POLYPEPTIDES HAVING 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 ALPHA-AMYLASE ACTIVITY AND NUCLEIC ACIDS ENCODING SAME Background of the Invention Field of the Invention The present invention relates to isolated polypeptides having alpha-amylase activity and isolated nucleic acid sequences encoding the polypeptides. 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 of the Related Art For a number of years alpha-amylases have been used for a variety of different purposes, the most important of which are starch liquefaction, textile desizing, starch modification in the paper and pulp industry, and for brewing and baking. A further use of alpha-amylases, which is becoming increasingly important is the removal of starchy stains during washing with a detergent at alkaline pH. Examples of commercial alpha-amylase products are TERMAMYL®, DURAMYL™, 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 in the range of from pH 5 to pH 7.5, and do not display optimal activity in detergent solutions at alkaline pH. All commercial alpha-amylases have been of bacterial, especially of Bacillus sp., or fungal origin, such as Aspergillus. It is an object of the present invention to provide improved polypeptides having alpha-amylase activity and nucleic acid encoding the polypeptides. Summary of the Invention The present invention relates to isolated polypeptides having alpha-amylase activity selected from the group consisting of: (a) a polypeptide having an amino acid sequence which has at least 75% identity with amino acids 16 to 500 of SEQ ID NO. 2; (b) a polypeptide encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (i) nucleotides 46 to 1500 of SEQ ID NO. 1, (ii) the genomic DNA sequence comprising nucleotides 46 to 1500 of SEQ ID NO. 1, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii); (c) a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 comprising a substitution, deletion, and/or insertion of one or more amino acids; (d) an allelic variant of (a) or (b); and (e) a fragment of (a), (b), or (d) that has alpha-amylase activity. 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 Figures 1A-1D show the cDNA sequence and the deduced amino acid sequence of an Agrotis segetum alpha-amylase (SEQ ID NOS. 1 and 2, respectively). Figure 2 shows a restriction map of pBANeό. Figure 3 shows a restriction map of pMBinl2. Detailed Description of the Invention Polypeptides Having Alpha-Amylase Activity The term "alpha-amylase activity" is defined herein as a 1 ,4-alpha-D-glucan glucanohydrolase activity which catalyses the conversion of polysaccharide containing alpha-(l-4)-linked glucose units in the presence of water to maltooligosaccharides. For purposes of the present invention, alpha-amylase activity is determined employing p- nitrophenyl-α,D-maltoheptaoside as the substrate. Following the cleavage, alpha- glucosidase digests the substrate to liberate a free p-nitrophenolate anion which has a yellow color and thus can be measured by visible spectrophometry at 405 nm. (400-420 nm.). One unit of alpha-amylase activity is defined as 1.0 μmole of p-nitrophenolate anion produced per minute at 25°C, pH 9. In a first embodiment, the present invention relates to isolated polypeptides having an amino acid sequence which has a degree of identity to amino acids 16 to 500 of SEQ ID NO. 2 (i.e., the mature polypeptide) of at least about 15%, preferably at least about 80%), more preferably at least about 85%>, even more preferably at least about 90%, most preferably at least about 95%), and even most preferably at least about 97%, which have alpha-amylase activity (hereinafter "homologous polypeptides"). In a preferred embodiment, the homologous polypeptides have an amino acid sequence which differs by five amino acids, preferably by four amino acids, more preferably by three amino acids, even more preferably by two amino acids, and most preferably by one amino acid from amino acids 16 to 500 of SEQ ID NO. 2. For purposes of the present invention, the degree of identity between two amino acid sequences is determined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, WI) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters were Ktuple=l, gap penalty=3, windows=5. and diagonals=5. Preferably, the polypeptides of the present invention comprise the amino acid sequence of SEQ ID NO. 2 or an allelic variant thereof; or a fragment thereof that has alpha-amylase activity. In a more preferred embodiment, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO. 2. In another preferred embodiment, the polypeptide of the present invention comprises amino acids 16 to 500 of SEQ ID NO. 2, or an allelic variant thereof; or a fragment thereof that has alpha-amylase activity. In another preferred embodiment, the polypeptide of the present invention comprises amino acids 16 to 500 of SEQ ID NO. 2. In another preferred embodiment, the polypeptide of the present invention consists of the amino acid sequence of SEQ ID NO. 2 or an allelic variant thereof; or a fragment thereof that has alpha-amylase activity. In another preferred embodiment, the polypeptide of the present invention consists of the amino acid sequence of SEQ ID NO. 2. In another preferred embodiment, the polypeptide consists of amino acids 16 to 500 of SEQ ID NO. 2 or an allelic variant thereof; or a fragment thereof that has alpha-amylase activity. In another preferred embodiment, the polypeptide consists of amino acids 16 to 500 of SEQ ID NO. 2. A fragment of SEQ ID NO. 2 is a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of this amino acid sequence. Preferably, a fragment contains at least 400 amino acid residues, more preferably at least 430 amino acid residues, and most preferably at least 460 amino acid residues. 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 in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene. In a second embodiment, the present invention relates to isolated polypeptides having alpha-amylase activity which are encoded by nucleic acid sequences which hybridize under very low stringency conditions, preferably low stringency conditions, more preferably 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 (i) nucleotides 46 to 1500 of SEQ ID NO. 1, (ii) the genomic DNA sequence comprising nucleotides 46 to 1500 of SEQ ID NO. 1, (iii) a subsequence of (i) or (ii), or (iv) a complementary strand of (i), (ii), or (iii) (J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York). The subsequence of SEQ ID NO. 1 may be at least 100 nucleotides or preferably at least 200 nucleotides. Moreover, the subsequence may encode a polypeptide fragment which has alpha-amylase activity. The polypeptides may also be allelic variants or fragments of the polypeptides that have alpha-amylase activity. The nucleic acid sequence of SEQ ID NO. 1 or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO. 2 or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having alpha- amylase activity from strains of different genera or species according to methods well known in 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, in 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 in 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, biotin, or avidin). Such probes are encompassed by the present invention. Thus, a genomic DNA or cDNA library prepared from such other organisms may be screened for DNA which hybridizes with the probes described above and which encodes a polypeptide having alpha-amylase activity. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA which is homologous with SEQ ID NO. 1 or a subsequence thereof, the carrier material is used in a Southern blot. For purposes of the present invention, hybridization indicates that the nucleic acid sequence hybridizes to a labeled nucleic acid probe corresponding to the nucleic acid sequence shown in SEQ ID NO. 1, its complementary strand, or a subsequence thereof, under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions are detected using X-ray film. In a preferred embodiment, the nucleic acid probe is a nucleic acid sequence which encodes the polypeptide of SEQ ID NO. 2, or a subsequence thereof. In another preferred embodiment, the nucleic acid probe is SEQ ID NO. 1. In another preferred embodiment, the nucleic acid probe is the mature polypeptide coding region of SEQ ID NO. 1. In another preferred embodiment, the nucleic acid probe is the nucleic acid sequence contained in plasmid pJeRS2805 which is contained in Escherichia coli DSM 12876, wherein the nucleic acid sequence encodes a polypeptide having alpha-amylase activity. In another preferred embodiment, the nucleic acid probe is the mature polypeptide coding region contained in plasmid pJeRS2805 which is contained in Escherichia coli DSM 12876. For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium- high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures. For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2%> SDS preferably at least at 45°C (very low stringency), more preferably at least at 50°C (low stringency), more preferably at least at 55°C (medium stringency), more preferably at least at 60°C (medium-high stringency), even more preferably at least at 65°C (high stringency), and most preferably at least at 70°C (very high stringency). For short probes which are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post- hybridization at about 5°C to about 10°C below the calculated T m using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 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 in length, the carrier material is washed once in 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 variants of the polypeptide having an amino acid sequence of SEQ ID NO. 2 comprising a substitution, deletion, and/or insertion of one or more amino acids. The amino acid sequences of the variant polypeptides may differ from the amino acid sequence of SEQ ID NO. 2 or the mature polypeptide thereof by an insertion or deletion of one or more amino acid residues and/or the substitution of one or more amino acid residues by different amino acid residues. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxy 1-terminal extensions, such as an amino-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-histidine tract, an antigenic epitope or a binding domain. Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions which do not generally alter the specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Nal, Ala/Glu, and Asp/Gly as well as these in reverse. In a fourth embodiment, the present invention relates to isolated polypeptides having immunochemical identity or partial immunochemical identity to the polypeptide having the amino acid sequence of SEQ ID NO. 2 or the mature polypeptide thereof. The immunochemical properties are determined by immunological cross-reaction identity tests by the well-known Ouchterlony double immunodiffusion procedure. Specifically, an antiserum containing polyclonal antibodies which are immunoreactive or bind to epitopes of the polypeptide having the amino acid sequence of SEQ ID NO. 2 or the mature polypeptide thereof are prepared by immunizing rabbits (or other rodents) according to the procedure described by Harboe and Ingild, In N.H. Axelsen, J. Krøll, and B. Weeks, editors, A Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter 23, or Johnstone and Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications, 1982 (more specifically pages 27-31). A polypeptide having immunochemical identity is a polypeptide which reacts with the antiserum in an identical fashion such as total fusion of precipitates, identical precipitate morphology, and/or identical electrophoretic mobility using a specific immunochemical technique. A further explanation of immunochemical identity is described by Axelsen, Bock, and Krøll, In N.H. Axelsen, J. Krøll, and B. Weeks, editors, A Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter 10. A polypeptide having partial immunochemical identity is a polypeptide which reacts with the antiserum in a partially identical fashion such as partial fusion of precipitates, partially identical precipitate morphology, and/or partially identical electrophoretic mobility using a specific immunochemical technique. A further explanation of partial immunochemical identity is described by Bock and Axelsen, In N.H. Axelsen, J. Krøll, and B. Weeks, editors. A Manual of Quantitative Immunoelectrophoresis, Blackwell Scientific Publications, 1973, Chapter 1 1. The antibody may also be a monoclonal antibody. Monoclonal antibodies may be prepared and used, e.g., according to the methods of E. Harlow and D. Lane, editors, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York. The polypeptides of the present invention have at least 20%>, preferably at least 40%, more preferably at least 60%>, even more preferably at least 80%>, even more preferably at least 90%>, and most preferably at least 100%> of the alpha-amylase activity of the mature polypeptide of SEQ ID NO. 2. A polypeptide of the present invention may be obtained from microorganisms or organisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by the nucleic acid sequence is produced by the source or by a cell in which the nucleic acid sequence from the source has been inserted. In a preferred embodiment, the polypeptide is secreted extracellularly. A polypeptide of the present invention may be an insect polypeptide, such as a Coleopteran, Dipteran, or Lipoderpteran polypeptide. The polypeptide may be obtained from any insect. In a preferred embodiment, the polypeptide is an Agrotus polypeptide. In a more preferred embodiment, the polypeptide is an. Agrotus segetum. polypeptide, the polypeptide with the amino acid sequence of SEQ ID NO. 2. A polypeptide of the present invention may be a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus polypeptide, e.g., a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thu ingiensis polypeptide; or a Streptomyces polypeptide, e.g., a Streptomyces lividans or Streptomyces murinus polypeptide; or a gram negative bacterial polypeptide, e.g., an E. coli or a Pseudomonas sp. polypeptide. A polypeptide of the present invention may be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or more preferably a filamentous fungal polypeptide such as an Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma polypeptide. In a preferred embodiment, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis polypeptide. In another preferred embodiment, the polypeptide is an Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide. 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). Insects may be obtained from natural habitats or from commercially available sources. 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 in the art (see, e.g., Sambrook et al., 1989, supra). As defined herein, an "isolated" polypeptide is a polypeptide which is essentially free of other non-alpha-amylase polypeptides, e.g., at least about 20% pure, preferably at least about 40%> pure, more preferably about 60%) pure, even more preferably about 80%> pure, most preferably about 90% pure, and even most preferably about 95% pure, as determined by SDS-PAGE. Polypeptides encoded by nucleic acid sequences of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus 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 in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator. 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 in SEQ ID NO. 1. In another more preferred embodiment, the nucleic acid sequence is the sequence contained in plasmid pJeRS2805 that is contained in Escherichia coli DSM 12876. In another preferred embodiment, the nucleic acid sequence is the mature polypeptide coding region of SEQ ID NO. 1. In another more preferred embodiment, the nucleic acid sequence is the mature polypeptide coding region contained in plasmid pJeRS2805 that is contained in Escherichia coli DSM 12876. The present invention also encompasses nucleic acid sequences which encode a polypeptide having the amino acid sequence of SEQ ID NO. 2 or the mature polypeptide thereof, which differ from SEQ ID NO. 1 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO. 1 which encode fragments of SEQ ID NO. 2 that have alpha-amylase activity. A subsequence of SEQ ID NO. 1 is a nucleic acid sequence encompassed by SEQ ID NO. 1 except that one or more nucleotides from the 5' and/or 3' end have been deleted. Preferably, a subsequence contains at least 1200 nucleotides, more preferably at least 1290 nucleotides, and most preferably at least 1320 nucleotides. The present invention also relates to mutant nucleic acid sequences comprising at least one mutation in the mature polypeptide coding sequence of SEQ ID NO. 1, in which the mutant nucleic acid sequence encodes a polypeptide which consists of amino acids 16 to 500 of SEQ ID NO. 2. The techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al, 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used. The nucleic acid sequence may be cloned from an Agrotus organism, 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 in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleic acid sequence will be replicated. The nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof. 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 46 to 1500) of at least about 65%, preferably about 70%>, preferably about 80%), more preferably about 90%, even more preferably about 95%, and most preferably about 97%) homology, which encode an active polypeptide. For purposes of the present invention, the degree of homology between two nucleic acid sequences is determined by the Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80: 726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, WI) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters were Ktuple=3, gap penalty=3, and windows=20. 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 in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in 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, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions which do not give rise to another amino 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 amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al, 1991, Protein Expression and Purification 2: 95-107. It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino 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 in the art, such as site- directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for alpha-amylase activity to identify amino 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 photoaffinity labelling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al, 1992, Journal of Molecular Biology 224: 899- 904; Wlodaver et al, 1992, FEBS Letters 309: 59-64). The present invention also relates to isolated nucleic acid sequences encoding a polypeptide of the present invention, which hybridize under very low stringency conditions, preferably low stringency conditions, more preferably 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 the nucleic acid sequence of SEQ ID NO. 1 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 very low, low, medium, medium-high, high, or very high stringency conditions with (i) nucleotides 46 to 1500 of SEQ ID NO. 1, (ii) the genomic DNA sequence comprising nucleotides 46 to 1500 of SEQ ID NO. 1, (iii) a subsequence of (i) or (ii), or (iv) a complementary strand of (i), (ii), or (iii); and (b) isolating the nucleic acid sequence. The subsequence is preferably a sequence of at least 100 nucleotides such as a sequence which encodes a polypeptide fragment which has alpha- amylase activity. The present invention also relates to a fragment of a nucleic acid sequence of the invention which is combined with another nucleic acid sequence encoding a second polypeptide such that the resulting fused or hybrid polypeptide has increased activity at alkaline pH of at least 10%> at pH values of 8.0 or above compared to the second 1 j - polypeptide. Methods for Producing Mutant Nucleic Acid Sequences The present invention further relates to methods for producing a mutant nucleic acid sequence, comprising introducing at least one mutation into the mature polypeptide coding sequence of SEQ ID NO. 1 or a subsequence thereof, wherein the mutant nucleic acid sequence encodes a polypeptide which consists of amino acids 16 to 500 of SEQ ID NO. 2 or a fragment thereof which has alpha-amylase activity. The introduction of a mutation into the nucleic acid sequence to exchange one nucleotide for another nucleotide may be accomplished by site-directed mutagenesis using any of the methods known in the art. Particularly useful is the procedure which utilizes a supercoiled, double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, extend during temperature cycling by means of Pfu DNA polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicks is generated. Following temperature cycling, the product is treated with Dpnl which is specific for methylated and hemimethylated DNA to digest the parental DNA template and to select for mutation-containing synthesized DNA. Other procedures known in the art may also be used. These other procedures include gene shuffling, e.g., as described in 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 in a suitable host cell under conditions compatible with the control sequences. Expression will be understood to include any step involved in the production of the polypeptide including, 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 combined and juxtaposed in a manner that would not otherwise exist in nature. The term nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. The term "coding sequence" is defined herein as a nucleic acid sequence which directly specifies the amino acid sequence of its protein product. The boundaries of a genomic 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 in a variety of ways to provide for expression of the polypeptide. Manipulation of the nucleic acid sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing recombinant DNA methods are well known in the art. The term "control sequences" is defined herein to include all components which are necessary or advantageous for the expression of a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide. The term "operably linked" is defined herein as a configuration in 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. 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 in 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 in a bacterial host cell, are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis 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 in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242: 74-94; and in Sambrook et al, 1989, supra. Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3 -phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al, 1992, Yeast 8: 423-488. 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 in the host cell of choice may be used in the present invention. Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3 -phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al. , 1992, supra. The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention. Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3- phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP). The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention. Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin- like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15 : 5983-5990. The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide 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 in order to enhance secretion of the polypeptide. However, any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention. Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prs A. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137. Effective signal peptide coding regions for filamentous fungal host cells are the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. In a preferred embodiment, the signal peptide coding region is nucleotides 1 to 45 of SEQ ID NO. 1 which encode amino acids 1 to 15 of SEQ ID NO. 2. Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described by Romanos et al, 1992, supra. The control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region. 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 in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the 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 in 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. The present invention also relates to nucleic acid constructs for altering the expression of an endogenous gene encoding a polypeptide of the present invention. The constructs may contain the minimal number of components necessary for altering expression of the endogenous gene. In one embodiment, the nucleic acid constructs preferably contain (a) a targeting sequence, (b) a regulator}' sequence, (c) an exon, and (d) a splice-donor site. Upon introduction of the nucleic acid construct into a cell, the construct inserts by homologous recombination into the cellular genome at the endogenous gene site. The targeting sequence directs the integration of elements (a)-(d) into the endogenous gene such that elements (b)-(d) are operably linked to the endogenous gene. In another embodiment, the nucleic acid constructs contain (a) a targeting sequence, (b) a regulatory sequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) a splice-acceptor site, wherein the targeting sequence directs the integration of elements (a)-(f) such that elements (b)-(f) are operably linked to the endogenous gene. However, the constructs may contain additional components such as a selectable marker. In both embodiments, the introduction of these components results in production of a new transcription unit in which expression of the endogenous gene is altered. In essence, the new transcription unit is a fusion product of the sequences introduced by the targeting constructs and the endogenous gene. In one embodiment in which the endogenous gene is altered, the gene is activated. In this embodiment, homologous recombination is used to replace, disrupt, or disable the regulatory region normally associated with the endogenous gene of a parent cell through the insertion of a regulatory sequence which causes the gene to be expressed at higher levels than evident in the corresponding parent cell. The activated gene can be further amplified by the inclusion of an amplifiable selectable marker gene in the construct using methods well known in the art (see, for example, U.S. Patent No. 5,641,670). In another embodiment in which the endogenous gene is altered, expression of the gene is reduced. The targeting sequence can be within the endogenous gene, immediately adjacent to the gene, within an upstream gene, or upstream of and at a distance from the endogenous gene. One or more targeting sequences can be used. For example, a circular plasmid or DNA fragment preferably employs a single targeting sequence, while a linear plasmid or DNA fragment preferably employs two targeting sequences. The regulatory sequence of the construct can be comprised of one or more promoters, enhancers, scaffold-attachment regions or matrix attachment sites, negative regulatory elements, transcription binding sites, or combinations of these sequences. The constructs further contain one or more exons of the endogenous gene. An exon is defined as a DNA sequence which is copied into RNA and is present in a mature mRNA molecule such that the exon sequence is in-frame with the coding region of the endogenous gene. The exons can, optionally, contain DNA which encodes one or more amino acids and/or partially encodes an amino acid. Alternatively, the exon contains DNA which corresponds to a 5' non-encoding region. Where the exogenous exon or exons encode one or more amino acids and/or a portion of an amino acid, the nucleic acid construct is designed such that, upon transcription and splicing, the reading frame is in- frame with the coding region of the endogenous gene so that the appropriate reading frame of the portion of the mRNA derived from the second exon is unchanged. The splice-donor site of the constructs directs the splicing of one exon to another exon. Typically, the first exon lies 5' of the second exon, and the splice-donor site overlapping and flanking the first exon on its 3' side recognizes a splice-acceptor site flanking the second exon on the 5' side of the second exon. A splice-acceptor site, like a splice-donor site, is a sequence which directs the splicing of one exon to another exon. Acting in conjunction with a splice-donor site, the splicing apparatus uses a splice- acceptor site to effect the removal of an intron. Expression Vectors The present invention also relates to recombinant expression vectors comprising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at such sites. Alternatively, the nucleic acid sequence of the present invention may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression. The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the nucleic acid sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i. e. , a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used. The vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dαl genes from Bacillus subtilis or Bacillus licheniformis, or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRPl, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5' -phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus. The vectors of the present invention preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. 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 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 addit ona nuc e c ac sequences ena e t e vector to e ntegrate nto t e ost ce 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 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 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 in the host cell in question. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUBl 10, pE194, pTA1060, and pAMBl permitting replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75: 1433). More than one copy of a nucleic acid sequence of the present invention may be inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent. The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al, 1989, supra). Host Cells The present invention also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a nucleic acid sequence of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non- unicellular microorganism, e.g., a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria including, but not limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans and 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 Spizizin, 1961, Journal of Bacteriology 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278). The host cell may be a eukaryote, such as a mammalian, insect, plant, or fungal cell. In a preferred embodiment, the host cell is a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawks worth et al, In, Ainsworth and Bisby 's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al, 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al, 1995, supra). In a more preferred embodiment, the fungal host cell is a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F.A., Passmore, S.M., and Davenport, R.R., eds, Soc. App. Bacteriol Symposium Series No. 9, 1980). In an even more preferred embodiment, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell. In a most preferred embodiment, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. In another most preferred embodiment, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred embodiment, the yeast host cell is a Yarrowia lipolytica cell. In another more preferred embodiment, the fungal host cell is a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al, 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative. In an even more preferred embodiment, the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma. In a most preferred embodiment, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred embodiment, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In an even most preferred embodiment, the filamentous fungal parent cell is a Fusarium venenatum (Nirenberg sp. nov.) cell. In another most preferred embodiment, the filamentous fungal host cell is a Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell. Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al, 1984, Proceedings of the National Academy of Sciences USA 81 : 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al, 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al, 1983, Journal of Bacteriology 153: 163; and Hinnen et al, 1978, Proceedings of the National Academy of Sciences USA 75: 1920. Methods of Production The present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. The present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide, wherein the host cell comprises a mutant nucleic acid sequence having at least one mutation in the mature polypeptide coding region of SEQ ID NO. 1, wherein the mutant nucleic acid sequence encodes a polypeptide which consists of amino acids 16 to 500 of SEQ ID NO. 2, and (b) recovering the polypeptide. The present invention further relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a homologously recombinant cell, having incorporated therein a new transcription unit comprising a regulatory sequence, an exon, and/or a splice donor site operably linked to a second exon of an endogenous nucleic acid sequence encoding the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. The methods are based on the use of gene activation technology, for example, as described in U.S. Patent No. 5,641,670. In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell ly sates. The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide as described herein. The resulting polypeptide may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray- drying, evaporation, or precipitation. The polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). Plants The present invention also relates to a transgenic plant, plant part, or plant cell which has been transformed with a nucleic acid sequence encoding a polypeptide having alpha-amylase activity of the present invention so as to express and produce the polypeptide in recoverable quantities. The polypeptide may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the recombinant polypeptide may be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor. The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn). Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana. Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers. Also specific plant tissues, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. Also included within the scope of the present invention are the progeny of such plants, plant parts and plant cells. The transgenic plant or plant cell expressing a polypeptide of the present invention may be constructed in accordance with methods known in the art. Briefly, the plant or plant cell is constructed by incorporating one or more expression constructs encoding a polypeptide of the present invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell. Conveniently, the expression construct is a nucleic acid construct which comprises a nucleic acid sequence encoding a polypeptide of the present invention operably linked with appropriate regulatory sequences required for expression of the nucleic acid sequence in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used). The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences is determined, for example, on the basis of when, where, and how the polypeptide is desired to be expressed. For instance, the expression of the gene encoding a polypeptide of the present invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al, 1988, Plant Physiology 86: 506. For constitutive expression, the 35S-CaMV promoter may be used (Franck et al, 1980, Cell 21 : 285-294). Organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al, 1994, Plant Mol Biol 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al, 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al, 1998, Journal of Plant Physiology 152: 708-711), a promoter from a seed oil body protein (Chen et al, 1998, Plant and Cell Physiology 39: 935-941), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al, 1993, Plant Physiology 102: 991- 1000, the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene promoter from rice (Kagaya et al, 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al, 1993, Plant Molecular Biology 22: 573-588). A promoter enhancer element may also be used to achieve higher expression of the enzyme in the plant. For instance, the promoter enhancer element may be an intron which is placed between the promoter and the nucleotide sequence encoding a polypeptide of the present invention. For instance, Xu et al, 1993, supra disclose the use of the first intron of the rice actin 1 gene to enhance expression. The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art. The nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et αl, 1990, Science 244: 1293; Pofrykus, 1990, Bio/Technology 8: 535; Shimamoto et αl., 1989, Nature 338: 274). Presently, Agrobαcterium tumefαciens-mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38). However it can also be used for transforming monocots, although other transformation methods are generally preferred for these plants. Presently, the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DΝA) of embryonic calli or developing embryos (Christou, 1992, Plant Journal 2: 275- 281; Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al, 1992, Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al, 1993, Plant Molecular Biology 21 : 415-428. Following transformation, the transformants having incorporated therein the expression construct are selected and regenerated into whole plants according to methods well-known in the art. The present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a transgenic plant or a plant cell comprising a nucleic acid sequence encoding a polypeptide having alpha-amylase activity of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. Signal Peptide The present invention also relates to nucleic acid constructs comprising a gene encoding a protein operably linked to a nucleic acid sequence consisting of nucleotides 1 to 15 of SEQ ID NO. 1 encoding a signal peptide consisting of amino acids 1 to 15 of SEQ ID NO. 2, wherein the gene is foreign to the nucleic acid sequence. The present invention also relates to recombinant expression vectors and recombinant host cells comprising such nucleic acid constructs. The present invention also relates to methods for producing a protein comprising (a) cultivating such a recombinant host cell under conditions suitable for production of the protein; and (b) recovering the protein. The nucleic acid sequence may be operably linked to foreign genes with other control sequences. Such other control sequences are described supra. As noted earlier, where both signal peptide and propeptide regions are present at the amino terminus of a protein, the propeptide region is positioned next to the amino terminus of a protein and the signal peptide region is positioned next to the amino terminus of the propeptide region. The protein may be native or heterologous to a host cell. The term "protein" is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins. The term "protein" also encompasses two or more polypeptides combined to form the encoded product. The proteins also include hybrid polypeptides which comprise a combination of partial or complete polypeptide sequences obtained from at least two different proteins wherein one or more may be heterologous or native to the host cell. Proteins further include naturally occurring allelic and engineered variations of the above mentioned proteins and hybrid proteins. Preferably, the protein is a hormone or variant thereof, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter. In a more preferred embodiment, the protein is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. In an even more preferred embodiment, the protein is an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta- galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase or xylanase. The gene may be obtained from any prokaryotic, eukaryotic, or other source. 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 alpha-amylase activity of the composition has been increased, e.g., with an enrichment factor of 1.1. The composition may comprise a polypeptide of the invention as the major enzymatic component, e.g., a mono-component composition. Alternatively, the composition may comprise multiple enzymatic activities, such as an 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 Humicola insolens, or Fusarium, preferably Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, or Fusarium venenatum. The polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the polypeptide composition may be in the form of a granulate or a microgranulate. The polypeptide to be included in the composition may be stabilized in accordance with methods known in 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 in the art. Uses The present invention is also directed to methods for using the polypeptides having alpha-amylase activity. Owing to their activity at alkaline pH values, the polypeptides of the present invention having alkaline alpha-amylase activity are well suited for use in a variety of industrial processes, in particular the polypeptides find potential applications as a component in detergents, e.g., laundry and hard surface cleaning detergent compositions, but it may also be useful in the production of sweeteners and ethanol from starch. Thus, it may be used in conventional starch-converting processes, such as liquefaction and saccharification processes described in U.S. Patent No. 3,912,590 and EP patent publications Nos. 252,730 and 63,909. The polypeptides of the present invention having alkaline alpha-amylase activity may also be used in the production of lignocellulosic materials, such as pulp, paper and cardboard, from starch reinforced waste paper and cardboard, especially where repulping occurs at pH above 7 and where amylases can facilitate the disintegration of the waste material through degradation of the reinforcing starch. The polypeptides of the present invention are especially useful in a process for producing a papermaking pulp from starch-coated printed paper. The process may be performed as described in WO 95/14807, comprising the following steps: (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 polypeptides of the present invention having alkaline alpha-amylase activity may also be very useful in modifying starch where enzymatically-modified starch is used in papermaking together with alkaline fillers such as calcium carbonate, kaolin and clays. With the polypeptides of the present invention modification of the starch in the presence of the filler is possible thus allowing for a simpler integrated process. The polypeptides of the present invention having alkaline alpha-amylase activity may also be very useful in textile desizing. In the textile processing industry, α-amylases are traditionally used as auxiliaries in the desizing process to facilitate the removal of starch-containing size which has served as a protective coating on weft yarns during weaving. Complete removal of the size coating after weaving is important to ensure optimum results in the subsequent processes, in which the fabric is scoured, bleached and dyed. Enzymatic starch break-down is preferred because it does not involve any harmful effect on the fiber material. In order to reduce processing costs and increase mill through-put, 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 polypeptides of the present invention having alkaline alpha-amylase activity as they have an improved performance in alkaline solutions. The polypeptides may be used alone or in combination with a cellulase when desizing cellulose-containing fabric or textile. The polypeptides of the present invention having alkaline alpha-amylase activity may also be very useful in a beer-making process; the polypeptides will typically be added during the mashing process. The polypeptides of the present invention having alkaline alpha-amylase activity may also be very useful in the formulation or manufacture of food and feed stuffs including, but not limited to animal feeds, breads, cakes, sauces, etc. Detergent Compositions The present invention also relates to detergent compositions comprising a polypeptide of the present invention having alkaline alpha-amylase activity. The detergent compositions 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 embodiment, the present invention provides a detergent additive comprising a polypeptide of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., laccase, and/or 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 in effective amounts. Proteases: Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583. Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in 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™, SAVINASE™, PRIMASE™, DURALASE™, ESPERASE™, and KANNASE™ (available from Novo Nordisk A/S, Bagsvaerd, Denmark); MAXATASE™, MAXACAL™, MAXAPEM™, PROPERASE™, PURAFECT™, PURAFECT OxP™, FN2™, and FN3™ (Genencor International Inc., Palo Alto, CA). Lipases: Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humicola (synonym Thermomyces), e.g., from Humicola lanuginosa (T lanuginosus) as described in EP 258 068 and EP 305 216 or from Humicola insolens as described in WO 96/13580; a Pseudomonas lipase, e.g., from Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (EP 218 272), Pseudomonas cepacia (EP 331 376), Pseudomonas stutzeri (GB 1,372,034), Pseudomonas fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), Pseudomonas wisconsinensis (WO 96/12012); and a Bacillus lipase, e.g., from Bacillus subtilis (Dartois et al, 1993, Biochemica et Biophysica Ada 1131, 253-360), Bacillus stearothermophilus (JP 64/744992) or Bacillus 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 protein engineered mutants are included. Amylases include, for example, α-amylases obtained from Bacillus, e.g., a special strain of Bacillus licheniformis, described in more detail in GB 1,296,839. Examples of useful amylases are the variants described in 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, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum 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 color 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 Coprinus cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include GUARDZYME™ (Novo Nordisk A/S). The detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive 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, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries. Non-dusting granulates may be produced, e.g., as disclosed in U.S. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216. The detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent may be aqueous, typically containing up to 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 zwitterionic. The surfactants are typically present at a level of from 0.1 %> to 60%o by weight. When included therein the detergent will usually contain from about 1% to about 40%) of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic 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, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives of glucosamine ("glucamides"). The detergent may contain 0-65 % of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst). The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), 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 hydrogen peroxide source such as perborate or percarbonate, which may be combined with a peracid- forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleaching system may comprise peroxyacids of, for example, the amide, imide, or sulfone type. The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, 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. In the detergent compositions any enzyme, in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per liter of wash liquor, preferably 0.05-5 mg of enzyme protein per liter of wash liquor, in particular 0.1-1 mg of enzyme protein per liter of wash liquor. The enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference. Dishwash Detergent Compositions The polypeptide of the present invention may also be used in dishwash detergent compositions, including the following: 1) POWDER AUTOMATIC DISHWASHING COMPOSITION 3) POWDER AUTOMATIC DISHWASHING COMPOSITION (5) POWDER AUTOMATIC DISHWASHING COMPOSITION (6) POWDER AND LIQUID DISHWASHING COMPOSITION WITH CLEANING SURFACTANT SYSTEM (7) NON-AQUEOUS LIQUID AUTOMATIC DISHWASHING COMPOSITION (8) NON-AQUEOUS LIQUID DISHWASHING COMPOSITION (9) THIXOTROPIC LIQUID AUTOMATIC DISHWASHING COMPOSITION (10) LIQUID AUTOMATIC DISHWASHING COMPOSITION (11) LIQUID AUTOMATIC DISHWASHING COMPOSITION CONTAINING PROTECTED BLEACH PARTICLES (11) Automatic dishwashing compositions as described in (1), (2), (3), (4), (6) and (10), wherein perborate is replaced by percarbonate. (12) Automatic dishwashing compositions as described in (1) - (6) which additionally contain a manganese catalyst. The manganese catalyst may, e.g., be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching", Nature 369: 637-639 (1994). The present invention is further described by the following examples which should not be construed as limiting the scope of the invention. Examples Materials Chemicals used as buffers and substrates were commercial products of at least reagent grade. Methods Assays for Alpha- Amylase Activity 1. Phadebas assay Alpha-amylase activity is determined by a method employing Phadebas® tablets as substrate. Phadebas tablets (Phadebas® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-colored starch polymer which has been mixed with bovine serum albumin and a buffer substance in tablet form. For every single measurement one tablet is suspended in a tube containing 5 ml of 50 mM Britton-Robinson 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 sodium hydroxide). The assay is performed in a water bath at the temperature of interest. The alpha-amylase to be tested is diluted in x ml of 50 mM Britton-Robinson buffer. 1 ml of the alpha-amylase solution is added to the 5 ml of 50 mM Britton-Robinson buffer. The starch is hydrolyzed by the alpha-amylase yielding soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of the alpha-amylase activity. It is important that the measured 620 nm absorbance after 10 or 15 minutes of incubation (testing time) is in the range of 0.2 to 2.0 absorbance units at 620 nm. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law). The dilution of the enzyme must therefore be adjusted to fit this criterion. Under a specified set of conditions (temp., pH, reaction time, buffer conditions) 1 mg of a given α- amylase will hydrolyze a certain amount of substrate and a blue colour will be produced. The color intensity is measured at 620 nm. The measured absorbance is directly proportional to the specific activity (activity/mg of pure alpha-amylase protein) of the alpha-amylase in question under the given set of conditions. 2. Alternative method Alpha-amylase activity is determined by a method employing p-nitrophenyl-α,D- maltoheptaoside as the substrate. Following the cleavage, the alpha-glucosidase included in the kit digests the substrate to liberate a free p-nitrophenolate anion which has a yellow color and thus can be measured by visible spectrophometry at 405nm. (400-420 nm.). Kits containing p-nitrophenyl-α,D-maltoheptaoside and alpha-glucosidase are manufactured by Boehringer-Mannheim (Indianapolis, IN). To prepare the substrate solution, one bottle of substrate (BM 1442309) is added to 5 ml of buffer (BM 1442309). To prepare the alpha-glucosidase solution, one bottle of alpha-glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309). The working solution is made by mixing 5 ml of alpha-glucosidase solution with 0.5 ml of substrate solution. The assay is performed by transferring 20 μl of enzyme solution to a 96 well microtitre plate and incubating at 25 °C followed by 200 μl of working solution. The solution is mixed and pre-incubated for 1 minute and the absorption is measured every 15 seconds for 3 minutes at 405 nm. From the slope of the time dependent absorption-curve, the specific activity (activity per mg enzyme) of the α-amylase under the given conditions can be calculated using TERMAMYL™ as a standard. Example 1 : Agrotis segetum Directional cDNA Library Construction Total RNA was prepared from Agrotis segetum larvae by extraction with guanidinium thiocyanate followed by ultracentrifugation through a 5.7 M CsCl cushion (Chirgwin et al, 1979, Biochemistry 18: 5294-5299) using the following modifications. The larvae were frozen and ground in liquid N 2 to a fine powder with a mortar and a pestle, followed by grinding in a precooled coffee mill, and immediately suspended in 5 volumes of RNA extraction buffer (4 M guanidinium thiocyanate, 0.5%> sodium laurylsarcosine, 25 mM sodium citrate pH 7.0, 0.1 M β-mercaptoethanol). The mixture was stirred for 30 minutes at room temperature and centrifuged (20 minutes at 10,000 rpm, Beckman) to pellet the cell debris. The supernatant was collected, carefully layered onto a 5.7 M CsCl cushion (5.7 M CsCl, 10 mM EDTA, pH 7.5, 0.1% diethylpyrocarbonate (DEPC); autoclaved prior to use) using 26.5 ml supernatant per 12.0 ml of CsCl cushion, and centrifuged to obtain the total RNA (Beckman, SW28 rotor, 25,000 rpm, room temperature, 24 hours). After centrifugation the supernatant was carefully removed and the bottom of the tube containing the RNA pellet was cut off and rinsed with 70%> ethanol. The total RNA pellet was transferred to an Eppendorf tube, suspended in 500 μl of 10 mM Tris-1 mM EDTA pH 7.6 (TE) (if difficult, heat occasionally for 5 minutes at 65 °C), phenol extracted, and precipitated with ethanol for 12 hours at -20°C (2.5 volumes of ethanol, 0.1 volume of 3 M sodium acetate pH 5.2). The RNA was collected by centrifugation, washed in 70% ethanol, and resuspended in a minimum volume of DEPC-treated deionized water (see Sambrook et al, 1989, supra). The RNA concentration was determined by measuring OD 260/280 . The poly(A) + RNA was isolated by oligo(dT)-cellulose affinity chromatography (Aviv & Leder, 1972, Proceedings of the National Academy of Sciences USA 69: 1408- 1412). A total of 0.2 g of oligo(dT) cellulose (Boehringer Mannheim, Indianapolis, IN) was pre-swollen in 10 ml of IX of column loading buffer (20 mM Tris-Cl, pH 7.6, 0.5 M NaCl, 1 mM EDTA, 0.1% SDS), loaded onto a DEPC-treated, plugged plastic column (Poly Prep Chromatography Column, BioRad, Hercules, CA; see Sambrook et al, 1989, supra), and equilibrated with 20 ml of IX loading buffer. The total RNA (1-2 mg) was heated at 65°C for 8 minutes, quenched on ice for 5 minutes, and after addition of 1 volume of 2X column loading buffer to the RNA sample loaded onto the column. The eluate was collected and reloaded 2-3 times by heating the sample as above and quenching on ice prior to each loading. The oligo(dT) column was washed with 10 volumes of lx loading buffer, then with 3 volumes of medium salt buffer (20 mM Tris- Cl, pH 7.6, 0.1 M NaCl, 1 mM EDTA, 0.1% SDS), followed by elution of the poly(A) + RNA with 3 volumes of elution buffer (10 mM Tris-Cl, pH 7.6, 1 mM EDTA, 0.05% SDS) preheated to 65°C, by collecting 500 μl fractions. The OD 260 was read for each collected fraction, and the mRNA containing fractions were pooled and ethanol precipitated at -20°C for 12 hours. The poly(A) + RNA was collected by centrifugation, resuspended in DEPC-treated water and stored in 5-10 μg aliquots at -80°C. Double-stranded cDNA was synthesized from 5 μg of Agrotis segetum poly(A) + RNA by the RNase H method (Gubler and Hoffman 1983, supra; Sambrook et al , 1989, supra) using a hair-pin modification. The poly(A) ^ RNA (5 μg in 5 μl of DEPC-treated deionized water) was heated at 70°C for 8 minutes in a pre-siliconized, RNase-free Eppendorf tube, quenched on ice, and combined in a final volume of 50 μl with reverse transcriptase buffer (50 mM Tris-Cl pH 8.3, 75 mM KC1, 3 mM MgCl 2 , 10 mM DTT) containing 1 mM of dATP, dGTP and dTTP, and 0.5 mM of 5-methyl-dCTP, 40 units of human placental ribonuclease inhibitor, 4.81 μg of oligo(dT) 18 -NøtI primer and 1000 units of Superscript II RΝase H - reverse transcriptase. First-strand cDΝA was synthesized by incubating the reaction mixture at 45 °C for 1 hour. After synthesis, the mRΝA:cDΝA hybrid mixture was gel filtrated through a Pharmacia MicroSpin S-400 HR spin column according to the manufacturer's instructions. After the gel filtration, the hybrids were diluted in 250 μl of second strand buffer (20 mM Tris-Cl pH 7.4, 90 mM KC1, 4.6 mM MgCl 2 , 10 mM (ΝH 4 ) 2 SO 4 , 0.16 mM BNAD + ) containing 200 μM of each dNTP, 60 units of E. coli DNA polymerase I (Pharmacia, Uppsala, Sweden), 5.25 units of RNase H, and 15 units of E. coli DNA ligase. Second strand cDNA synthesis was performed by incubating the reaction tube at 16°C for 2 hours, and an additional 15 minutes at 25°C. The reaction was stopped by addition of EDTA to 20 mM final concentration followed by phenol and chloroform extractions. The double-stranded cDNA was ethanol precipitated at -20°C for 12 hours by addition of 2 volumes of 96% ethanol and 0.2 volume of 10 M ammonium acetate, recovered by centrifugation, washed in 70%> ethanol, dried (SpeedVac), and resuspended in 30 μl of Mung bean nuclease buffer (30 mM sodium acetate pH 4.6, 300 mM NaCl, 1 mM ZnSO 4 , 0.35 mM dithiothreitol, 2% glycerol) containing 25 units of Mung bean nuclease. The single-stranded hair-pin DNA was clipped by incubating the reaction at 30°C for 30 minutes, followed by addition of 70 μl of 10 mM Tris-Cl, pH 7.5, 1 mM EDTA, phenol extraction, and ethanol precipitation with 2 volumes of 96% ethanol and 0.1 volume 3 M sodium acetate pH 5.2 on ice for 30 minutes. The double-stranded cDNAs were recovered by centrifugation (20,000 rpm, 30 minutes), and blunt-ended with T4 DNA polymerase in 30 μl of T4 DNA polymerase buffer (20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol) containing 0.5 mM of each dNTP, and 5 units of T4 DNA polymerase by incubating the reaction mixture at +16°C for 1 hour. The reaction was stopped by addition of EDTA to 20 mM final concentration, followed by phenol and chloroform extractions and ethanol precipitation for 12 h at -20°C by adding 2 volumes of 96%) ethanol and 0.1 volume of 3M sodium acetate pH 5.2. After the fill-in reaction the cDNAs were recovered by centrifugation as above, washed in 10% ethanol, and the DNA pellet was dried in a SpeedVac. The cDNA pellet was resuspended in 25 μl of ligation buffer (30 mM Tris-Cl, pH 7.8, 10 mM MgCl 2 , 10 mM dithiothreitol, 0.5 mM ATP) containing 2 μg Ec RI adaptors (0.2 μg/μl, Pharmacia, Uppsala, Sweden) and 20 units of T4 ligase by incubating the reaction mix at 16°C for 12 hours. The reaction was stopped by heating at 65°C for 20 minutes, and then placed on ice for 5 minutes. The adapted cDNA was digested with Notl by addition of 20 μl of autoclaved water, 5 μl of 10X Notl restriction enzyme buffer and 50 units of Notl, followed by incubation for 3 hours at 37°C. The reaction was stopped by heating the sample at 65°C for 15 minutes. The cDΝAs were size-fractionated by agarose gel electrophoresis on a 0.8%> SeaPlaque GTG low melting temperature agarose gel (FMC, Rockland, MΕ) in 50 mM Tris-50 mM boric acid-1 mM disodium ΕDTA buffer (TBΕ) to separate unligated adaptors and small cDΝAs. The gel was run for 12 hours at 15 V, and the cDΝA was size-selected by staining the molecular weight marker lane only with ethidium bromide and cutting the area between 0.5 Kb and 6.0 Kb from the cDΝA lane. The cDΝA was extracted from the gel using the QIAΕX II gel band purification kit (Qiagen Inc., Valencia, CA) as follows. The trimmed gel slice was weighed in a 2 ml Biopure Εppendorf tube, then 300 μl of QXI Buffer (Qiagen Inc., Valencia, CA) was added for each 100 mg of gel slice, the pH was adjusted to 5.0 with 0.1M sodium acetate and the gel slice was dissolved by incubation at 25°C for 10 minutes, until the agarose was completely solubilized. The melted sample was incubated with 30 μl of QIAΕX II beads (Qiagen Inc., Valencia, CA) for 10 minutes at 50°C. Following a brief centrifugation the beads were washed with 0.5 ml PΕ and the sample was air dried. The cDΝA was eluted by incubating the beads twice with 25 μl of 50°C ΕB Buffer for 5 minutes. The eluted cDNA was stored at -20°C until library construction. A plasmid DNA preparation for a EcoRI-Notl insert-containing pYΕS2.0 cDΝA clone, was purified using a QIAGEN Tip- 100 according to the manufacturer's instructions (QIAGEN, Valencia, CA.) A total of 10 μg of purified plasmid DNA was digested to completion with Notl and EcoRI in a total volume of 60 μl by addition of 6 μl of lOx ΝΕBuffer for EcoRI (New England Biolabs, Beverly, MA), 40 units of Notl, and 20 units of EcoRI followed by incubation for 6 hours at 37°C. The reaction was stopped by heating the sample at 65°C for 20 minutes. The digested plasmid DΝA was extracted once with phenol-chloroform, then with chloroform, followed by ethanol precipitation for 12 hours at -20°C by adding 2 volumes of 96%> ethanol and 0.1 volume of 3 M sodium acetate pH 5.2. The precipitated DΝA was resuspended in 25 μl of lx TΕ pH 7.5, loaded on a 0.8% SeaKem agarose gel in TBΕ buffer, and run on the gel for 3 hours at 60 V. The digested vector was cut out from the gel, and the DΝA was extracted from the gel using the GFX gel band purification kit (Amersham-Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. After measuring the DΝA concentration by OD 260/280 , the eluted vector was stored at -20°C until library construction. One ligation was carried out in 100 μl of ligation buffer (30 mM Tris-Cl pH 7.8, 10 mM MgCl 2 , 10 mM DTT, 0.5 mM ATP) containing 40 μl of double-stranded cDΝA, 5 units of T4 ligase, and 50 ng of EcoRI-Notl cleaved pYΕS2.0 vector (Invitrogen, San Diego, CA). The ligation reaction was performed by incubation at 16°C for 24 hours. The salts and ligase were removed by passing the ligation through Qiaquick PCR spin column (Qiagen Inc., Valencia, CA.). The sample was eluted from the column in 50 μl of EB Buffer (Qiagen Inc., Valencia, CA.) and placed in a speed vacuum until the volume was reduced to 6 μl. Four separate electroporations (200 W, 2.5 kV, 25 mF) were performed using 1 μl of the ligation mixtures in 40 μl of electrocompetent E. coli DH10B cells (Life Technologies, Gaithersburg, MD). After addition of 1 ml of SOC pH 7 medium (composed per liter of 20 g of tryptone, 5 g of yeast extract, 0.5 g of sodium chloride, and 10 ml of 250 mM potassium chloride) to each transformation mix, the cells were grown at 37°C for 1 hour, 250 μl from each electroporation was plated on LB plates supplemented with ampicillin at 100 μg per ml and grown at 37°C for 12 hours. Four Agrotis segetum cDΝA libraries containing a total of 5,500 clones were established in E. coli DH10B. The colonies were scraped from the plates and plasmid DΝA containing the directional, double-stranded cDNA was purified using a Qiagen tip 500 (Qiagen Inc., Valencia, CA). The DNA was resuspended in 300 μl of TE at a concentration of 2.2 μg/μl and stored at -20°C. Example 2: Screening the Agrotis segetum cDNA library One μl of plasmid from the Agrotis segetum cDNA library was electroporated (200 W, 1.5 kV, 25 mF) to 50 μl electrocompetent Saccharomyces cerevisiae JG169 cells. One ml of ice cold 1M sorbitol was added to the transformation mix and 25 μl, 50 μl and 100 μl was plated on Synthetic Complete (SC) medium lacking uridine and grown at 30°C for 24 hours to establish a titer. SC medium lacking uridine is composed per liter of 20 mg of adenine sulfate, 20 mg of L-tryptophan, 20 mg of L-histidine-HCl, 20 mg of L-arginine-HCl, 20 ng of methionine, 30 mg of L-tyrosine, 30 mg of L-leucine, 30 mg of L-isoleucine, 30 mg of L-lysine-HCl, 50 mg of L-phenylalanine, 100 mg of L-glutamic acid, 100 mg of L-aspartic acid, 150 mg of L-valine, 200 mg of L-threonine, and 400 mg of L-serine. Each plate yielded a total of 1600, 3200 and 6400 colonies respectively. Two additional electroporations were done as described above and the transformation mix was diluted with ice cold 1 M sorbitol such that 184 plates containing ca. 443 colonies per plate were obtained. All of the plates, including those used to establish a titer, were replica plated to SC-galactose plates (-URA) containing 0.1%) AZCL-amylose granuoles (Megazyme, Dublin, Ireland) for alpha-amylase screening. Following a 24 incubation at 30°C, the plates were overlayed with 1%> agarose containing 0.25 M glycine, pH 10.0 and 0.1%o AZCL-amylose. The plates were incubated overnight at 37°C and colonies surrounded by a blue zone were isolated on SC-glucose (-URA) plates. All isolates were re-screened on SC GAL (-URA) plates containing AZCL-Amylose as described above. One Saccharomyces cerevisiae transformant was isolated and designated #152, which continued to produce a strong blue zone. The plasmid containing the alpha-amylase gene therein was isolated and transformed into E. coli DH10B cells using standard techniques. A transformant containing p.TeRS2805 was isolated and designated NN049487. Example 3: DNA sequence analysis of Agrotis segetum alpha-amylase gene DNA sequencing of the alpha-amylase gene contained in pJeRS2805 described in Example 2 was performed with an Applied Biosystems Model 373 A Automated DNA Sequencer (Applied Biosystems, Inc., Foster City, CA) on both strands using the primer walking technique with dye-terminator chemistry (Giesecke et al, 1992, Journal of Virology Methods 38: 47-60). Oligonucleotide sequencing primers were designed to complementary sequences in the alpha-amylase gene and were synthesized on an Applied Biosystems Model 394 DNA/RNA Synthesizer according to the manufacturer's instructions. The cDNA nucleotide sequence of the gene encoding the Agrotis segetum alpha- amylase and the deduced amino acid sequence thereof is shown in Figure 1 (SEQ ID NOS. 1 and 2, respectively). Sequence analysis of the cloned insert revealed a large open reading frame of 1500 nucleotides (excluding the stop codon) encoding a protein of 500 amino acids (SEQ ID NO. 2). The G+C content of this open reading frame is 53.56%). Based on the rules of van Heijne (van Heijne, 1984, Journal of Molecular Biology 173: 243-251), the first 15 amino acids likely comprise a secretory signal peptide which directs the nascent polypeptide into the endoplasmic reticulum. A comparative alignment of alpha-amylase sequences was undertaken using the Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, WI) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters were Ktuple=l, gap penalty=3, windows=5, and diagonals=5. The comparative alignment showed that the deduced amino acid sequence of the Agrotis segetum alpha-amylase shares 66.39% identity with the alpha-amylase from Penaeus vannamei (Penoeid shrimp) alpha-amylase (Accession number Q26193). Example 4: Construction of an Agrotis segetum alpha-amylase expression vector for an Aspergillus host pMBinl2 was constructed as described below to contain the TAKA/NA2-tpi leader hybrid promoter, the Agrotis segetum alpha-amylase gene bordered by Swal and Pad sites, the AMG terminator, and the full-length Aspergillus nidulans amdS gene as a selectable marker. PCR was employed to insert a Swal site at the 5' end and a Pad site at the 3' end of the alpha-amylase gene from pJeRS2805 using primers 1 and 2 below. The primers were synthesized with an Applied Biosystems Model 394 DNA RNA Synthesizer (Applied Biosystems, Inc., Foster City, CA) according to the manufacturer's instructions. Primer 1 : 5'-ATTTAAATATGTTCCGTCTCAATCCTTTGC3' (SEQ ID NO. 3) Primer 2: 5'-TTAATTAATTACAGTCTCGATTCAGATCC-3' (SEQ ID NO. 4) Four amplification reactions (50 μl) were prepared using approximately 0.06 μg of plasmid DNA from pJeRS2805 as the template. Each reaction contained the following components: 0.06 μg of plasmid DNA, 100 pmol of the forward primer, 100 pmol of the reverse primer, 2.5 mM each of dATP, dCTP, dGTP, and dTTP, IX Pwo DNA polymerase buffer, and 5.0 U of Pwo DNA polymerase (Roche Diagnostics GmbH, Mannheim, Germany). The reactions were incubated in an Ericomp TwinBlock™ System (Ericomp, Inc., San Diego, CA) programmed as follows: One cycle at 95°C for 5 minutes followed by 30 cycles each at 95°C for 1 minute, 55°C for 1 minute and 72°C for 2 minutes, with a final cycle at 72°C for 5 minutes. The PCR products were electrophoresed on a 1% agarose gel to confirm the presence of a 1.5 kb alpha-amylase gene fragment. The products were excised from the gel and purified using a QIAEX II Gel Extraction Kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. A single deoxyadenosine was added to the 3' ends of the PCR products by incubating with 2.5 U Taq DNA polymerase (Perkin-Elmer Corp., Branchburg, NJ) in IX Taq DNA polymerase buffer and 2.5 mM each of dATP, dCTP, dGTP, and dTTP. Each reaction was heat denatured for 3 minutes at 95°C, followed by a 5 minute incubation at 72°C. The PCR products were subsequently subcloned into pCRII using a TA Cloning Kit (Invitrogen, San Diego, CA) according to the manufacturer's instructions. The transformants were then screened by extracting plasmid DNA from the transformants using a QIAwell-8 Plasmid Kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's instructions, and restriction digesting the plasmid DNA with Swal/ Pad followed by agarose electrophoresis to confirm the presence of a 1.5 kb fragment. To insure that no PCR errore occurred, the PCR products were sequenced with an Applied Biosystems Model 373A Automated DNA Sequencer (Applied Biosystems, Inc., Foster City, CA) on both strands using the primer walking technique with dye- terminator chemistry (Giesecke et al, 1992, Journal of Virol Methods 38: 47-60) using the M13 reverse (-48) and M13 forward (-20) primers (New England Biolabs, Beverly, MA) and primers unique to the DNA being sequenced. A pCRII plasmid containing the verified alpha-amylase gene sequence was then digested with SwallPacl and separated on a 1%) agarose gel. The 1.5 kb fragment was purified using a QIAEX II Gel Extraction Kit according to the manufacturer's instructions. pBaneό (Figure 2), which contains the TAKA/NA2-tpi leader hybrid promoter, a polylinker containing Swal and Pad, the AMG terminator, and the Aspergillus nidulans amdS gene, was digested with Swal and Pad. The alpha-amylase gene, excised from pCRII as described above, was subcloned into the SwallPacl digested pBANeό, resulting in the expression plasmid pMBinl2 (Figure 3) in which transcription of the alpha- amylase gene is under the control of the TAKA/NA2-tpi leader hybrid promoter. Example 5: Expression of the Agrotis segetum alpha-amylase gene in Aspergillus oryzae Plasmid pMBinl2 was introduced into an alkaline protease-deficient Aspergillus oryzae host JaL228 using the following protoplast transformation methods. The transformation was conducted with protoplasts at a concentration of ca. 2xl0 7 protoplasts per ml. One hundred μl of protoplasts were placed on ice with ca. 10 μg of pMBinl2; 250 μl of 60% PEG 4000, lOmM Tris-HCl, pH 7.5, 10 mM CaCl 2 was added, and the protoplasts were incubated at 37°C for 30 minutes. Seven ml of 50°C amdS overlay was added, the solution was mixed gently and poured onto COVE transformation plates. The amdS overlay contains per liter 20 ml of Cove salts (20X), 273.8 g of sucrose, 8g of Noble agar, 10 mM acetamide, and 15 mM CsCl. A 20X Cove salt solution is composed per liter of 26 g of KCl, 26 g of MgSO 4 , 76 g of KH 2 PO 4 and 50 ml of Cove trace elements. COVE plates were composed per liter of 0.52 g of KCl, 0.52 g of MgSO 4 -7H 2 0, 1.52 g of KH 2 PO 4 , 1 ml of trace metals described below, 342.3 g of sucrose, 25 g of Noble agar, 10 ml of 1 M acetamide, and 10 ml of 3 M CsCl. The trace metals solution (1000X) was composed per liter of 22 g of ZnSO 4 -7H 2 O, 11 g of H 3 BO 3 , 5 g of MnCl 2 -4H 2 O, 5 g of FeSO 4 -7H 2 O, 1.6 g of CoCl 2 -5H 2 O, 1.6 g of (NH 4 ) 6 Mo 7 O 24 , and 50 g of Na 4 EDTA. Plates were incubated 5 days at 34°C. Transformants were transferred to plates of the same medium and incubated 3 days at 34°C. Totally, 27 transformants were recovered by their ability to grow on COVE medium using acetamide as sole nitrogen source. The 27 transformants were grown for 6 days at 34°C, 200 rpm in shake flasks containing 20 ml of MY50 maltose medium. MY50 was composed per liter of 50 g of maltose, 2.0 g of MgSO 4 -7H 2 O, 10 g of KH 2 PO 4 , 2 g of citric acid, 10 g of yeast extract, 2.0 g of urea, 2 g of K 2 SO 4 and 0.5 ml of trace elements solution adjusted to pH 6.0. The trace metals solution was composed per liter of 14.3 g of ZnSO 4 -7H 2 O, 2.5g of CuSO 4 -5H 2 O, 0.5g of NiCl 2 -6H 2 O, 13.8 g of FeSO 4 -7H 2 O, 8.5 g of MnSO 4 -H 2 O, 3 g of citric acid. Each of the shake flasks were assayed for alpha-amylase activity using Phadebas Tablets as the substrate as described above. The supernatant was diluted 1 :100 in 1 M Tris-2 mM CaCl 2 pH 9.0 and serial dilutions from 1 :2 to 1 :16 were made. Each of the serial dilutions were diluted an additional 1 :10 in reaction buffer (50 μl of diluted supernatant plus 450 μl of reaction buffer). Reaction buffer was made in 5 ml quantities containing one Phadebas Tablet dissolved in 0.5 M Tris-10 mM CaCl 2 pH 9.0 and is preheated to 37°C prior to use. Following the addition of sample, the reaction was incubated for 45 minutes at 37°C. The reaction was terminated by placing on ice and then centrifuged briefly at 4°C. The supernatant was removed and the absorbance was read at 620 nm. All of the transformants assayed produced detectable alpha-amylase activity. Deposit of Biological Material The following biological material has been deposited under the terms of the Budapest Treaty with the Deutsche Sammlung von Microorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany and given the following accession number: Deposit Accession Number Date of Deposit E. coli NN049487 (pJeRS2805) DSM 12876 June 10, 1999 The strain has 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 in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action. 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. Applicant's or agent's file International application reference number 5899.204-WO To be assigned INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13 bis) A. The indications made below relate to the microorganism referred to in the description on page 56 , line 27 . B. IDENTIFICATION OF Further deposits are identified on an additional sheet D Name of depository institution Deutsche Sammlung von Microorganismen und Zellkulturen GmbH (DSM) Address of depository institution (including postal code and country) Mascheroder Weg 1 b D-38124 Braunschweig Germany Date of deposit Accession Number June 10, 1999 DSM 12876 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet D Until the publication of the mention of grant of a European patent or, where applicable, for twenty years from the date of filing if the application has been refused, withdrawn or deemed withdrawn, a sample of the deposited microorganizm is only to be provided to an independent expert nominated by the person requesting the sample (cf. Rule 28(4) EPC). And as far as Australia is concerned, the expert option is likewise requested, reference being had to Regulation 3.25 of Australia Statutory Rules 1991 No. 71. Also, for Canada we request that only an independent expert nominated by the Commissioner is authorized to have access to a sample of the microorganizm deposited. D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indication listed below will be submitted to the International Bureau Later (specify the general nature of the indications e.g. "Accession Number of Deposit") For receiving Office use only For International Bureau use only This sheet was received with the international application This sheet was received with the International Bureau on: Authorized officer Authorized officer Form PCT/RO/134 (July 1992)

Claims: Claims What is claimed is: 1. An isolated polypeptide having alpha-amylase activity, selected from the group consisting of: (a) a polypeptide having an amino acid sequence which has at least 75% identity with amino acids 16 to 500 of SEQ ID NO. 2; (b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (i) nucleotides 46 to 1500 of SEQ ID NO. 1, (ii) the genomic DNA comprising nucleotides 46 to 1500 of SEQ ID NO. 1, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii); (c) a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 comprising a substitution, deletion, and/or insertion of one or more amino acids; (d) an allelic variant of (a) or (b); and (e) a fragment of (a), (b), or (d) that has alpha-amylase activity. 2. The polypeptide of claim 1, having an amino acid sequence which has at least 75% identity with amino acids 16 to 500 of SEQ ID NO. 2. 3. The polypeptide of claim 2, having an amino acid sequence which has at least 80%) identity with amino acids 16 to 500 of SEQ ID NO. 2. 4. The polypeptide of claim 3, having an amino acid sequence which has at least 85%o identity with amino acids 16 to 500 of SEQ ID NO. 2. 5. The polypeptide of claim 4, having an amino acid sequence which has at least 90% identity with amino acids 16 to 500 of SEQ ID NO. 2. 6. The polypeptide of claim 5, having an amino acid sequence which has at least 95% identity with amino acids 16 to 500 of SEQ ID NO. 2. 7. The polypeptide of any of claims 1-6, comprising the amino acid sequence of SEQ ID NO. 2. 8. The polypeptide of any of claims 1-7, consisting of the amino acid sequence of SEQ ID NO. 2 or a fragment thereof. 9. The polypeptide of claim 8, consisting of the amino acid sequence of SEQ ID NO. 2. 10. The polypeptide of claim 9, which consists of amino acids 16 to 500 of SEQ ID NO. 2. 11. The polypeptide of claim 1 , which is encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (i) nucleotides 46 to 1500 of SEQ ID NO. 1, (ii) the genomic DNA comprising nucleotides 46 to 1500 of SEQ ID NO. 1, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii). 12. The polypeptide of claim 11, which is encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (i) nucleotides 46 to 1500 of SEQ ID NO. 1, (ii) the genomic DNA comprising nucleotides 46 to 1500 of SEQ ID NO. 1, or (iii) a complementary strand of (i) or (ii). 13. The polypeptide of claim 1, which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with (i) nucleotides 46 to 1500 of SEQ ID NO. 1, (ii) the genomic DNA comprising nucleotides 46 to 1500 of SEQ ID NO. 1, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii). 14. The polypeptide of claim 13, which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with (i) nucleotides 46 to 1500 of SEQ ID NO. 1, (ii) the genomic DNA comprising nucleotides 46 to 1500 of SEQ ID NO. 1, or (iii) a complementary strand of (i) or (ii). 15. The polypeptide of claim 1, wherein the polypeptide is a variant of the polypeptide having an amino acid sequence of SEQ ID NO. 2 comprising a substitution, deletion, and/or insertion of one or more amino acids. 16. The polypeptide of claim 1, which is encoded by the nucleic acid sequence contained in plasmid pJeRS2805 which is contained in E. coli DSM 12876. 17. The polypeptide of any of claims 1-16 which has at least 20% of the alpha- amylase activity of SEQ ID NO. 2. 18. A polypeptide having the same alpha-amylase activity as the polypeptide of any of claims 1-17. 19. An isolated nucleic acid sequence comprising a nucleic acid sequence which encodes the polypeptide of any of claims 1-18. 20. 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, in which the mutant nucleic acid sequence encodes a polypeptide consisting of amino acids 16 to 500 of SEQ ID NO. 2. 21. An isolated nucleic acid sequence produced by (a) hybridizing a DNA under medium stringency conditions with (i) nucleotides 46 to 1500 of SEQ ID NO. 1, (ii) the genomic DNA comprising nucleotides 46 to 1500 of SEQ ID NO. 1, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii); and (b) isolating the nucleic acid sequence. 22. The isolated nucleic acid sequence of claim 21 produced by (a) hybridizing a DNA under high stringency conditions with (i) nucleotides 46 to 1500 of SEQ ID NO. 1, (ii) the genomic DNA comprising nucleotides 46 to 1500 of SEQ ID NO. 1, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii); and (b) isolating the nucleic acid sequence. 23. A nucleic acid construct comprising the nucleic acid sequence of claim 19 operably linked to one or more control sequences that direct the production of the polypeptide in a suitable expression host. 24. A recombinant expression vector comprising the nucleic acid construct of claim 23. 25. A recombinant host cell comprising the nucleic acid construct of claim 24. 26. A method for producing a mutant nucleic acid sequence, comprising (a) introducing at least one mutation into the mature polypeptide coding sequence of SEQ ID NO. 1, wherein the mutant nucleic acid sequence encodes a polypeptide consisting of amino acids 16 to 500 of SEQ ID NO. 2; and (b) recovering the mutant nucleic acid sequence. 27. A mutant nucleic acid sequence produced by the method of claim 26. 28. A method for producing a polypeptide, comprising (a) cultivating a strain comprising the mutant nucleic acid sequence of claim 27 encoding the polypeptide to produce a supernatant comprising the polypeptide; and (b) recovering the polypeptide. 29. A method for producing the polypeptide of any of claims 1-18 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. 30. 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 in the mature polypeptide coding sequence of SEQ ID NO. 1, wherein the mutant nucleic acid sequence encodes a polypeptide consisting of amino acids 16 to 500 of SEQ ID NO. 2, and (b) recovering the polypeptide. 31. A method for producing the polypeptide of any of claims 1-18 comprising (a) cultivating a homologously recombinant cell, having incorporated therein a new transcription unit comprising a regulatory sequence, an exon, and/or a splice donor site operably linked to a second exon of an endogenous nucleic acid sequence encoding the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. 32. A method for producing a mutant of a cell, which comprises disrupting or deleting a nucleic acid sequence encoding the polypeptide of any of claims 1-18 or a control sequence thereof, which results in the mutant producing less of the polypeptide than the cell. 33. A mutant cell produced by the method of claim 32. 34. The mutant cell of claim 33, which further comprises a nucleic acid sequence encoding a heterologous protein. 35. A method for producing a heterologous polypeptide comprising (a) cultivating the mutant of claim 34 under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. 36. A nucleic acid construct comprising a gene encoding a protein operably linked to a nucleic acid sequence encoding a signal peptide consisting of nucleotides 1 to 45 of SEQ ID NO. 1, wherein the gene is foreign to the nucleic acid sequence. 37. A recombinant expression vector comprising the nucleic acid construct of claim 36. 38. A recombinant host cell comprising the nucleic acid construct of claim 36. 39. A method for producing a protein comprising (a) cultivating the recombinant host cell of claim 38 under conditions suitable for production of the protein; and (b) recovering the protein. 40. A detergent composition comprising a carrier and the polypeptide of any of claims 1-18. 41. A fragment of the nucleic acid sequence of any of claims 19-22 and 27 that when combined with a sequence encoding a second polypeptide confers on that second polypeptide an increase activity at alkaline pH of at least 10%> at pH values of 8.0 or above.

Inventors: CONNELLY MARIAH BINDELL, SHUSTER JEFFREY, CONNELLY, MARIAH, BINDELL, SHUSTER, JEFFREY
Applicants: NOVO NORDISK BIOTECH INC, NOVO NORDISK BIOTECH, INC.
Application year: 2000
Main classification: C12N9
IPC codes: C11D3, C12N9