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

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 α-amylase activity and isolated nucleic acid sequences encoding the polypeptides. Further, the invention relates to variants of the α-amylases 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 α-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 α-amylases, which is becoming increasingly important, is the removal of starchy stains during washing with a detergent at alkaline pH. Examples of commercial α-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 optimal activity m detergent solutions at alkaline pH. WO 95/26397 discloses an α-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. M. Takagi et al . , J. Ferment. Bioeng., vol 81, No. 6, 557- 559 (1996) describe an alkaliphilic alpha-amylase-pullulanase from Bacillus sp . 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 α-amylase derived from Bacillus sp . strain KSM- AP1378 with the deposited no. FERM BP-3048 suitable for detergents . It is an object of the present invention to provide novel α-amylases with improved performance m alkaline solutions, especially in alkaline detergent solutions at pH around 9-11. Summary of the Invention The present invention relates to isolated polypeptides having α-amylase activity and one or more characteristics or properties selected from the group consisting of: (a) a polypeptide having an amino acid sequence which has at least 85% identity with amino acids for mature polypeptide amino 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 (ι) 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 , (m) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i) , (ii) , or (iii) ; (c) an allelic variant of (a) or (b) ; (d) a fragment of (a) , (b) or (c) that has α-amylase activity; (e) a polypeptide having improved wash performance in alkaline detergent solutions, especially in alkaline detergent solutions at a pH around 9-11, and more preferably, at a pH around 9-10.5; (f) a polypeptide having a temperature optimum determined using the Phadebas method (pH 9.0) m the range between 55 to 65°C; and (g) a polypeptide having a pi between 7-8 determined by isoelectric focusing (Pharmacia, Ampholine, pH 35-9.3) . 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 α- 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 The present invention is further illustrated with reference to the accompanying drawings, in which: Figure 1 shows the alignment of a number of Baci llus α- amylases . Figure 2 shows the pH Profile of the α-amylases AAI-6 and AAI-10 compared to α-amylases SP722 and SP690. The activity s shown in absolute values as Abs650/mg. The pH profile is measured at 37°C. Figure 3 shows the Temperature Profile of the α-amylases 7AAI-6 and AAI-10 at pH 9.0 compared to the reference α- amylases SP722 and SP690. Figure 4 shows the wash performance of AAI-6 and AAI-10 m the AP Model Detergent 97 m comparison to SP722 , SP690 and Termamyl ® . Figure 5 shows the wash performance of AAI-6 and AAI-10 m Omo Multi Acao m comparison to SP722 , SP690 and Termamyl ® . Figure 6 shows the wash performance of AAI-6 and AAI-10 m Omo Concentrated m comparison to SP722 , SP690 and Termamyl ® . Figure 7 shows the wash performance of AAI-6 and AAI-10 m Ariel Futur liquid m comparison to SP722 , SP690 and Termamyl ® . Detailed Description of the Invention Microbial source The alkaline α-amylases of the invention may be derived from a strain of Bacillus . Preferred strains are of Bacillus sp . DSM 12650 (the AAI-6 α-amylase) or DSM 12651 (the AAI-10 α-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 . Escherichia coli strains termed NN049469 and NN049468 containing the α-amylase genes m plasmids pLιH1300 (AAI 6) and pLιH1298 (AAI10), 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 DSM12763 and DSM12762, respectively. The above deposited Bacillus sp . donor organisms were collected Iceland m 1997. Polypeptides Having α-amylase Activity α-Amylases (α-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 polysacchaπdes . For purposes of the present invention, α- 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 polypeptides) of at least about 85%, preferably at least about 90%, preferably at least about 93%, more preferably at least about 95%, even more preferably at least 97%, and most preferably 99% which have α-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. The ammo acid 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. 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. Alternatively, Gap from GCG version 9 may be used with a translated version 8 peptide scoring matrix, a gap creation penalty of 30, a gap extension penalty of 1 using ntol ' s matrix (http : //plasmιd/-bιoweb/matrιx/) without end gap penalty. Homology to known Bacillus sp. α-amylases A homology search of known sequences showed homologies for the sequences of the invention with a number of Bacillus amylases m the range 67-81 % on ammo acid determined as described above . Specifically, homologous α-amylases to the sequences of the invention (i.e., SEQ ID NO: 2 and SEQ ID NO: 4) are SP690 (SEQ ID NO: 1 of US patent no. 5,856,164 which is about 81% homologous), SP722 (SEQ ID NO: 2 of US patent no. 5,856,164 which is about 81% homologous) and the mature part (ammo acids 31-516) of the α-amylase obtained from Bacillus sp . KSM-AP1378 disclosed as SEQ ID NO: 2 of WO 97/00324 which is about 81% homologous to the sequences of the invention. Preferably, the polypeptides of the present invention comprise the ammo acid sequence of SEQ ID NO : 2 or SEQ ID NO : 4 or an allelic variant thereof; or a fragment thereof that has σ-amylase activity. SEQ ID NO: 2 or SEQ ID NO: 4 show the mature part the alkaline α-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 polymorphism within populations. Gene mutations can be silent (no change m 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 am o 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 protem; small deletions, typically of one to about 30 ammo acids; small ammo- or carboxyl -terminal extensions, such as an ammo-terminal methionine 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 antigemc epitope or a binding domam. Examples of conservative substitutions are within the group of basic ammo acids (arginme, lysine and histidine) , acidic ammo acids (glutamic acid and aspartic acid) , polar ammo acids (glutamine and asparagine) , hydrophobic ammo acids (leucine, isoleucine and valine) , aromatic ammo acids (phenylalanme, tryptophan and tyrosme), and small ammo acids (glycine, alamne, serine, threonine and methionine) . Ammo acid substitutions, which do not generally alter the specific activity are known m 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, eu/lle, Leu/Val , Ala/Glu, and Asp/Gly as well as these m reverse. In a second embodiment, the present invention relates to isolated polypeptides having α-amylase activity which are encoded by nucleic acid sequences which hybridize under medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with a nucleic acid probe which hybridizes under the same conditions with (l) the nucleic acid sequence of SEQ ID NO : 1 or SEQ ID NO: 3, (il) the cDNA sequence of SEQ ID NO : 1 or SEQ ID NO : 3 , (m) a subsequence of (I) or (ii), or (iv) a complementary strand of (l) , (ii) , or (in) (J. Sambrook, E.F. Fπtsch, and T. Ma atus, 1989, Mol ecular Cloning, A Laboratory Manual , 2d edition, Cold Spring Harbor, New York) . The subsequence of SEQ ID NO:l or SEQ ID NO : 3 may be at least 100 nucleotides or preferably at least 200 nucleotides. Moreover, the subsequence may encode a polypeptide fragment, which has α-amylase activity. The polypeptides may also be allelic variants or fragments of the polypeptides that have α-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 α-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 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 32 P, 3 H, 35 S, biot , 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 α-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 m SEQ ID NO : 1 or SEQ ID NO: 3, its complementary strand, or a subsequence 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 m plasmids pLιH1300 (AAI6) or pLιH1298 (AAI10) , respectively, which are contained m Escherichia coli DSM12763or Escheri chia coli DSM12762, respectively, or, wherem the nucleic acid sequence encodes a polypeptide having acid α-amylase activity of the invention and shown m SEQ ID NO: 2 or SEQ ID NO : 4, respectively. For long probes of at least 100 nucleotides in length, medium to very high stringency conditions are defined as prehybπdization and hybridization at 42 °C m 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 m 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 m length, stringency conditions are defined as prehybridization, hybridization, and washing post -hybridization at 5°C to 10°C below the calculated T m using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sci ences USA 48:1390) m 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 temperature optimum (see Fig. 3) determined using the Phadebas method (pH 9.0) m the range between 55 and 65°C, more precisely at about 60°C. A pi for AAI-6 determined using lsoelectπc focusing was found to be between 7-7.3, and the pi for AAI-10 was found to be between 7-8. 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 α-amylase activity of the mature polypeptide of SEQ ID NO: 2 or 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 m 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 m which the nucleic acid sequence from the source has been inserted. A polypeptide of the present invention may be a bacterial polypeptide. For example, tne 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 len tus , Bacillus li chen! formis , Bacillus megateriu , Bacillus stearother ophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide; or a Streptomyces polypeptide, e . g . , a St e to-nyces lividans or Streptomyces murinus polypeptide; or a gram negative bacterial polypeptide, e . g. , an E . coli or a Pseudomonas sp . polypeptide. In another preferred embodiment, the polypeptide is a Bacillus sp . polypeptide, more preferred embodiment, the polypeptide is a Bacillus sp . DSM 12650 and Bacillus sp . DSM 12651 polypeptide, e . g. , the polypeptide with the amino acid sequence of SEQ ID NO: 2 or 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 in the art. The nucleic acid sequence may then be derived by similarly 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-α-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 ligating 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 9, 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 α-amylases Altered properties of variants of the invention The following discusses the relationship between mutations, especially substitutions and deletions, which may be introduced into in the AAI-6 or AAI10 alpha-amylases of the invention, and desirable alterations in properties relative to those of the parent alpha-amylases. Invention also relates to a mutant of the α-amylases shown in SEQ ID NO: 2 (AAI-10) or SEQ ID NO: 4 (AAI-6) . The mutant α-amylase of to the invention is characterized by the fact that one or more of the methionine amino acid residues is exhanged with any amino acid residue except for Cys and Met. Thus, according to the invention the amino acid residues to replace the methionine amino 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 α-amylase of the invention is characterized by the fact that one or more of the methionine amino acid residues is (are) exchanged with a Leu, Thr, Ala, Gly, Ser, lie, or Asp amino acid residue, preferably a Leu, Thr, Ala, or Gly amino acid residue. In this embodiment a very satisfactory activity level and stability in the presence of oxidizing agents is obtained. Specifically this means that one or more of the methionines in the following position may be replaced or deleted using any suitable technique known in the art, including especially site directed mutagenesis and gene shuffling. Contemplated positions in SEQ ID NO: 2 are: 10, 105, 202, 208, 246, 286, 309, 430, 440 and positions in SEQ ID NO: 4 are: 10, 105, 202, 208, 246, 286, 309, 430, 440, 454. In a preferred embodiment of the mutant α-amylase of the invention is characterized by the fact that the methionine ammo acid residue at position 202 is exchanged with any of am o 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 mclude: deletion of D183 -G1844-N195F, deletion of D183-G184+G186R, deletion of D183 -G184+G186R+N195F and G186R+N195F . Increased thermostability Mutations resulting m variants (of both AAI-10 and AAI-6) of the invention, e.g., having increased thermostability, m particular at acidic pH and/or at low Ca 2+ concentration include mutations at the following positions (usmg the AAI-10 alpha- amylase numbering, i.e., SEQ ID NO: 2) : R158, S174, A186, N195, N197, H210, E212, 1214, N215, E216, K269, N270. In the context of the invention the term "acidic pH" means a pH below 7.0, especially below the pH range, m 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 Ca 2+ in the corn. Normally a dosage corresponding to 1 mM (40ppm) is added which together with the level m corn gives between 40 and 60 ppm free Ca 2+ . In the context of the invention the term "high temperatures" means temperatures between 95°C and 160°C, especially the temperature range m 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 Ca 2+ concentration can be increased even more by combining other mutations including the above-mentioned mutations and/or V206 with each other. Said "other" mutations are the following (relative to the AAI-10 alpha-amylase, SEQ ID NO: 2) : N195, E212, E216, K269 and V206. Said mutation may further be combined with deletions m one, preferably two or even three or more positions as described m WO 96/23873 ( i . e . , m positions R181, G182, D183, G184 m SEQ ID NO: 2 herein) . According to the present invention, variants of the parent AAI-10 and AAI-6 alpha-amylases may comprise mutations m one, two, three, four, five, six, seven or more of the above positions . It should be emphasized that not only the AAI-10 and AAI-6 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 ammo acid residues, respectively, at positions corresponding to N195, V206, E212 and E216, respectively, m SEQ ID NO: 2 or SEQ ID NO: 4 constitute ammo acid residues, which are conserved m numerous Termamyl- like alpha-amylases, i.e., Termamyl ® (B . licheniformis alpha- amylase) . Thus, for example, the corresponding positions of residues m AAI-10 and AAI-6 and Termamyl can be see the alignment m Fig. 1 and m Table 1 and Table 2, below. Table 1. Termamyl -like alpha-amylase B . li cheniformis (Termamyl) N190 1201 H205 E211 N265 SP690 N195 V206 H210 E216 N270 SP722 N195 V206 H210 E216 N270 AAI-10 (SEQ ID NO: 2) N195 V206 H210 E216 N270 AAI-6 (SEQ ID NO: 4) N195 V206 H210 E216 N270 Table 2 Termamyl -like alpha-amylase SP690 R181, G182, T183, G184, K185, A186 SP722 R181, G182, D183, G184, K185, A186 AAI-10 (SEQ ID NO: 2 ) R181, G182, D183, G184, K185, A186 AAI-6 (SEQ ID NO. 4) R181, G182, D183, G184, K185, A186 Mutations of these conserved ammo acid residues are very important m 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 Ca 2+ concentrations (relative to SEQ ID NO: 2 or SEQ ID NO: 4 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: V206A,R,D,N,C,E,Q,G,H,,I,L,K,M,F,P,S,T,W,Y; 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 or SEQ ID NO: 4 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*/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. In a preferred embodiment the variant comprises the following mutations: N195F/K264S in SEQ ID NO: 2 or SEQ ID NO : 4 or in corresponding positions in 85% homologous alpha-amylases as defined herein. In another embodiment the variant of the invention comprises the following mutations: R181*/G182*/N195F in SEQ ID NO: 2 or SEQ ID NO: 4 in corresponding positions in another homologous alpha-amylases. Said variant may further comprise a substitution in position E216Q. Improved Ca 2 " stability of AAI-10 and AAI-6 variant at pH 8-10.5 Improved Ca 2+ stability means the stability of the enzyme under Ca 2+ 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 Ca 2+ 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 m addition to those outlined above. Thus, it may be advantageous that one or more prolme residues present 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 ala ne, glycine, serine, threonine, valme or leucine. Analogously, it may be preferred that one or more cysteine residues present among the ammo acid residues with which the parent alpha-amylase is modified is/are replaced with a non- cyste e residue such as serine, alamne, threonine, glycine, valme or leucine. 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 an ammo acid fragment corresponding to the ammo acid fragment 190-214 of SEQ ID NO: 2 or SEQ ID NO: 4 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 an ammo acid fragment corresponding to the ammo acid fragment 190-214 of SEQ ID NO: 2 or SEQ ID NO: 4 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 in any of the variants described herein. Mutations of the invention may suitably include mutations in the following positions: Y135, V17, M202, 1214. Cloning a DNA sequence encoding an α-amylase The DNA sequence encoding a parent alpha-amylase as defined above may be isolated from any cell or microorganism producing the alpha-amylase in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the alpha-amylase to be studied. Then, if the amino acid sequence of the alpha-amylase is known, homologous, labelled oligonucleotide probes may be synthesized and used to identify alpha-amylase-encoding clones from a genomic library prepared from the organism in question. Alternatively, a labelled oligonucleotide probe containing sequences homologous to a known alpha-amylase gene could be used as a probe to identify alpha-amylase-encoding clones, using hybridization and washing conditions of lower stringency. Yet another method for identifying alpha-amylase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming alpha- amylase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for alpha-amylase, thereby allowing clones expressing the alpha-amylase to be identified. 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 oligo- nucleotides 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-encoding sequence, is created m a vector carrying the alpha-amylase gene. Then the synthetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the smgle-stranded DNA. The remaining 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 disclose the introduction of oligonucleotides encoding multiple mutations by performing minor alterations of the cassette. However, an even greater variety of mutations can be introduced at any one time by the Mormaga method, because a multitude of oligonucleotides, of various lengths, can be introduced. Another method for introducing mutations mto 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 syn- thesized 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 mto an expression plasmid. Random Mutagenesis Random mutagenesis is suitably performed either as localised or region-specific random mutagenesis at least three parts of the gene translating to the ammo acid sequence shown m question, or within the whole gene. The random mutagenesis of a DNA sequence encoding a parent alpha-amylase may be convemently performed by use of any method known m 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. wherem 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 m step (a) m 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 mutagenizmg 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 mutagenizmg agents. The mutagenizmg agent may, e . g . , be one that induces transitions, transversions, inversions, scrambling, deletions, and/or insertions. presence of the mutagenizmg 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 ammo acids are avoided. The doped or spiked oligonucleotide can be incorporated mto the DNA encoding the alpha-amylase enzyme by any published technique, usmg 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 m 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 the choice of a doping scheme is based on genetic as well as protein-structural constraints. The doping scheme may be made usmg the DOPE program (see "Material and Methods" section) , wnich, 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- corporation of nucleotides (Deshler 1992; Leung et al . , Technique, Vol.l, 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 mto 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 mto 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 bacteriophage, which as such may be incubated with or otherwise exposed to the mutagemsmg agent. The DNA to be mutagenized may also be present m 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 m 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 m 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 m 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 m the art. Alternatively, the DNA sequence encoding the part of the DNA sequence to be modified may be isolated, e . g. , by insertion mto 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 m the art including the methods, e . g. , described m 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 alternative methods known m tne art, can be expressed, m 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 mto which it is to be introduced. Thus, the vector may be an autonomously replicatmg 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, mimchromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced mto a host cell, is integrated mto the host cell genome and replicated together with the chromosome (s) mto 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 m a bacterial host, are the promoter of the lac operon of E. coli , the Streptomyces coelicolor agarase gene dagrA promoters, the promoters of the Bacillus licheniformis alpha- amylase gene (amyL) , the promoters of the Bacillus stearothermophilus maltogemc 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, Rhizo ucor miehei aspartic proteinase, 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, m eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the alpha-amylase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter. The vector may further comprise a DNA sequence enabling the vector to replicate m the host cell m 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 m the host cell, such as the dal genes from B . subtilis or B . licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycm, chloramphemcol or tetracyclm resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, maD and sC, a marker giving rise to hygromycm resistance, or the selection may be accomplished by co-transformation, e.g., as described m WO 91/17243. While intracellular 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 mto 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 in the recombinant production of an alpha-amylase variant of the invention. The cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell . Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e . g. , by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells. 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 amyloliquefaci ens, Bacillus coagulans , Bacillus circulanε, 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 trans- formation or by using competent cells in 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 centrifugation 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 In another more preferred embodiment, the nucleic acid sequence is the sequence contained m plasmid pLIH1300 (AAI 6) or plasmid LιH1298 (AAI 10) , respectively, that is contained m Escherichia coli DSM12763 and Escherichia coli DSM 12762, respectively. In another preferred embodiment, the nucleic acid sequence is the mature polypeptide coding region of SEQ ID NO: 1 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 or SEQ ID NO: 4 which differ from SEQ ID NO: 1 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 α-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 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 screenmg 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 Applica tion , 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 stram 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 mto a vector molecule, and incorporation of the recombinant vector mto 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 sequences 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 85% homology on DNA level, preferably about 90%, more preferably about 93%, more preferably about 95%, even more preferably about 97%, and most preferably about 99% homology, which encodes 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 GCGvδ 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 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 Purifi cation 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 α- amylase 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, Science 255: 306-312; Smith et al . , 1992, Journal of Mol ecular 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 α-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 mto the mature polypeptide coding sequence of SEQ ID NO : 1 or SEQ ID NO: 3 or a subsequence thereof, wherem 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 α-amylase activity. The introduction of a mutation mto 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-contammg synthesized DNA. Other procedures known m the art may also be used. These other procedures include gene shuffling, e . g. , as described WO 95/22625 (from Affymax 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 m 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 includmg, but not limited to, transcription, post-transcriptional 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 a manner which would not otherwise exist nature. The term nucleic ac d 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 prote 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 mto 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 mclude 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 the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. 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 subtili s levansucrase gene ( sacB) , Bacillus licheniformis alpha-amylase gene ( amyL) , Bacillus stearothermophilus maltoge c amylase gene ( a yM) , Bacillus amyloliquefaciens alpha-amylase gene ( amyQ) , Bacillus licheniformi s penicillinase 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 National 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 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 coding region that codes for an ammo acid sequence linked to the ammo terminus of a polypeptide and directs the encoded polypeptide mto the cell's secretory pathway. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked m 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 coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region m order to enhance secretion of the polypeptide. However, any signal peptide coding region which directs the expressed polypeptide mto 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 maltogemc amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilism, Bacillus licheniformi s beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS , nprM) , and Bacillus subtilis prsA . Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109- 137. 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 metallothionein 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 mto an appropriate vector for expression. In creating the expression vector, the coding sequence is located m 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 tne 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 replicatmg 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 mimchromosome, or an artificial chromosome. The vector may contain any means for assuring self -replication. Alternatively, the vector may be one which, when introduced mto the host cell, is integrated mto the genome and replicated together with the chromosome (s) mto 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 mto 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 subtili s or Bacillus licheniformis , or markers, which confer antibiotic resistance such as ampicillin, kanamycm, chloramphemcol or tetracyclme resistance. Suitable markers for yeast host cells are ADE2 , HIS3, LEU2 , LYS2 , MET3 , TRP1, and URA3. A selectable marker for use m a filamentous fungal host cell may be selected from the group including, but not limited to, amdS (acetamidase) , argB (ornithme carbamoyltransferase) , bar (phosphmothricm acetyltransferase) , hygB (hygromycm phosphotransferase) , maD (nitrate reductase), pyrG (orotιdme-5 ' -phosphate αecarboxylase) , sC (sulfate adenyltransferase) , trpC 'anthra late synthase) , as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus . 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 in 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) in the chromosome (s) . To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 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 integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell . Furthermore, the integrational elements may be non-encoding or encoding 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 m 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 2mιcron origin of replication, ARS1, ARS4 , the combination of ARS1 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 Na tional Academy of Sciences USA 75: 1433). More than one copy of a nucleic acid sequence of the present invention may be inserted mto 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 mto 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 m 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 m 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 amyloliquefaciens, Bacillus brevis, Bacillus circulans , Bacillus clausii , Bacillus coagulans, Bacillus lautus, Bacillus lentus , Bacillus li cheniformis , Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis ; or a Streptomyces cell, e . g. , Streptomyces lividans or Streptomyces murinus , 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 Spizizm, 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, wherem the host cell comprises a mutant nucleic acid sequence having at least one mutation m 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 a polypeptide of the present invention. Preferably, the compositions are enriched in a polypeptide of the present invention. In the present context, the term "enriched" indicates that the α-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 aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha- galactosidase, beta-galactosidase , glucoamylase, alpha- glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, 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 Humi cola insolens , or Fusarium, preferably Fusarium bactridioides, Fusarium cerealis , Fusarium crookwellense , Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi , Fusarium oxysporum, Fusarium reti culatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides , or Fusarium venenatum . The polypeptide compositions may be prepared in accordance with methods known the art and may be the form of a liquid or a dry composition. For instance, the polypeptide composition may be m the form of a granulate or a microgranulate. 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 α- amylases of the invention are well suited for use m a variety of industrial processes, particular the enzyme finds potential applications as a component m detergents, e.g., laundry, dish wash 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 the production of sweeteners and ethanol, such as fuel, drinking and industrial ethanol, from starch or whole grains. Starch Conversion Conventional starch-conversion processes, such as liquefaction and sacchaπfication processes are described m, 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 α-amylases 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 repulpmg occurs at pH above 7 and where amylases can facilitate the disintegration of the waste material through degradation of the reinforcing starch. The α- amylases of the invention is especially useful a process for producing a papermakmg pulp from starch-coated printed 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 α-amylases of the invention may also be very useful m modifying starch where enzymatically modified starch is used m papermakmg together with alkaline fillers such as calcium carbonate, kaolin and clays. With the alkaline a-amylases of the invention it 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 Tne α-amylases of the invention may also be very useful m textile desizmg In the textile processing industry, a-amylases are traditionally used as auxiliaries in the desizmg process to facilitate the removal of starch- containing size, which has served as a protective coating on weft yarns during weaving Complete removal of the size coating after weaving is important to ensure optimum results in the subsequent processes, m which the fapπc is scoured, bleached and dyed Enzymatic starch breakdown is preferred because it does not involve any harmful effect on the f iber material . In order to reduce processing cost and increase mill throughput , the desizing 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 in alkaline solutions . The alpha-amylases may be used alone or in combination with a cellulase when desizing cellulose-containing fabric or textile . Desi zing and bleaching processes are well known in the art . For instance , such processes are described in WO 95/21247 , US patent 4 , 643 , 736 , EP 119 , 920 hereby in corporate by reference . Commercially available products for desizing include Aquazyme ® and Aquazyme ® Ultra from Novo Nordisk A/S . Beer making The α-amylases of the invention may also be very useful in a beer-making process; the α-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 rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations . 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 m effective amounts. Proteases : Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protem engineered mutants are included. The protease may be a serine 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, subtilis Carlsberg, subtilism 309, subtilism 147 and subtilism 168 (described m WO 89/06279) . Examples of trypsm- like proteases are trypsm (e.g. of porcine or bovine origin) and the Fusarium protease described 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 protem engineered mutants are included. Examples of useful lipases mclude lipases from Humi cola (synonym Thermomyces) , e.g. from H. lanuginosa ( T. lanuginosus) as described m EP 258 068 and EP 305 216 or from H. insolens 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. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp . strain SD 705 (WO 95/06720 and WO 96/27002) , P. wi scons inens is (WO 96/12012) , a Bacillus lipase, e.g. from B. subtilis (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 in 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 (α and/or β) include those of bacterial or fungal origin. Chemically modified or protem engineered mutants are included. Amylases include, for example, α-amylases obtained from Bacillus, e.g. a special strain of B . licheniformis, described m more detail m GB 1,296,839. Examples of useful amylases are the variants described m WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in 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 Purastar™ (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, Humi cola , Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporu disclosed in 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 in 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 in 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) , Clazinase™, and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation) . Peroxidases/Oxidases : Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus , e.g. from C. cinereus , and variants thereof as those described in WO . 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. as a granulate, a liquid, a slurry, etc. Preferred detergent additive formulations are granulates, m particular non-dusting granulates, liquids, particular stabilized liquids, or slurries . Non-dustmg granulates may be produced, e.g., as disclosed m 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 nonylphenols 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 triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given m GB 1483591. Liquid enzyme preparations may, for m- stance, 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 cationic and/or zwitteriomc. 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-olefmsulfonate, alkyl sulfate (fatty alcohol sulfate) , alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccimc 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, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylammeoxide, ethoxylated fatty acid monoethanolamide, 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, tπphosphate, phosphonate, carbonate, citrate, nitrilotπacetic acid, ethylenediammetetraacetic acid, diethylenetπammepentaacetic acid, alkyl- or alkenylsuccimc acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst) . The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly (vmylpyrrolidone) , poly (ethylene glycol), poly (vinyl alcohol), poly (vmylpyridme- N-oxide) , poly (vmylimidazole) , polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers. The detergent may contain a bleaching system which may comprise a H 2 0 2 source such as perborate or percarbonate which may be combined with a peracid- forming bleach activator such as tetraacetylethylenediamme or nonanoyloxybenzenesulfonate . 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 usmg 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 boromc acid derivative such as 4-formylphenyl boromc acid, and the composition 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 redeposition agents, dyes, bactericides, optical brighteners, 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 protem per liter of wash liqour, preferably 0.05-5 mg of enzyme prote per liter of wash liqour, m particular 0.1-1 mg of enzyme protem per liter of wash liqour. 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 in 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 triphosphate 20 - 40% Sodium carbonate 0 - 20% Sodium perborate 2 - 9% Tetraacetyl ethylene dia ine (TAED) 1 - 4% Sodium sulphate 5 - 33% Enzymes 0.0001 - 0.1% 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 co6 - 25% polymer) Enzymes 0.0001 - 0.1% Perfume 0.1 - 0.5% Water 5 - 10 ) 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% Sodium polyacrylate 0 - 8% Enzymes 0.0001 - 0.1% 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 diamine (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% 6) POWDER AND LIQUID DISHWASHING COMPOSITION WITH CLEANING SURFACTANT SYSTEM A blend of C 12 -C 15 ethoxylated alcohols with an average degree of 0 - 6.5% ethoxylation of 9 A blend of C 13 -C 15 ethoxylated alcohols with an average degree of 0 - 4% ethoxylation of 30 Sodium disilicate 0 - 33% Sodium tripolyphosphate 0 - 46% Sodium citrate 0 - 28% Citric acid 0 - 29% Sodium carbonate 0 - 20% Sodium perborate monohydrate 0 - 11.5% Tetraacetyl ethylene diamine (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 C 16 -C 18 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 polyglycol 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 C- 2 _ C 14 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 tristearate 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 amine (MEA) 0 - 1.86% 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 diamine (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 triphosphate 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% 11) Automatic dishwashing compositions as described m 1), 2), 3), 4), 6) and 10), wherem perborate is replaced by per- carbonate . 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 α-amylase activity m detergent compositions; in particular laundry detergent compositions and dish wash detergent compositions, hard surface cleaning compositions, and m composition for desizmg 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: α-amylase disclosed SEQ ID NO: 1 of US patent no. 5,856,164. SP722: α-amylase disclosed m SEQ ID NO: 2 of US patent no. US patent no. 5,856,164. Termamyl ® : α-amylase from Bacillus li cheniformis disclosed in SEQ ID NO: 1 of US patent no. 5,830,837. AAI-10: α-amylase of the invention shown in SEQ ID NO: 2. AAI-6: α-amylase of the invention shown in SEQ ID NO: 4. Model detergent: A/P (Asia/Pacific) Model Detergent has the following composition: 20% STPP (sodium tripolyphosphate) , 25% Na 2 S0 4 , 15% Na 2 C0 3 , 20% LAS (linear alkylbenzene sulfonate, Nansa 80S) , 5% C 12 -C 15 alcohol ethoxylate (Dobanol 25-7), 5% Na 2 Si 2 0 5 , 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 NN17562 DSM 12650 25 th January 1999; NN17563 DSM 12651 25 tn January 1999; NN049469 DSM12763 7 th April 1999; NN049468 DSM12762 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 where 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: Strain SHa273 disclosed m WO 95/10603 E. coli strain SJ2 (Diderichsen et al . (1990)), J. Bacteriol . , vol. 172, pp. 4315-4321. Plasmids : The gene bank vector was pSJ1678 which is further disclosed in WO 94/19454 which is hereby incorporated by reference. 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 Na 2 HP0 4 , 12 H 2 0 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 m 5 minutes. Assays for α-Amylase Activity 1. Phadebas assay α-amylase activity is determined by a method employing Phadebas ® tablets as substrate. Phadebas tablets (Phadebas ® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross- linked insoluble blue-colored starch polymer, which has been mixed with bovme serum albumin and a buffer substance and tabletted. For every single measurement one tablet is suspended m a tube containing 5 ml 50 mM Bπtton-Robmson buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl 2 , pH adjusted to the value of interest with NaOH) . The test is performed m a water bath at the temperature of mterest . The α- amylase to be tested is diluted m x ml of 50 mM Britton- Robinson buffer. 1 ml of this α-amylase solution is added to the 5 ml 50 mM Britton-Robmson buffer. The starch is hydrolyzed by the α-amylase giving soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of the α-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 α-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 directly proportional to the specific activity (activity/mg of pure α-amylase protem) of the α- amylase m question under the given set of conditions. 2. Alternative method α-amylase activity is determined by a method employing the PNP-G7 substrate. PNP-G7 which is a abbreviation for p- mtrophenyl-α, D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase . Following the cleavage, the α-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 α-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 α- Glucosidase one bottle of α-Glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309) . The working solution is made by mixing 5 ml α-Glucosidase solution with 0.5 ml substrate. The assay is performed by transforming 20μl enzyme solution to a 96 well microtitre plate and incubating at 25°C. 200 μl working solution, 25 °C is added. The solution is mixed and pre- mcubated 1 minute and absorption is measured every 15 sec. over 3 minutes at OD 405 nm. From the slope of the time dependent absorption-curve, the specific activity (activity per mg enzyme) of the α-amylase 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 alpha-amylases m question: Method 1. One assay which measures the stability at reduced pH, pH 5.0, m 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 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 m a water bath at 95°C for 30 minutes. Stability determination All the stability trials are made usmg 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, 37°C. The activity measured before incubation (0 minutes) is used as reference (100%) . The decline in percent is calculated as a function of the incubation time. Specific activity determination. The specific activity is determined using the Phadebas assay (Pharmacia) as activity/mg enzyme. Jet cooking and liquefaction with alpha-amylase and variants Liquefaction experiments are run in 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 (Neocuproine method) as a function of time. Cornstarch slurries are prepared by suspending about 11.8 kg Cerestar C*Pharm GL 03406 (89 % starch) in 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 CaCl 2 . 2H 2 0. An amount of enzyme corresponding to 50 NU/g DS is added, and the conductivity adjusted to 300mS using NaCl. 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 liquefaction) pH (initial) 5.5 After jetting, the liquefied starch is collected and transported in sealed thermos- lasks from the pilot plant to the laboratory, where secondary liquefaction is continued at 95°C. 10 ml samples are taken at 15 minutes intervals from 15-90 minutes. 2 drops of 1 N HCl 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 Neocuproine 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 CaCl 2 , pH 6.5 Enzyme solutions: parent AAI-10 or AAI-6 (1 g/1) Dosages : 0.05, 0.1, 0.2, 0.5 and 2.0 g/1 of the enzyme solutions in the impregnation liquor. Incubation : 2 hours at 55 or 65°C 22 hours at 30°C Wash: 10 minutes in water Drying : Room temperature Procedures : Evaluation of Desizing 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 mto 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 m 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 alpha-amylase variants having an improved stability at high pH compared to the parent enzyme, and 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 S- Schuell, Dassel, Germany) - and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates with 10 micro g/ml kanamycin 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 in order to be able to localize positive variants on the filter and the nitrocellulose filter with bound variants is transferred to a container with glycin-NaOH buffer, pH 8.6-10.6 and incubated at room temperature (can be altered from 10°-60°C) for 15 min. 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 in glycin-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 . , kanamycin 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 m 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 re-screened twice under the same conditions as the first screen. EXAMPLES Example 1 Isolation of genomic DNA of Strain DSM 12560 and DSM 12651 The strains Bacillus sp . DSM 12650 (the AAI-6 α-amylase) and Bacillus sp . DSM 12651 (the AAI-10 α-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 strains above were partially digested with restriction enzyme Sau3A, and size-fractionated by electrophoresis on a 0.7 % agarose gel. Fragments between 2 and 10 kb in 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 transformants: DNA libraries in E. coli SJ2 , constructed as described above, were screened on LB agar plates (described in Ausbel et al.(1995)) containing 0.5 % AZCL-amylose (Megazyme) and 10 micro g/ml Chloramphe col and incubated overnight at 37°C. Clones expressing amylase activity appeared with blue diffusion haloes. One such clone expressing AAI-6 amylase was named LiH1300. Another clone expressing AAI-10 amylase was named LiH1298. The DNA was further characterized by DNA sequencing of part of the cloned Sau3A DNA fragments. Example 2 Determination of the DNA sequence of the gene encoding alpha amylase from strain DSM 12650 (AAI-6) . The clone constituting a large chromosomal fragment containing the gene encoding the amylolytic activity inserted into plasmid pSJ1678, pLιH1300, was used as template in an attempt to specifically PCR amplify internal DNA fragments of the amylase encoding gene by the use of degenerate primers directed towards the conserved regions m known Bacillus α- 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 this initial sequence the DNA sequence of the complete coding region of AAI-6 α-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 12651 (AAI-10) : The clone constituting a large chromosomal fragment containing the gene encoding the amylolytic activity inserted mto plasmid pSJ1678, pLιH1298, was used as template m an attempt to specifically PCR amplify internal DNA fragments of the 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/l ) 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 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 this initial sequence the DNA sequence (SEQ ID NO: 1) of the complete coding region of AAI-10 α-amylase (SEQ ID NO: 4) was determined by a standard primers-walking approach. Example 4 Subclonmg of DSM 12650 (AAI-6) α-amylase mto pTVBllO. pTVBllO is a plasmid replicatmg 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 chlorampemcol , obtained from plasmid pC194 (Hormouchi, S. and Weisblum, B. (1982), J. Bact. 150: 815-825). The plasmid harbors a truncated version of the Bacillus licheniformi s 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 AAI-6 amylase the mature gene was fused precisely to the amyL signal sequence such that transcriptions is initiated by the amyL promoter and translocation is directed by the amyL signal sequence. Primers 197clonmgN and 197clonmgC were used to amplify an approximately 1480 bp fragment by PCR on plasmid pLιH1300 using the Pwo polymerase under conditions recommended by the manufacturer (Boehringer Mannheim) . The resulting approximately 1480 bp fragment was digested with restriction endonucleases Pstl and Sfil and ligated with plasmid digested w th the same enzymes, Protease and amylase deleted Bacillus subtilis strain SHa273 (disclosed in WO95/10603) was transformed with the ligation mixture and the DNA sequence of an amy-plus transformant was verified. This plasmid is denoted pTVB230. Oligonucleotides : 197clonmgC: 5' CCG AGC TCG GCC GGC TGG GCC GTC GAC TTA TCG TTT CAC CCA TAC GG (SEQ ID NO: 11) 197clonmgN: 5' CAT TCT GCA GCA GCG GCG CAT CAC GAT GGG ACG AAC GG (SEQ ID NO: 12) Example 5 Subclonmg of AAI-10 alpha amylase mto pTVBllO. DNA sequencing revealed a rather high identity between alpha-amylases from stains AAI-6 and AAI-10. Consequently the same oligonucleotides and strategy was utilized for the cloning of AAI-10 alpha-amylase to expression vector pTVBllO resulting m plasmid pTVB229, which was then fermented usmg standard techniques. Example 6 Purification of the AAI-6 α-amylase The culture broth was flocculated by adding 0.01 ml 50% (w/w) CaCl 2 ,2H 2 0, O.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 m 1.2 M ammonium sulphate, 10 mM Tris-HCl, pH 7.0. The amylase was eluted usmg 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 Purification of AAI-10 α-amylase The culture broth was flocculated as described above. 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 m 1.2 M ammonium sulphate, 10 mM Tris-HCl, pH 7.0. 20% of the activity was eluted with 5 mM Tris-HCl, pH 7.0 and the rest with isopropanol . The combined pool was fractionated on a Q-Sepharose column (100 ml), pH 9.7. 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 9.4. The amylase containing pool was finally fractionated on a S Sepharose column, pH 5.5. Purity of the amylase preparation was above 95% judged by SDS-PAGE. Example 8 Characterization of the AAI-6 and AAI-10 α-amylases : The α-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 Isoelectπc Point was determined using isoelectric focusing (Pharmacia, Ampholme, pH 3.5-9.3). The result of the pH-optimum determination and temperature optimum determination is shown Figure 2 and Figure 3, respectively. Example 9 Washing test Washing performance was evaluated by washing soiled test swatches at 15 and 30 minutes at 25 and 40°C respectively m detergent solutions with the AAI-6 and AAI-10 α-amylase of the invention. The detergents used are disclosed in Table 3 below. The A/P Model Detergent is described in the Materials section above. The other detergents are commercially available detergents. Commercial detergents containing amylase were inactivated by microwaves before wash. The purified recombinant AAI-6 and AAI-10 α-amylases of Example 6 and 7 were 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 3. Detergents and wash conditions. Area Detergent Det . Inacti Enzyme Temp. Time pH Water Ca : Dose Vation dose hardnes Mg g/1 tπg/1 °c min s °dH _ _ __ __ ____ _ 2 : 1 A/P Model 3 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 α-amylase of the invention is effective in both detergents at highly alkaline pH. Example 10 Desizmg test with the AAI-10 The parent AAI-10 alpha-amylase are used for desizmg of fabrics using the TEGEWA method (Method and standard scales obtainable from Verband TEGEWA, Karlstrasse 21, Frankfurt a.M., Germany) . The conditions are described m the "Materials & Methods" section. The AAI-10 alpha-amylase is used m desizmg tests carried out at 30 and 55°C. The enzyme is dosed from 0.05 to 2 g/1 m the impregnation solution. The after-washing procedure is carried out by washing the fabrics m water - instead of the usually hot soda wash. The effect of is measured usmg the Violet Scale (TEGEWA) , TEGEWA scale: 1-9. EXAMPLE 11 Construction of variants of AAI-10 Variants of AAI-10 are constructed as described Example 4 of WO 99/23211. Example 12 Testing the washing performance of AAI-10 variants The wash performance of AAI-10 variants of the invention are tested as described Example 9. Example 13 Testing the specific activity of AAI-10 variants The specific activity of AAI-10 variants is determined as described m Example 8. Example 14 Testing the Calcium stability of AAI-10 variants The calcium stability of AAI-10 variants is tested using the assays described in the "Materials and Method" section. The invention described and claimed herein is not to be limited in 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 within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control. Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. References Ausubel, F. M. et al . (eds.); Current protocols in Molecular Biology; 1995; John Wiley and Sons. Sambrook et al . ; Molecular Cloning: A Laboratory Manual; 1989,; Cold Spring Harbor Lab.; Cold Spring Harbor; NY. 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 brevis; 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: Claims What is claimed is: 1. An isolated polypeptide having α-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 85% 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 low 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 : 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 α-amylase activity; (e) a polypeptide having improved wash performance m alkaline detergent solutions at a pH around 9-11; (f) a polypeptide with a temperature optimum determined using the Phadebas method (pH 9.0) m the range between 55 to 65 °C; and (g) a polypeptide with a pi between 7-8 determined by isoelectric focusing (Pharmacia, Amphol e, pH 3.5-9.3) . 2. The polypeptide of claim 1, having an ammo acid sequence which has at least 85% 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 amino 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 amino 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, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i) , (ii), or (iii) . 7. The polypeptide of claim 1, which is encoded by the nucleic acid sequence contained in plasmid ppLiH1300 or pLiH1298 contained in E. coli DSM12769 or E. coli DSM 12768, respectively. 8. A polypeptide having the same α-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 in 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 , (n) the cDNA sequence of SEQ ID NO : 1 , (in) a subsequence of (I) or (n) of at least 100 nucleotides, or (iv) a complementary strand of (I) , (n) , or (in) ; 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 mto the mature polypeptide coding sequence of SEQ ID NO : 1 or SEQ ID NO: 3, wherem 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. 16. A 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. 23. 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, wherem 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 , wherem said alpha-amylase has one or more mutations, m particular substitution or deletions, m the following positions (relative to SEQ ID NO: 2 or SEQ ID NO: 4) : 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: V206A,R,D,N,C,E,Q,G,H,,I,L,K,M,F,P,S,T,W,Y; 4: E212A,R,D,N,C,Q,G,H, I , , 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 mutations: -R181*/G182*/N195A,R E,Q,G,H,I,L,K,M,F,P,S,T,W, Y,V, -G182*/T183*/N195A,R E.Q.G.H L,K,M F,P,S,T,W,Y,V -T183*/G184*/N195A,R E.Q.G.H L,K,M F,P,S,T,W,Y,V -T183*/G184*/R181A,N E,Q,G,H L,K,M F,P,S,T,W,Y,V -R181*/G182*/V206A,R C,E,Q,G I,L,K M,F,P,S,T,W,Y -G182*/T183*/V206A,R C,E,Q,G I,L,K M,F,P,S,T,W,Y -T183*/G184*/V206A,R C,E,Q,G I . L-K M,F,P,S,T,W, Y -R181*/G182*/E212A,R C,Q,G,H L,K,M F,P,S,T,W,Y,V -G182*/T183*/E212A,R C.Q.G . H L,K,M F,P,S,T,W,Y,V -T183*/G184*/E212A,R C,Q,G,H L,K,M F,P,S,T,W,Y,V -R181*/G182*/E216A,R C,Q,G,H L,K,M F,P,S,T,W,Y,V -G182*/T183*/E216A,R CQ.G.H L,K,M F,P,S,T,W,Y,V -T183*/G184*/E216A,R C,Q,G,H L,K,M F,P,S,T,W,Y,V -R181*/G182*/K269A,R C,E,Q,G I, ,M F,P,S,T,W,Y,V -G182*/T183*/K269A,R C,E,Q,G I,L,M F,P,S,T,W,Y,V -T183*/G184*/K269A,R C,E,Q,G I,L,M F,P,S,T,W,Y,V -N195A,R,D,C,E,Q,G,H K,M,F,P T,W,Y V /V206A,R,D,N,C,E,Q,G L,K,M,F S,T,W Y; -N195A,R,D,C,E,Q,G,H K,M,F,P T,W,Y v /E212A,R,D,N,C,Q,G,H K,M,F,P T,W, Y V; -N195A,R,D,C,E,Q,G,H K,M,F,P T,W,Y V /E216A,R,D,N,C,Q,G,H K,M,F,P T,W,Y V; -N195A,R,D,C,E,Q,G,H K,M,F,P T,W,Y V /K269A,R,D,N,C,E,Q,G L,M,F,P T,W,Y V; -V206A,R,D,N,C,E,Q,G L,K,M,F s,τ,w Y /E212A,R,D,N,C,Q,G,H K,M,F,P T,W,Y V; -V206A,R,D,N,C,E,Q,G L,K,M,F s,τ,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 m position E216Q. 21. Use of the polypeptide of claim 1-8 or variant of claims 20-22 m a detergent composition, m particular a laundry detergent composition and a dish wash detergent composition and hard surface cleaning compositions. 24. Use of the polypeptide of any of claims 1- or variant of claims 20-22 m a desizmg composition. 25. Use of the polypeptide of any of claims 1- 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.

Inventors: ANDERSEN CARSTEN, BISGAARD-FRANTZEN HENRIK, BORCHERT TORBEN VEDEL, HOECK LISBETH HEDEGAARD, NIELSEN BJARNE ROENFELDT, NIELSEN VIBEKE SKOVGAARD, OUTTRUP HELLE, SVENDSEN ALLAN, ANDERSEN, CARSTEN, BISGAARD-FRANTZEN, HENRIK, BORCHERT, TORBEN, VEDEL, HOECK, LISBETH, HEDEGAARD, NIELSEN, BJARNE, ROENFELDT, NIELSEN, VIBEKE, SKOVGAARD, OUTTRUP, HELLE, SVENDSEN, ALLAN
Applicants: NOVONORDISK AS, NOVO NORDISK A/S
Application year: 2000
Main classification: C12N9
IPC codes: C11D3, C12N1, C12N5, C12N9, C12N15, C12P7, C12P19, D06L1