁₂-C₁₅ alcohol ethoxylate (Dobanol 25-7), 5% Na₂Sι₂0₅, 0.3% NaCl. Omo Multi Acao (Brazil) ,

Omo concentrated powder (EU) (Unilever) Ariel Futur liquid (EU) (Procter and Gamble)

Deposit of Biological Material The following biological material has been deposited under the terms of the Budapest Treaty with the Deutshe Sammmlung von Microorganismen und Zellkulturen GmbH (DSMZ) , Mascheroder Weg lb, D-38124 Braunschweig DE, and given the following accession number : Deposit Accession Number Date of Deposit

NN017557 DSM 12648 25 January 1999

NN017560 DSM 12649 25 January 1999

NN049467 DSM12761 7^th April 1999

NN049470 DSM12764 7^th April 1999 The strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposit represents a substantially pure culture of the deposited strain. The deposit is available as required by foreign patent laws m countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention m derogation of patent rights granted by governmental action Host organism

Bacillus subtilis strain SHa273 is disclosed m WO 95/10603

E. coli strain SJ2 (Diderichsen et al . (1990)), J. Bacteπol . , vol. 172, pp. 4315-4321.

Plasmids :

The gene bank vector pSJ1678 is further disclosed m WO 94/19454, which is hereby incorporated by reference. pTVBHO is a plasmid replicating m Bacillus subtilis by the use of origin of replication from pUBHO (Gryczan, T.J.

(1978) J. Bact . 134:318-329). The plasmid further encodes the cat gene, conferring resistance towards chlorampenicol , obtained from plasmid pC194 (Hoπnouchi, S. and Weisblum, B. (1982), J. Bact. 150: 815-825). The plasmid harbors a truncated version of the Bacillus licheniformis alpha-amylase gene, amyL, such that the amyL promoter, signal sequence and transcription terminator are present , but the plasmid does not provide an amy-plus phenotype (halo formation on starch containing agar) .

pTVB299: see the construction m Example 10

Methods General molecular biology methods :

Unless otherwise mentioned the DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al . (1989); Ausubel et al . (1995) ; Harwood and Cutting (1990) . Fermentation of alpha-amylases and variants

Fermentation may be performed by methods well known m the art or as follows.

A B . subtilis strain harboring the relevant expression plasmid is streaked on a LB-agar plate with 10 micro g/ml Kanamycm from -80°C stock, and grown overnight at 37°C. The colonies are transferred to 100 ml BPX media supplemented with 10 micro g/ml kanamycm m a 500 ml shaking flask. Composition of BPX medium: Potato starch 100 g/1

Barley flour 50 g/1

BAN 5000 SKB 0.1 g/1

Sodium casemate 10 g/1

Soy Bean Meal 20 g/1 Na₂HP0₄, 12 H₂0 9 g/1

Pluronic™ 0.1 g/1

The culture is shaken at 37°C at 270 rpm for 5 days.

Cells and cell debris are removed from the fermentation broth by centrifugation at 4500 rpm m 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 20mM Acetate pH 5.5. The UF-filtrate is applied on a S-sepharose F.F. and elution is carried out by step elution with 0.2M NaCl m the same buffer. The eluate is dialysed against lOmM Tris, pH 9.0 and applied on a Q-sepharose F.F. and eluted with a linear gradient from 0-0.3M NaCl over 6 column volumes. The fractions, which 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 5 minutes . Assays for Alpha-Amylase Activity 1. Phadebas assay

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- 1inked insoluble blue-colored starch polymer, which has been mixed with bovine serum albumin and a buffer substance and tabletted. For every single measurement one tablet is suspended m a tube containing 5 ml 50 mM Britton-Robmson buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl_^, pH adjusted to the value of interest with NaOH) . The test is performed m a water bath at the temperature of interest . The alpha-amylase to be tested is diluted m x ml of 50 mM Britton- Robmson buffer. 1 ml of this alpha-amylase solution is added to the 5 ml 50 mM Britton-Robmson 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.

It is important that the measured 620 nm absorbance after 10 or 15 minutes of incubation (testing time) is m 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 colour will be produced. The colour intensity is measured at 620 nm. The measured absorbance is αirectly proportional to the specific activity (activity/mg of pure alpha-amylase protein) of the alpha-amylase m question under the given set of conditions.

2. Alternative method (PNP-G7 assay) 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 m the kit digest the substrate to liberate a free PNP molecule which has a yellow colour and thus can be measured by visible spectophometry at λ=405nm. (400- 420 nm. ) . Kits containing PNP-G7 substrate and alpha-Glucosidase is manufactured by Boehrmger-Mannheim (cat. No. 1054635) .

To prepare the substrate one bottle of substrate (BM 1442309) is added to 5 ml buffer (BM1442309) . To prepare the -

Glucosidase one bottle of alpha-Glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309) . The working solution is made by mixing 5 ml alpha-Glucosidase solution with 0.5 ml substrate.

The assay is performed by transforming 20 micro 1 enzyme solution to a 96 well microtitre plate and incubating at 25°C. 200 micro 1 working solution, 25°C is added. The solution is mixed and pre-mcubated 1 mmute and absorption is measured every 15 sec. over 3 minutes at OD 405 nm.

The slope of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the alpha-amylase m question under the given set of conditions .

Measurement of the calcium- and pH-dependent stability Normally industrial liquefaction processes runs using pH 6.0- 6.2 as liquefaction pH and an addition of 40 ppm free calcium m order to improve the stability at 95°C-105°C. Some of the herein proposed substitutions have been made m order to improve the stability at 1. lower pH than pH 6.2 and/or 2. at free calcium levels lower than 40 ppm free calcium.

Two different methods can be used to measure the alterations m stability obtained by the different substitutions m the AA560 alpha-amylase:

Method 1. One assay which measures the stability at reduced pH, pH 5.0 , the presence of 5 ppm free calcium.

10 micro g of the variant are incubated under the following conditions: A 0.1 M acetate solution, pH adjusted to pH 5.0, containing 5ppm calcium and 5% w/w common corn starch (free of calcium) . Incubation is made m a water bath at 95°C for 30 minutes .

Method 2. One assay, which measure the stability m the absence of free calcium and where the pH is maintained at pH 6.0. This assay measures the decrease m calcium sensitivity: 10 micro g of the variant were incubated under the following conditions: A 0.1 M acetate solution, pH adjusted to pH 6.0, containing 5% w/w common corn starch (free of calcium) . Incubation was made a water bath at 95 °C for 30 minutes.

Stability determination All the stability trials are made using the same set up. The method is:

The enzyme is incubated under the relevant conditions (1-4) .

Samples are taken at 0, 5, 10, 15 and 30 minutes and diluted 25 times (same dilution for all taken samples) m assay buffer (0.1M 50mM Bπtton buffer pH 7.3) and the activity is measured using the Phadebas assay (Pharmacia) under standard conditions pH 7 . 3 , 3 7 ° C .

The activity measured before incubation (0 minutes) is used as reference (100%) . The decline m percent is calculated as a function of the incubation time.

Specific activity determination.

The specific activity is determined using the Phadebas assay (Pharmacia) as activity/mg enzyme.

Jet cooking and liquefaction with AA560 and variants

Liquefaction experiments are run m the mini - et system using a dosage of 50 NU/g DS at pH 5.5 with 5 ppm added Ca⁺⁺, to compare the performance of formulated alpha-amylase variant with that of parent alpha-amylase. The reaction is monitored by measuring the DE increase (Neocuprome method) as a function of

Cornstarch slurries are prepared by suspending about 11.8 kg Cerestar C*Pharm GL 03406 (89 % starch) m deionized water and making up to 30 kg. The pH is adjusted to 5.5 at ambient temperature, after the addition of 0.55 g CaCl₂. 2H₂0.

An amount of enzyme corresponding to 50 NU/g DS is added, and the conductivity adjusted to 300mS using NaCl. The standard conditions are as follows:

Substrate concentration 35 % w/w (initial) 31.6-31.9 % w/w (final)

Temperature 105°C, 5 minutes (Primary liquefaction)

95°C, 90 minutes (Secondary lique action) pH (initial) 5.5

After jetting, the liquefied starch is collected and transported m sealed thermos-flasks from the pilot plant to the laboratory, where secondary liquefaction is continued at 95°C.

10 ml samples are taken at 15 mmute intervals from 15-90 minutes. 2 drops of 1 N HC1 are added to inactivate the enzyme. From these samples, 0.3-0.1 g (according to the expected DE) are weighed out and diluted to 100 ml. Reducing sugars are then determined according to the Neocuprome method (Determination of reducing sugar with improved precision. Dygert, Li , Florida and Thomas (1965) . Anal. Biochem 13, 368) and DE values determined. The development of DE as a function of time is compared.

Desizing materials:

Standard fabrics : TS526, twill weave, 100% cotton. Impregnation : 55°C, 60 ppm CaCl₂, pH 6.5 Enzyme solutions: parent AA560 (1 g/1) Dosages : 0.05, 0.1, 0.2, 0.5 and 2.0 g/1 of the enzyme solutions m the impregnation liquor .

Incubation: 2 hr at 55 or 65°C

22 hr at 30°C

Wash: 10 minutes m water Drying : Room temperature Procedures : Evaluation of Desizmg Results - Violet

Scale (TEGEWA) .

General Method For Random Mutagenesis By Use Of The DOPE Program

The random mutagenesis may be carried out by the following steps :

1. Select regions of interest for modification m the parent enzyme,

2. Decide on mutation sites and non-mutated sites m the selected region, 3. Decide on which kind of mutations should be carried out, e . g. , with respect to the desired stability and/or performance of the variant to be constructed,

4. Select structurally reasonable mutations, 5. Adjust the residues selected by step 3 with regard to step 4.

6. Analyze by use of a suitable dope algorithm the nucleotide distribution.

7. If necessary, adjust the wanted residues to genetic code realism, e . g. , taking into account constraints resulting from the genetic code, e.g., m order to avoid introduction of stop codons; the skilled person will be aware that some codon combinations cannot be used practice and will need to be adapted 8. Make primers

9. Perform random mutagenesis by use of the primers

10. Select resulting glucoamylase variants by screening for the desired improved properties.

Dope Algorithm

Suitable dope algorithms for use m step 6 are well known m the art. One such algorithm is described by Tomandl , D. et al . ,

1997, Journal of Computer-Aided Molecular Design 11:29-38.

Another algorithm is DOPE (Jensen, LJ, Andersen, KV, Svendsen, A, and Kretzschmar, T (1998) Nucleic Acids Research 26:697-702).

Filter screening assays

The assay can be used to screening of AA560 variants having an improved stability at high pH compared to the parent enzyme and AA560 alpha-amylase variants having an improved stability at high pH and medium temperatures compared to the parent enzyme depending of the screening temperature setting

High pH filter assay 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 micro g/ml kanamycm at 37°C for at least 21 hours. The cellulose acetate layer is located on the TY agar plate.

Each filter sandwich is specifically marked with a needle after plating, but before incubation order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with glyc -NaOH buffer, pH 8.6-10.6 and incubated at room temperature (can be altered from 10°-60°C) for 15 mm. The cellulose acetate filters with colonies are stored on the TY- plates at room temperature until use. After incubation, residual activity is detected on plates containing 1% agarose, 0.2% starch m glycm-NaOH buffer, pH 8.6-10.6. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours . at room temperature. 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 rescreened twice under the same conditions as the first screen.

Low calcium filter assay The Bacillus library 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 a relevant antibiotic, e . g . , kanamycm or chloramphenicol , at 37°C for at least 21 hours. The cellulose acetate layer is located on the TY agar plate. Each filter sandwich is specifically marked with a needle after plating, but before incubation m order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with carbonate/bicarbonate buffer pH 8.5-10 and with different EDTA concentrations (0.001 mM - 100 mM) . The filters are incubated at room temperature for 1 hour. The cellulose acetate filters with colonies are stored on the TY-plates at room temperature until use. After incubation, residual activity is detected on plates containing 1% agarose, 0.2% starch carbonate/bicarbonate buffer pH 8.5-10. The assay plates with nitrocellulose filters are marked the same way as the filter sandwich and incubated for 2 hours, at room temperature. 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 rescreened twice under the same conditions as the first screen.

EXAMPLES

Example 1

Isolation of genomic DNA from DSM 12648 and DSM 12649

The strains Bacillus sp . DSM 12649 (the AA560 alpha- amylase) and Bacillus sp . DSM 12648 (the AA349 alpha-amylase) were propagated m liquid TY medium (as described m Ausubel et al.(1995)). After 16 hours incubation at 37°C and 300 rpm, the cells were harvested, and genomic DNA isolated by the method described by Pitcher et al . (1989) .

Genomic library construction Genomic DNA of strain DSM 12649 was partially digested with restriction enzyme Sau3A, and size-fractionated by elec- trophoresis on a 0.7 % agarose gel. Fragments between 2 and 10 kb m size was isolated by electrophoresis onto DEAE-cellulose paper (Dretzen et al . (1981) .

Isolated DNA fragments were ligated to BamHI digested pSJ1678 plasmid DNA, and the ligation mixture was used to transform E. coli SJ2.

Transformation

E. coli SJ2 host cells were prepared for and transformed by electroporation using a gene PULSER™ electroporator from BIO- RAD as described by the supplier.

Identification of positive transformant :

A DNA library m E. coli SJ2 , constructed as described above, was screened on LB agar plates (described m Ausbel et al.(1995)) containing 0.5 % AZCL-amylose (Megazyme) and 10 micro g/ml Chloramphenicol and incubated overnight at 37 ° C. Clones expressing amylase activity appeared with blue diffusion haloes. One such clone was named LιH1274. The DNA was further characterized by DNA sequencing of part of the cloned Sau3A DNA fragment .

Example 2

Determination of the DNA sequence of the gene encoding alpha- amylase from strain DSM 12648 (AA349)

The clone constituting a large chromosomal fragment containing the gene encoding the amylolytic activity inserted into plasmid pSJ1678, pLιH1274, was used as template to specifically PCR amplify internal DNA fragments of the alpha- amylase encoding gene by the use of degenerate primers directed towards the conserved regions m known Bacillus alpha-amylases. The degenerate primers were directed towards the following regions/ammo acid sequences : For36 : GITA (L/V/I) W (I/L) (SEQ ID NO: 5) For97: VY(G/A)D(V/F/L)V(M/L/I/F)NH (SEQ ID NO : 6) For227: DG (F/I ) R (F/L/I/V) DA (A/V) KH (SEQ ID NO: 7)

Rev235 DG(F/I)R(F/L/I/V)DA(A/V)KH (SEQ ID NO: 8)

Rev328 VTFV(D/E)NHD (SEQ ID NO: 9) Rev410 GWTREG (SEQ ID NO: 10) The various combinations of forward (For) and reverse (Rev) primers were used m PCR and internal DNA fragments could be amplified.

The DNA fragments were purified by QIAquick spin colums (QUIGEN) and sequenced utilizing the same degenerate primers.

From sequence the DNA sequence (SEQ ID NO: 1) of the complete coding region encoding the mature AA349 alpha-amylase (SEQ ID NO: 2) was determined by a standard primers-walking approach.

Example 3

Determination of the DNA sequence of the gene encoding alpha amylase from strain DSM 12649 (AA560) :

A preparation of chromosomal DNA from strain DSM 12649 (Example 1) was utilized as template m a similar experiment to the one described above m Example 2 m order to determine the DNA sequence of the AA560 alpha-amylase (SEQ ID NO: 4) .

Example 4 Subcloning of the AA349 alpha-amylase into pTVBllO. pTVBHO is a plasmid replicating m Bacillus subtilis by the use of origin of replication from pUBHO (Gryczan, T.J. (1978) J. Bact. 134:318-329). The plasmid further encodes the cat gene, conferring resistance towards chlorampenicol , obtained from plasmid pC194 (Hormouchi, S. and Weisblum, B. (1982), J. Bact. 150: 815-825). The plasmid harbors a truncated version of the Bacillus licheniformis alpha-amylase gene, amyL, such that the amyL promoter, signal sequence and transcription terminator are present, but the plasmid does not provide an amy-plus phenotype (halo formation on starch containing agar) . In order to express high amount of the AA349 alpha-amylase the mature gene was fused precisely to the amyL signal sequence so that transcription is initiated by the amyL promoter and translocation is directed by the amyL signal sequence.

A Pstl site is found within the mature AA349 alpha- amylase. Since the cloning of the gene into pTVBllO would utilize the Pstl site m pTVBllO, the Pstl site located within the AA349 alpha-amylase gene was destroyed during the cloning

(by introduction of a silent mutation for ammo acid Alanine 88

(GCA to GCG) . Primers 188clonmgN and 188 (Pst-) were used to amplify an approximately 280 bp fragment by PCR on plasmid pLιH1274 using the Pwo polymerase under conditions recommended by the manufacturer (Boehrmger Mannheim) . This fragment was purified from agarose gel and used as a megaprimer (G. Sarkar and S.S. Sommer (1990) Biotechniques 8: 404-407) together with primer 188clonmgC to amplify the full length gene encoding the mature amylase m a second PCR.

The resulting approximately 1480 bp fragment was digested with restriction endonucleases Pstl and Sfil and ligated with plasmid pTVBllO digested with the same enzymes.

Protease and amylase deleted Bacillus subtili s strain SHa273 (mentioned m WO 95/10603) was transformed with the ligation mixture and the DNA sequence of an amy-plus transformant was verified. This plasmid is denoted pTVB231.

Oligonucleotides:

188 (Pst-) :

5' GGC GTT AAC CGC AGC TTG TAA C (SEQ ID NO: 11)

188clonmgC:

5' CCG AGC TCG GCC GGC TGG GCC GTC GAC TTA TTT GTT TAC CCA AAT AGA AAC (SEQ ID NO: 12) 188clonmgN:

5' CAT TCT GCA GCA GCG GCG CAC CAT AAT GGT ACG AAC G (SEQ ID NO: 13)

Example 5

Subclonmg of the AA560 alpha-amylase into pTVBllO.

DNA sequencing revealed a high DNA identity between alpha- amylases from stains DSM12648 (AA349) and DSM 12649 (AA560) . Consequently the same oligonucleotides and strategy was utilized for the cloning of AA560 alpha-amylase into expression vector pTVBllO resulting plasmid pTVB232, which was then fermented using standard techniques.

Example 6 Purification of the parent AA560 Alpha-Amylase

The culture broth was flocculated by adding 0.01 ml

50% (w/w) CaCl₂,2H₂0, 0.0125 ml 12% (w/w) Sodium alummate,

0.025 ml 10% C521 and 0.075 ml 0.1% A130 pr. ml culture broth.

A clear solution was obtained after centrifugation. The enzyme solution was added ammonium sulphate to a final concentration of 1.2 M and applied on a Butyl Toyo Pearl column (100 ml) previously equilibrated in 1.2 M ammonium sulphate, 10 mM Tris- HCl, pH 7.0. The amylase was eluted using 5 mM Tris-HCl, pH 7.0 and the eluted pool was dialysed against 5 mM Tris-HCl over night. The fraction was then subjected to ion exchange chromatography using a Q-Sepharose column (200 ml) previously equilibrated in 20 mM Tris-HCl, pH 9.0. Unbound material was washed out with the equilibration buffer, and the amylase was eluted using a linear gradient 0 - 1 M NaCl, 20 mM Tris-HCl, pH 9.0. Purity of the amylase preparation was above 95% judged by SDS-PAGE.

Example 7

Characterization of the parent AA560 Alpha-Amylase

The alpha-amylase activity was measured using both the Phadebas assay (37°C, pH 7.3) and the Alternative pNPG7 Assay (25°C, pH 7.1) described above. pH- and temperature profiles were made at selected pH- and temperature values. The pH- profile was measured at 37°C and the temperature profile was measured at pH 9.0 Isoelectric Point was determined using isoelectric focusing (Pharmacia, Ampholine, pH 3.5-9.3).

Table 1. Specific Activity and pi.

Specific activity

Enzyme NU/mg NU(T) /mg pi

Phadebas pNPG7

AA560 (SEQ ID NO: 4) 35, 000 9, 000 7-8

SP722 (SEQ ID NO: 2 of 35, 000 6, 000 7-9

US patent no. 5,856,164)

SP690 (SEQ ID NO: 1 of 35, 000 7, 000 5-6 US patent no . 5 , 856 , 164 )

E = 3.0 cm¹ *(g/l) ^l for AA560, SP722 and SP690

The result of the pH-optimum determination and temperature optimum determination is shown Figure 2 and Figure 3, respectively.

Example 8

Washing test with Parent AA560 Alpha-Amylase

Washing performance was evaluated by washing soiled test swatches for 15 and 30 minutes at 25°C and 40°C, respectively, in detergent solutions with the AA560 alpha-amylase of the invention.

The detergents used are disclosed m Table 2 below. The A/P Model Detergent is described the Materials section above. The other detergents are commercially available detergents. Commercial detergents containing alpha-amylase was inactivated by microwaves before wash.

The purified recombinant AA560 alpha-amylase of Example 6 was added to the detergent solutions at the concentration indicated below. The test swatches were soiled with orange rice starch (CS-28 swatches available from CFT, Center for Test Material, Holland) .

After washing, the swatches were evaluated by measuring the remission at 460 nm using an Elrepho Remission Spectrophotometer . The results are expressed as ΔR = remission of the swatch washed with the alpha-amylase minus the remission of a swatch washed at the same conditions without the alpha- amylase .

Table 2. Detergents and wash conditions.

Area Detergent Det Inacti Enzyme Temp Time pH Water Ca Dose vation dose hardnes Mg g/ 1 mg/ 1 °c Min s

°dH

A/P Model 3 25 15 10 . 5 2 : 1 detergent

97

Latin Omo Multi 3 25 15 10 . 6 2 : 1

America Acao

Europe Omo cone . 4 0.2 40 30 10.2 15 4 :1 Powder

Europe Ariel 5 0.2 40 30 9.0 15 4 :1 Futur liquid

The results are shown in Figures 4-7. The results demonstrate that the alpha-amylase of the invention is effective in all detergents at highly alkaline pH and show that the AA560 alpha-amylase has improved wash performance in comparison to SP690 and SP722, respectively.

Example 9

Desizing test with the AA560 Alpha-Amylase

The parent AA560 alpha-amylase was used for desizing of fabrics using the TEGEWA method (Method and standard scales obtainable from Verband TEGEWA, Karlstrasse 21, Frankfurt a.M., Germany) . The conditions are described in the "Materials & Methods" section.

The AA560 alpha-amylase was used in desizing tests carried out at 30 and 55°C. The enzyme was dosed from 0.05 to 2 g/1 in the impregnation solution. The after-washing procedure was carried out by washing the fabrics m water - instead of the usually hot soda wash.

The effect of was measured using the Violet Scale (TEGEWA) TEGEWA scale: 1-9.

Grade 1 = not desized, grade 9 = totally desized.

AA560: 30 °C, 22 hours and 55 °C, 2 hours

Example 10

Construction of the deposited material pTVB299

The mature gene has been subcloned into plasmid pzero-2 (Invitrogen, Gron gen, The Nederlands) . An approximately 1.5 kb Pstl-Sacl fragment containing the complete mature region of AA560 (obtained from pTVB232) was ligated with vector pZero-2, digested with the same restriction endonucleases. The ligatation mixture was transformed into competent E. coli cells Transformants were analyzed by PCR to verify the presence of the inserted fragment and the part of this fragment encoding the mature alpha-amylase was sequenced. The resulting plasmid, denoted pTVB299, is deposited at DSMZ as DSM12764.

Example 11

Construction of variants of the AA560 Alpha-Amylase

The gene encoding the AA560 alpha-amylase shown m SEQ ID NO: 2 is located m a plasmid pTVB223. The amylase is expressed from the amyL promoter m this construct m Bacillus subtilis . A variant of the invention with delta (D183-G184 ) mutations was constructed by the mega-primer method as described by Sarkar and Sommer, (1990), BioTechmques 8: 404-407.

Gene specific primer Bl and mutagenic primer 101458 were used to amplify by PCR an approximately 645 bp DNA fragment from a pTVB223 plasmid encoding AA560 shown m SEQ ID NO : 12) . The 645 bp fragment was purified from an agarose gel and used as a mega-primer together with primer Y2 m a second PCR carried out on the same template.

The resulting approximately 1080 bp fragment was digested with restriction enzymes BstEII and Afllll and the resulting approximately 510 bp DNA fragment was purified and ligated with the pTVB223 plasmid digested with the same enzymes. Competent Bacillus subtilis SHA273 (amylase and protease low) cells were transformed with the ligation and Chlorampenicol resistant transformants and was checked by DNA sequencing to verify the presence of the correct mutations on the plasmid. primer Bl :

5' CGA TTG CTG ACG CTG TTA TTT GCG 3' (SEQ ID NO: 14) primer Y2 : 5' CTT GTT CCC TTG TCA GAA CCA ATG 3' (SEQ ID NO : 15) primer 101458:

5' GT CAT AGT TGC CGA AAT CTG TAT CGA CTT C 3' (SEQ ID NO : 16) The resulting plasmid encoding the AA560 alpha-amylase with delta (D183-G184) +N195F was named pTVB232. The construction of other variants of the invention may be carried out m a similar manner.

Example 12

Testing the washing performance of AA560 variants The wash performance of AA560 variants of the invention is tested as described m Example 8.

Example 13

Testing the specific activity of AA560 variants The specific activity of AA560 variants is determined as described m Example 7. Example 14

Testing the Calcium stability of AA560 variants

The calcium stability of AA560 variants is tested using the assays described m the "Materials and Method" section.

The invention described and claimed herein is not to be limited scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be withm the scope of this invention. Indeed, various modifications of the invention m addition to those shown and described herein will become apparent to those skilled m the art from the foregoing description. Such modifications are also intended to fall withm the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which are incorporated by reference m their entireties.

References

Sambrook et al . ; Molecular Cloning: A Laboratory Manual; 1989,;

Cold Spring Harbor Lab.; Cold Spring Harbor; NY.

Ausubel, F. M. et al . (eds.); Current protocols in Molecular Biology; 1995; John Wiley and Sons.

Harwood C. R., and Cutting S. M. (eds.); Molecular Biological

Methods for Bacillus; 1990; John Wiley and Sons.

Diderichsen B., Wedsted U. , Hedegaard L., Jensen B. R. , Sjøholm

C . ; Cloning of aldB, which encodes alpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevi s ; J .

Bacteriol. , 1990, vol. 172, pp. 4315-4321.

Pitcher D. G., Saunders N. A., Owen R. J.; Rapid extraction of bacterial genomic DNA with guanidium thiocyanate; Lett. Appl .

Microbiol . ; 1989; vol. 8; pp. 151-156. Dretzen G., Bellard M. , Sassone-Corsi P., Chambon P.; A reliable method for the recovery of DNA fragments from agarose and acrylamide gels; Anal . Biochem. ; 1981; vol. 112; pp. 295-

298.

Claims

ClaimsWhat is claimed is:

1. An isolated polypeptide having alpha-amylase activity and one or more characteristics or properties selected from the group consisting of:

(a) a polypeptide having an ammo acid sequence which has at least 96% identity with ammo acids 1 to 485 of SEQ ID NO : 2 or SEQ ID NO: 4 ; (b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (l) the nucleic acid sequence of SEQ ID NO : 1 or SEQ ID NO: 3, (ii) the cDNA sequence of SEQ ID NO:l or SEQ ID NO: 3, (in) a subsequence of d) or (n) of at least 100 nucleotides, or dv) a complementary strand of d) , (n) , or (in) ;

(c) an allelic variant of (a) or (b) ;

(d) a fragment of (a) , (b) , or (c) that has alpha-amylase activity;

(e) a polypeptide with a pH optimum determined using the Phadebas method (37°C) m the range between pH 8 and 9;

(f) a polypeptide a temperature optimum determined using the Phasebas method (pH 9.0) m the range between 55 and 65°C;

(g) a polypeptide with a pi between 7-8 determined by isoelectric focusing (Pharmacia, Ampholme, pH 3.5-9.3); and (l) a polypeptide with improved washing and/or dishwashing performance between pH 9-11.

2. The polypeptide of claim 1, having an ammo acid sequence which has at least 96% identity with ammo acids 1 to 485 of SEQ ID NO: 2 or SEQ ID NO: 4

3. The polypeptide of claim 1, comprising the ammo acid sequence of SEQ ID NO : 2 or SEQ ID NO: 4..

4. The polypeptide of claim 1, consisting of the ammo acid sequence of SEQ ID NO : 2 or SEQ ID NO: 4 or a fragment thereof.

5. The polypeptide of claim 1, which consists of ammo acids 1 to 485 of SEQ ID NO : 2 or SEQ ID NO: 4.

6. The polypeptide of claim 1, which is encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (I) the nucleic acid sequence of SEQ ID NO : 1 or SEQ ID NO: 3, (ii) the cDNA sequence of SEQ ID NO : 1 or SEQ ID NO: 3, (m) a subsequence of (I) or (n) of at least 100 nucleotides, or (IV) a complementary strand of (l) , (ii) , or (in) .

7. The polypeptide of claim 1, which is encoded by the nucleic acid sequence contained m plasmid pLιH1274 or pTVB299 contained m E. coli DSM12761 or E . coli DSM12764, respectively.

8. A polypeptide having the same alpha-amylase activity as the polypeptide of any of claims 1-7.

9. An isolated nucleic acid sequence comprising a nucleic acid sequence which encodes the polypeptide of any of claims 1-

10. An isolated nucleic acid sequence comprising a nucleic acid sequence having at least one mutation m the mature polypeptide coding sequence of SEQ ID NO : 1 or SEQ ID NO: 3, m which the mutant nucleic acid sequence encodes a polypeptide consisting of ammo acids 1 to 485 of SEQ ID NO: 2 or SEQ ID NO: 4.

11. The isolated nucleic acid sequence of claim 9 or 10 produced by (a) hybridizing a DNA under medium stringency conditions with (l) the nucleic acid sequence of SEQ ID NO : 1 ,

(ii) the cDNA sequence of SEQ ID NO : 1 , (in) a subsequence of (I) or (ii) of at least 100 nucleotides, or (IV) a complementary strand of (I) , (ii) , or (m) ; and (b) isolating the nucleic acid sequence.

12. A recombinant expression vector comprising the nucleic acid construct of claims 9-11.

13. A recombinant host cell comprising the nucleic acid construct of claim 12.

14. A method for producing a mutant nucleic acid sequence, comprising (a) introducing at least one mutation into the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, wherein the mutant nucleic acid sequence encodes a polypeptide consisting of ammo acids 1 to 485 of SEQ ID NO: 2 or SEQ ID NO: 4; and (b) recovering the mutant nucleic acid sequence .

15. A mutant nucleic acid sequence produced by the method of claim 14.

method for producing a polypeptide, comprising (a) cultivating a strain comprising the mutant nucleic acid sequence of claim 15 encoding the polypeptide to produce a supernatant comprising the polypeptide; and (b) recovering the polypeptide .

17. A method for producing the polypeptide of any of claims 1- 8 comprising (a) cultivating a strain to produce a supernatant comprising the polypeptide; and (b) recovering the polypeptide.

18. A method for producing the polypeptide of any of claims 1- 8 comprising (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleic acid sequence encoding the polypeptide under conditions suitable for production of the polypeptide; and (b) recovering the polypeptide.

19. A method for producing a polypeptide comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide, wherein the host cell comprises a mutant nucleic acid sequence having at least one mutation m the mature polypeptide coding sequence of SEQ ID NO : 1 or SEQ ID NO: 3, wherein the mutant nucleic acid sequence encodes a polypeptide consisting of ammo acids 1 to 485 of SEQ ID NO : 2 or SEQ ID NO: 4, and (b) recovering the polypeptide.

20. A mutant of a polypeptide with alpha-amylase activity of any of claims 1 to 8 , wherein said alpha-amylase has one or more mutations, m particular substitution or deletions, m the following positions (relative to SEQ ID NO: 2) : 1: R181*, G182*, D183*, G184*; 2: N195A,R,D,C,E,Q,G,H, I,L,K,M,F, P, S,T,W, Y,V; 3: I206A,R,D,N,C,E,Q,G,H,L,K,M,F,P,S,T,W,Y,V; 4 : E212A,R,D,N,C,Q,G,H, I,L,K,M,F,P,S,T,W,Y,V; 5: E216A,R,D,N,C,Q,G,H, I,L,K,M,F,P,S,T,W,Y,V; 6: K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V; 7: R181A,N,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V.

21. The mutant of claim 20, selected from the group of mutants with the following mutantions: -R181*/G182*/N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

-G182*/T183*/N195A,R,D,C,E,Q,G,H,I L,K,M,F,P,S,T,W,Y,V

-T183*/G184*/R181A,N,D,C,E,Q,G,H, I L,K,M,F,P,S,T,W,Y,V -T183*/G184*/N195A,R,D,C,E,Q,G,H, I L,K,M,F,P,S,T,W,Y,V -R181*/G182*/V206A,R,D,N,C,E,Q,G,H I,L,K,M,F,P,S,T,W,Y -G182*/T183*/V206A,R,D,N,C,E,Q,G,H I,L,K,M,F,P,S,T,W,Y -T183*/G184*/V206A,R,D,N,C,E,Q,G,H I,L,K,M,F,P,S,T,W,Y -R181*/G182*/E212A,R,D,N,C,Q,G,H,I L,K,M,F,P,S,T,W,Y,V -G182*/T183*/E212A,R,D,N,C,Q,G,H,I L,K,M,F,P,S,T,W,Y,V -T183*/G184*/E212A,R,D,N,C,Q,G,H, I L,K,M,F,P,S,T,W,Y,V -R181*/G182*/E216A,R,D,N,C,Q,G,H,I L,K,M,F,P,S,T,W,Y,V -G182*/T183*/E216A,R,D,N,C,Q,G,H, I L,K,M,F,P,S,T,W,Y,V -T183*/G184*/E216A,R,D,N,C,Q,G,H,I L,K,M,F,P,S,T,W,Y,V -R181*/G182*/K269A,R,D,N,C,E,Q,G,H I,L,M,F,P,S,T,W,Y,V -G182*/T183*/K269A,R,D,N,C,E,Q,G,H I,L,M,F,P,S,T,W,Y,V -T183*/G184*/K269A,R,D,N,C,E,Q,G,H I,L,M,F,P,S,T,W,Y,V -N195A,R,D,C,E,Q,G,H, I,L,K,M,F,P,S T,W,Y,V; /V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P S,T,W,Y; -N195A,R,D,C,E,Q,G,H, I,L,K,M,F,P,S T,W,Y,V /E212A,R,D,N,C,Q,G,H, I,L,K,M,F,P,S T,W,Y,V; -N195A,R,D,C,E,Q,G,H, I,L,K,M,F,P,S T,W,Y,V /E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S T,W,Y,V; -N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S T,W, Y,V /K269A,R,D,N,C,E,Q,G,H, I,L,M,F,P,S T,W,Y,V; -V206A,R,D,N,C,E,Q,G,H,I,L,K,M,F,P S,T,W,Y /E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

-V206A,R,D,N,C,E,Q,G,H, I,L,K,M,F,P,S,T,W,Y

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

-V206A,R,D,N,C,E,Q,G,H, I,L,K,M,F,P,S,T,W,Y

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

-E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;

E212A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;

-E216A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V

/K269A,R,D,N,C,E,Q,G,H, I,L,M,F,P,S,T,W,Y,V.

22. The mutant of claims 20 or 21, further comprise a substitution in position_. E216Q.

23. Use of the polypeptide of any of claims 1-8 or variant of claims 20-22 in a detergent composition, in paticular a laundry detergent composition and a dishwash detergent composition.

24. Use of the polypeptide of any of claims 1-8 or variant of claims 20-22 in a desizing composition.

25. Use of the polypeptide of any of claims 1-8 or variant of claims 20-22 for liquefaction of starch.

26. Use of the polypeptide of any of claims 1-8 or variant of claims 20-22 for ethanol production.