Α-amylase variants

Abstract

To provide variants of an α-amylase, polynucleotides encoding the variants, and a method of producing the variants.SOLUTION: The present invention relates to variants of a parent α-amylase having an improved wash performance when compared to the parent α-amylase. The present invention also relates to polynucleotides encoding the variants, nucleic acid constructs, vectors and host cells comprising the polynucleotides, and a method of producing the variants of the present invention.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to α-amylase variants, polynucleotides encoding the variants, and methods for producing the variants. [Background technology]

[0003] Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycoside oligosaccharides and polysaccharides.

[0004] There is a long history of industrial use of α-amylases in a number of well-known applications, such as detergents, bakery, brewing, starch liquefaction and saccharification, e.g., in the preparation of isomerized sugar or as part of the production of ethanol from starch, etc. These and other uses of α-amylases are well known, and in particular utilize α-amylases of microbial origin, such as bacterial α-amylases.

[0005] The first bacterial α-amylase used was the α-amylase from B. licheniformis, also known as Termamyl, which has been extensively characterized and a crystal structure for this enzyme is available. Alkaline amylases such as AA560 form a particular group of α-amylases that have found use in detergents. Many of these known bacterial amylases have been modified to improve their performance in specific applications.

[0006] Bacillus amylases, such as Termamyl, AA560 (WO 2000 / 060060), and SP707 (described by Tsukamoto et al., 1988, Biochem. Biophys. Res. Comm. 151:25-31), form a particular group of α-amylases that find use in detergents. These amylases have been modified to improve their stability in detergents. For example, WO 96 / 23873 discloses that amino acids 181+182 or 183+184 of SP707 (SEQ ID NO: 7 in WO 96 / 23873) are deleted to improve the stability of this amylase. WO 96 / 23873 further discloses modifying SP707 amylase by substituting M202, for example, with leucine, to stabilize the molecule against oxidation. Therefore, it is known to modify amylases to improve certain properties.

[0007] To protect the environment, it is becoming increasingly important to lower the temperature during washing, dishwashing, and / or cleaning processes. However, the optimum temperature of most enzymes, including amylases, is higher than the temperatures typically used in low-temperature washing. α-Amylases are important enzymes used in detergent compositions, and their use is becoming increasingly important for removing starchy stains during laundry washing or dishwashing. Therefore, it is important to find α-amylase variants that retain their cleaning performance, stain removal efficacy, and / or activity even at low temperatures. However, despite the efficiency of current detergent enzyme compositions, many stains are difficult to completely remove. These problems are exacerbated by the increasing use of low wash temperatures (e.g., cold water) and short wash cycles. Therefore, it is desirable to obtain amylolytic enzymes that can function at low temperatures while maintaining or increasing other desirable properties, such as specific activity (amylolytic activity), stability, and / or cleaning performance.

[0008] Therefore, the present invention aims to provide α-amylase variants that can be used in washing, dishwashing and / or cleaning processes at low temperatures, such as temperatures between 5 and 40° C. Furthermore, the present invention also aims to provide α-amylase variants that have improved washing performance at low temperatures compared to the parent α-amylase or compared to the α-amylase of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 or 8. Summary of the Invention [Means for solving the problem]

[0009] The present invention relates to variants of a parent α-amylase, wherein (i) the variant comprises a modification at one or more positions corresponding to 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence according to SEQ ID NO: 1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence according to SEQ ID NO: 1; (ii) the variant has at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, but less than 100% sequence identity to the amino acid sequence according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8; and (iii) the variant has α-amylase activity.

[0010] The present invention also relates to polynucleotides encoding the variants of the present invention, nucleic acid constructs comprising polynucleotides encoding the variants of the present invention, expression vectors comprising polynucleotides encoding the variants of the present invention, and host cells comprising polynucleotides encoding the variants of the present invention.

[0011] The present invention also relates to a method for producing an α-amylase variant, comprising the steps of: (a) culturing a host cell of the invention under conditions suitable for expression of the variant; and (b) recovering the variant.

[0012] The present invention further relates to a method for obtaining an α-amylase variant, comprising the steps of introducing modifications into a parent α-amylase at one or more positions corresponding to 109, 7, 1, 391, 280, 284, 320 and 323 of the amino acid sequence according to SEQ ID NO: 1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence according to SEQ ID NO: 1, wherein each modification is independently a substitution or a deletion, and wherein the variant has α-amylase activity; and recovering the variant. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention relates to variants of a parent α-amylase, wherein (i) the variant comprises a modification at one or more positions corresponding to 109, 7, 1, 391, 280, 284, 320 and 323 of the amino acid sequence according to SEQ ID NO: 1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence according to SEQ ID NO: 1; (ii) the variant has at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, but less than 100% sequence identity to the amino acid sequence according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8; and (iii) the variant has α-amylase activity.

[0014] In one aspect, the present invention relates to variants of a parent α-amylase, wherein (i) the variant comprises a modification at one or more positions corresponding to positions selected from the group consisting of 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence according to SEQ ID NO: 1, and optionally at one or more positions corresponding to positions selected from the group consisting of 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence according to SEQ ID NO: 1; (ii) the variant has at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, but less than 100% sequence identity to the amino acid sequence according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8; and (iii) the variant has α-amylase activity.

[0015] definition Allelic variant: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation and can also result from polymorphism in a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode a polypeptide having an altered amino acid sequence. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0016] The term "α-amylase" (α-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) constitutes a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycosidic oligosaccharides and polysaccharides. For purposes of the present invention, α-amylase activity is determined according to the procedures described in the Examples section. In one embodiment, a variant of the invention has at least 20%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100%, of the α-amylase activity of the mature polypeptide of SEQ ID NO: 1.

[0017] As used herein, the term "amino acid" includes the 20 standard genetically encoded amino acids and their corresponding "D" stereoisomers (as compared to the natural "L" forms), ω-amino acids, other naturally occurring amino acids, unusual amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derivatized amino acids. Chemical derivatives of one or more amino acids can be achieved by reaction with a functional side group. Such derivatized molecules include, for example, molecules in which a free amino group has been derivatized to form an amine hydrochloride, p-toluenesulfonyl, carboxybenzoxy, t-butyloxycarbonyl, chloroacetyl, or formyl group. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters, or other types of esters and hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. Chemical derivatives also include peptides containing natural amino acid derivatives of the 20 standard amino acids. For example: 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine, and ornithine can be substituted for lysine. Derivatives also include peptides containing one or more additions or deletions, so long as the required activity is maintained. Other included modifications are terminal modifications, such as amidation, amino-terminal acylation (e.g., acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g., with ammonia or methylamine), etc.

[0018] Where an amino acid is specifically recited (such as "alanine" or "Ala" or "A"), the term refers to both l-alanine and d-alanine unless otherwise specified. Other unconventional amino acids may also be suitable components of the polypeptides of the invention, so long as the desired functional properties are retained by the polypeptide. For the peptides shown, each encoded amino acid residue is represented, where appropriate, by a single-letter symbol corresponding to the trivial name of the conventional amino acid. In one embodiment, the polypeptides of the invention comprise or are composed of L-amino acids.

[0019] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before emerging as the mature, spliced ​​mRNA.

[0020] The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0021] The term "control sequence" refers to a nucleic acid sequence required for expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from different genes) to the polynucleotide encoding the variant, or may be native or foreign to each other. 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, they comprise a control sequence, 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 to the coding region of the polynucleotide encoding the variant.

[0022] The term "enhanced washing performance" or "improved washing performance" refers to the ability of the polypeptides of the invention to provide improved cleaning benefits (e.g., stain removal) in a washing process, such as laundry or dishwashing, compared to the parent α-amylase of SEQ ID NO: 1. Washing performance can be determined using methods well known in the art, such as using the Automated Mechanical Stress Assay (AMSA). Those skilled in the art will recognize that enhanced washing performance can be achieved under only some, or perhaps all, of the washing conditions, for example, wash temperatures above 20°C (e.g., 40°C).

[0023] As used herein, the term "enzyme detergency benefit" refers to the advantageous effect that an enzyme can impart to a detergent compared to the same detergent without the enzyme. Important detergency benefits that enzymes can provide are soil removal with no or little visible soil after washing and / or cleaning; prevention or reduction of redeposition of soil liberated in the washing process (an effect also known as anti-redeposition); and complete or partial restoration of whiteness to fabrics that were originally white but have acquired a grayish or yellowish appearance after repeated use and washing (an effect also known as whitening). Textile care benefits that are not directly related to catalytic soil removal or prevention of soil redeposition are also important for enzyme detergency benefits. Examples of such fabric treatment benefits are preventing or reducing dye transfer from one fabric to another or to other parts of the same fabric (an effect also known as dye transfer or backstain prevention); removing protruding or broken fibers from the fabric surface to reduce pilling or removing existing pilling or fuzz (an effect also known as anti-pilling); improving fabric softness; clarifying the color of the fabric; and removing particulate soils trapped in the fibers of the fabric or garment. Enzyme bleaching is an additional enzyme cleaning benefit, where catalytic activity is generally used to catalyze the formation of bleaching components such as hydrogen peroxide or other peroxides.

[0024] The term "expression" includes any step involved in the production of a variant, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0025] The term "expression vector" includes a linear or circular DNA molecule containing a polynucleotide encoding a variant and operably linked to a control sequence that provides for its expression.

[0026] The term "fragment" refers to a polypeptide lacking one or more (e.g., multiple) amino acids at the amino and / or carboxyl terminus of the polypeptide of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, or 8; wherein the fragment has α-amylase activity. In one embodiment, the fragment contains at least 200 contiguous amino acid residues of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, or 8, e.g., at least 300, or at least 350, or at least 400, or at least 450 contiguous amino acid residues of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, or 8.

[0027] The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc., with a nucleic acid construct or expression vector comprising a polynucleotide of the invention. 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.

[0028] As used herein, the term "intensity value" refers to a measurement of washing performance. It is measured as luminance, which is the intensity of light reflected from a sample when illuminated with white light. When a sample is dirty, the intensity of the reflected light is lower than that from a clean sample. Therefore, the intensity of the reflected light can be used to measure washing performance, with higher intensity values ​​correlating with better washing performance. Color measurements are taken with a professional flatbed scanner (Kodak iQsmart, Kodak) used to capture images of the washed fabrics. To extract light intensity values ​​from the scanned image, 24-bit pixel values ​​from the image are converted to red, green, and blue (RGB) values. The intensity value (Int) is calculated by adding the RGB values ​​together as a vector and then calculating the length of the resulting vector.

number

[0029] The term "Δ strength" or "Δ strength value" is defined herein as the result of measuring the strength of a test material, such as a swatch CS-28 (Center For Test materials BV, PO Box 120, 3133 KT Vlaardingen, the Netherlands) or a hard surface. The swatch is measured along with a portion of a swatch washed under identical conditions as a background. Δ strength is the strength value of the test material washed with amylase minus the strength value of the test material washed without amylase.

[0030] As used herein, the term "improved property" refers to a characteristic of a variant that is improved compared to the parent. Such improved properties include, but are not limited to, cleaning performance, thermal activity, heat resistance, and stability under storage conditions and chemical stability. The improved property can be any of those, such as stability, as defined and described herein.

[0031] The term "improved cleaning performance" is defined herein to indicate an alteration in the cleaning performance of an amylase of the invention relative to the cleaning performance of the amylase of SEQ ID NO: 2 or 1, which may be manifested, for example, as improved soil removal. Improved cleaning performance is determined according to Example 1. Improved cleaning performance is indicated by an improvement factor (IF) of greater than 1.0, preferably greater than 1.05, under one or more of the conditions listed in Example 1 at 20°C in model detergent A with an α-amylase variant concentration of 0.2 mg / L, or at 40°C in model detergent A with an α-amylase variant concentration of 0.05 mg / L, or at 20°C in model detergent J with an α-amylase variant concentration of 0.2 mg / L, or at 30°C in model detergent J with an α-amylase variant concentration of 0.05 mg / L, or at 20°C in detergent K with an α-amylase variant concentration of 0.2 mg / L. Washing conditions are described in the Examples section.

[0032] The term "washing performance" typically includes cleaning, such as hard surface cleaning in dishwashing, but also includes fabric cleaning performance, such as laundry, and industrial and institutional cleaning. Improved washing performance can be measured by comparing the delta strengths described in the definitions herein.

[0033] The term "isolated" refers to a substance in a form or setting that does not occur in nature. Non-limiting examples of isolated substances include: (1) any non-naturally occurring substance; (2) any substance, including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide, or cofactor, from which one or more or all of the naturally occurring components with which it is associated in nature have been at least partially removed; (3) any substance that has been artificially modified relative to the substance found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components with which it is associated in nature (e.g., multiple copies of the gene encoding the substance; use of a stronger promoter than that naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample. In one aspect, the present invention relates to an isolated α-amylase variant.

[0034] Isolated polynucleotide: The term "isolated polynucleotide" refers to a polynucleotide that has been artificially modified. In one embodiment, an isolated polynucleotide is at least 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, at least 90% pure, and at least 95% pure, as measured by agarose gel electrophoresis. The polynucleotide may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combination thereof.

[0035] Isolated variant: The term "isolated variant" refers to a variant that has been artificially modified. In one embodiment, the variant is at least 1% pure, as measured by SDS-PAGE, such as at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, and at least 90% pure.

[0036] Low temperature: "Low temperature" is a temperature of 5 to 40°C, preferably 5 to 35°C, preferably 5 to 30°C, more preferably 5 to 25°C, more preferably 5 to 20°C, most preferably 5 to 15°C, and especially 5 to 10°C. In a preferred embodiment, "low temperature" is a temperature of 10 to 35°C, preferably 10 to 30°C, or 10 to 25°C, or 10 to 20°C, or 10 to 15°C.

[0037] The term "mature polypeptide" refers to a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell can produce a mixture of two additional different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide.

[0038] The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having α-amylase activity.

[0039] The term "variant" refers to a polynucleotide that encodes the variant.

[0040] The term "mutation" in the context of the polypeptides of the invention means that one or more amino acids in a reference amino acid sequence (i.e., SEQ ID NO: 1) are modified by substitution with a different amino acid or deletion. Furthermore, a mutation can represent the insertion of one or more extra amino acids into the reference amino acid sequence.

[0041] The term "nucleic acid construct" refers to a single- or double-stranded nucleic acid molecule, which has been isolated from a naturally occurring gene or which has been modified to contain a segment of nucleic acid not otherwise found in nature, or which is synthetic and includes one or more regulatory sequences. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the regulatory sequences required for expression of a coding sequence of the invention.

[0042] The term "operably linked" refers to a structure in which a control sequence is positioned in an appropriate position relative to a coding sequence of a polynucleotide so that the control sequence effects the expression of the coding sequence.

[0043] The terms "parent" or "parent α-amylase" refer to an α-amylase that has been modified to provide an enzyme variant of the present invention. The parent may be a naturally occurring (wild-type) polypeptide or a variant thereof. For example, the parent may be the α-amylase of SEQ ID NO: 1 (known as SP722). Alternatively, the parent may refer to the α-amylase of SEQ ID NO: 2. The parent α-amylase may be any suitable α-amylase, such as those listed herein as SEQ ID NOs: 3, 4, 5, 6, 7, and 8.

[0044] The relatedness between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity."

[0045] For purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably the Needle program version 5.0.0 or later. The parameters used may be a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (equivalent residues × 100) / (length of alignment − total number of gaps in the alignment)

[0046] Alternatively, the parameters used can be a gap open penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix. The output of the Needle label "longest identity" (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (equivalent deoxyribonucleotides × 100) / (length of alignment − total number of gaps in alignment)

[0047] Starch removal process: The expression "starch removal process" relates to any kind of process in which starch is removed (or converted), such as in a washing process in which starch is removed from fabrics, for example fabric cleaning, such as in laundry. The starch removal process can also be hard surface cleaning, such as dish washing, or a general cleaning process, such as industrial or institutional cleaning. The expression also generally includes other starch removal processes or starch conversion, ethanol production, starch liquefaction, fabric desizing, paper and pulp production, beer production, and detergents.

[0048] Substantially pure polynucleotide: The term "substantially pure polynucleotide" refers to a polynucleotide preparation that is free of other extraneous or unwanted nucleotides and is in a form suitable for use in a genetically engineered polypeptide production system. Thus, a substantially pure polynucleotide contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5% by weight of other polynucleotide material with which it is naturally or recombinantly associated. However, a substantially pure polynucleotide may include naturally occurring 5'- and 3'-untranslated regions, such as promoters and terminators. Preferably, a substantially pure polynucleotide is at least 90% pure by weight, e.g., at least 92% pure, at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, and at least 99.5% pure. The polynucleotides of the present invention are preferably in a substantially pure form.

[0049] Substantially pure variant: The term "substantially pure variant" refers to a preparation containing at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5 wt% of other polypeptide material with which it is naturally or recombinantly related. Preferably, the variant is at least 92% pure, based on the total weight of polypeptide material present in the preparation, e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, at least 99.5% pure, and 100% pure. The variants of the invention are preferably in substantially pure form. This can be achieved, for example, by preparing the variant by well-known recombinant methods or by classical purification methods.

[0050] The term "subsequence" refers to a polynucleotide lacking one or more (e.g., multiple) nucleotides at the 5' and / or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment that has α-amylase activity.

[0051] Fabric: Fabric sample CS-28 (rice starch on cotton) is obtained from Center For Test materials BV, PO Box 120, 3133 KT Vlaardingen, the Netherlands.

[0052] As used herein, the term "fabric handling benefits" is defined as not directly related to catalytic soil removal or preventing soil redeposition, but is important to enzyme cleaning benefits.Examples of such fabric handling benefits include preventing or reducing dye transfer from one fabric to another or to another part of the same fabric (also known as dye transfer prevention or back-soiling prevention); removing protruding or broken fibers from the fabric surface to reduce pilling, or removing existing pilling or fluff (also known as anti-pilling); improving fabric softness; clarifying fabric color; and removing particulate soiling stuck in fabric fibers.Enzyme bleaching is another enzyme cleaning benefit, where catalytic activity is generally used to catalyze the formation of bleaching components such as hydrogen peroxide or other peroxides or other bleaching species.

[0053] The term "variant" refers to a polypeptide having α-amylase activity that contains a mutation (i.e., substitution, insertion, and / or deletion) at one or more (e.g., multiple) positions relative to the "parent" α-amylase of SEQ ID NO:1 or SEQ ID NOs:2, 3, 4, 5, 6, 7, or 8. A substitution refers to the replacement of an amino acid at a position with a different amino acid; a deletion refers to the removal of an amino acid at a position; and an insertion refers to the addition of an amino acid immediately adjacent to the amino acid at a position. Variants of the invention have at least 20%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100%, of the α-amylase activity of the mature polypeptide of SEQ ID NO:1 or SEQ ID NO:2.

[0054] The term "wild-type" α-amylase refers to an α-amylase expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus, as found in nature.

[0055] Conventional mutant determination The polypeptides of the invention having α-amylase activity correspond to variants of the α-amylases derived from Bacillus as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8. [ka]

[0056] For purposes of the present invention, the polypeptide disclosed in SEQ ID NO:1 is used to determine corresponding amino acid residues in other α-amylase polypeptides. However, one of skill in the art will recognize that the sequence of SEQ ID NO:2 can also be used to determine corresponding amino acid residues in other α-amylase polypeptides. The amino acid sequence of the other α-amylase is aligned with the mature polypeptide disclosed in SEQ ID NO:1, and based on this alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO:2 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the Needle program of the EMBOSS package, preferably version 5.0.0 or later (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277). The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0057] Identification of corresponding amino acid residues in other α-amylases can be performed using tools such as, but not limited to, MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Katoh et al., 2005, Nucleic Acids Research 33:511-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology 537:39-64; Katoh and Toh, 2010, Bioinformatics 26:1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680), using their respective default parameters, can be determined by alignment of multiple polypeptide sequences using a number of computer programs.

[0058] Where other α-amylases diverge from the mature polypeptide of SEQ ID NO:1 to the extent that traditional sequence-based comparisons do not permit detection of relationships, other pairwise sequence comparison algorithms can be used. The sensitivity of sequence-based searches can be enhanced by using search programs that utilize probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process, enabling the detection of distant homologs (Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402). Sensitivity can be further enhanced when a polypeptide family or superfamily has one or more representations in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) use information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as input to neural networks that predict the fold structure of a query sequence. Similarly, the method by Gough et al., 2000, J. Mol. Biol. 313:903-919, can be used to align sequences of unknown structure with superfamily models present in the SCOP database. These alignments can then be used to generate homology models for the polypeptide, which can be accurately evaluated using a variety of tools developed for that purpose.

[0059] For proteins of known structure, numerous tools and resources are available for searching and generating structural alignments. For example, the SCOP superfamily of proteins has been structurally aligned, and these alignments are accessible and downloadable. Two or more protein structures can be aligned using a variety of algorithms, such as distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combinatorial extension methods (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), which can be run incrementally against a query structure database along with the target structure to discover potential structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0060] In describing the α-amylase variants of the present invention, the following nomenclature is adopted for ease of reference: The accepted IUPAC one-letter or three-letter amino acid abbreviations are utilized.

[0061] Substitutions. The following nomenclature is used for amino acid substitutions: original amino acid, position, substituted amino acid. Thus, for example, a substitution of threonine with alanine at position 226 is designated "Thr226Ala" or "T226A." Multiple mutations are separated by a plus sign ("+"), e.g., "Gly205Arg+Ser411Phe" or "G205R+S411F," representing substitutions of glycine (G) with arginine (R) and serine (S) with phenylalanine (F) at positions 205 and 411, respectively.

[0062] Deletions. The following nomenclature is used for amino acid deletions: original amino acid, position, * Therefore, the deletion of glycine at position 181 is "Ser181 * " or "S181 *Multiple deletions are separated by a plus sign ("+"), e.g., "Ser181 * +Thr182 * " or "S181 * +T182 * "It is said that

[0063] Insertions. The following nomenclature is used for amino acid insertions: original amino acid, position, original amino acid, inserted amino acid. Thus, for example, the insertion of a lysine after a glycine at position 195 is designated as "Gly195GlyLys" or "G195GK." Multiple amino acid insertions are designated as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of a lysine and an alanine after a glycine at position 195 is designated as "Gly195GlyLysAla" or "G195GKA."

[0064] In such cases, the inserted amino acid residue is numbered by adding a subscript to the amino acid residue preceding the inserted amino acid residue. Thus, in the above example, the sequence would be:

[0065] [Table 1]

[0066] Multiple Modifications. Variants containing multiple modifications are separated by a plus sign ("+"), e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent substitutions of tyrosine and glutamic acid at positions 170 and 195 for arginine and glycine, respectively.

[0067] Different modifications. When different modifications can be introduced at one position, the different modifications are separated by a comma, for example, "Arg170Tyr,Glu" represents the substitution of arginine with tyrosine or glutamic acid at position 170. Thus, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variant: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".

[0068] Parent α-amylase The parent α-amylase can also be a polypeptide having at least 80% sequence identity to the polypeptide set forth in SEQ ID NO:1.

[0069] In one embodiment, the parent α-amylase has at least 80% sequence identity, such as at least 85%, at least 90%, e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 or 100%, to a polypeptide of SEQ ID NO: 1 having α-amylase activity. In one embodiment, the amino acid sequence of the parent α-amylase differs from the polypeptide of SEQ ID NO: 1 by 10 or fewer amino acids, e.g., by 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids and 1 amino acid.

[0070] Preferably, the parent α-amylase comprises or consists of the amino acid sequence of SEQ ID NO: 1. In another embodiment, the parent α-amylase is an allelic variant of the polypeptide of SEQ ID NO: 1.

[0071] The parent α-amylase can also be a polypeptide having at least 80% sequence identity to the polypeptide set forth in SEQ ID NO:2.

[0072] In one embodiment, the parent α-amylase has at least 80% sequence identity, such as at least 85%, at least 90%, e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 or 100%, to a polypeptide of SEQ ID NO: 2 having α-amylase activity. In one embodiment, the amino acid sequence of the parent α-amylase differs from the polypeptide of SEQ ID NO: 2 by 10 or fewer amino acids, e.g., by 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids and 1 amino acid.

[0073] Preferably, the parent α-amylase comprises or consists of the amino acid sequence of SEQ ID NO: 2. In another embodiment, the parent α-amylase is an allelic variant of the polypeptide of SEQ ID NO: 2.

[0074] The parent α-amylase may also be a polypeptide having at least 80% sequence identity to the polypeptide set forth in SEQ ID NO:3.

[0075] In one embodiment, the parent α-amylase has at least 80% sequence identity, such as at least 85%, at least 90%, e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 or 100%, to a polypeptide having α-amylase activity of SEQ ID NO: 3. In one embodiment, the amino acid sequence of the parent α-amylase differs from the polypeptide of SEQ ID NO: 3 by 10 or fewer amino acids, e.g., by 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids and 1 amino acid.

[0076] Preferably, the parent α-amylase comprises or consists of the amino acid sequence of SEQ ID NO: 3. In another embodiment, the parent α-amylase is an allelic variant of the polypeptide of SEQ ID NO: 3.

[0077] The parent α-amylase may also be a polypeptide having at least 80% sequence identity to the polypeptide set forth in SEQ ID NO:4.

[0078] In one embodiment, the parent α-amylase has at least 80% sequence identity, such as at least 85%, at least 90%, e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 or 100%, to a polypeptide of SEQ ID NO: 4 having α-amylase activity. In one embodiment, the amino acid sequence of the parent α-amylase differs from the polypeptide of SEQ ID NO: 4 by 10 or fewer amino acids, e.g., by 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids and 1 amino acid.

[0079] Preferably, the parent α-amylase comprises or consists of the amino acid sequence of SEQ ID NO: 4. In another embodiment, the parent α-amylase is an allelic variant of the polypeptide of SEQ ID NO: 4.

[0080] The parent α-amylase may also be a polypeptide having at least 80% sequence identity to the polypeptide set forth in SEQ ID NO:5.

[0081] In one embodiment, the parent α-amylase has at least 80% sequence identity, such as at least 85%, at least 90%, e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 or 100%, to a polypeptide having α-amylase activity of SEQ ID NO: 5. In one embodiment, the amino acid sequence of the parent α-amylase differs from the polypeptide of SEQ ID NO: 5 by 10 or fewer amino acids, e.g., by 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids and 1 amino acid.

[0082] Preferably, the parent α-amylase comprises or consists of the amino acid sequence of SEQ ID NO: 5. In another embodiment, the parent α-amylase is an allelic variant of the polypeptide of SEQ ID NO: 5.

[0083] The parent α-amylase may also be a polypeptide having at least 80% sequence identity to the polypeptide set forth in SEQ ID NO:6.

[0084] In one embodiment, the parent α-amylase has at least 80% sequence identity, such as at least 85%, at least 90%, e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 or 100%, to a polypeptide of SEQ ID NO: 6 having α-amylase activity. In one embodiment, the amino acid sequence of the parent α-amylase differs from the polypeptide of SEQ ID NO: 6 by 10 or fewer amino acids, e.g., by 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids and 1 amino acid.

[0085] Preferably, the parent α-amylase comprises or consists of the amino acid sequence of SEQ ID NO: 6. In another embodiment, the parent α-amylase is an allelic variant of the polypeptide of SEQ ID NO: 6.

[0086] The parent α-amylase may also be a polypeptide having at least 80% sequence identity to the polypeptide set forth in SEQ ID NO:7.

[0087] In one embodiment, the parent α-amylase has at least 80% sequence identity, such as at least 85%, at least 90%, e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 or 100%, to a polypeptide of SEQ ID NO: 7 having α-amylase activity. In one embodiment, the amino acid sequence of the parent α-amylase differs from the polypeptide of SEQ ID NO: 7 by 10 or fewer amino acids, e.g., by 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids and 1 amino acid.

[0088] Preferably, the parent α-amylase comprises or consists of the amino acid sequence of SEQ ID NO: 7. In another embodiment, the parent α-amylase is an allelic variant of the polypeptide of SEQ ID NO: 7.

[0089] The parent α-amylase may also be a polypeptide having at least 80% sequence identity to the polypeptide set forth in SEQ ID NO:8.

[0090] In one embodiment, the parent α-amylase has at least 80% sequence identity, such as at least 85%, at least 90%, e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 or 100%, to a polypeptide of SEQ ID NO: 8 having α-amylase activity. In one embodiment, the amino acid sequence of the parent α-amylase differs from the polypeptide of SEQ ID NO: 8 by 10 or fewer amino acids, e.g., by 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids and 1 amino acid.

[0091] Preferably, the parent α-amylase comprises or consists of the amino acid sequence of SEQ ID NO: 8. In another embodiment, the parent α-amylase is an allelic variant of the polypeptide of SEQ ID NO: 8.

[0092] The amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or fragments thereof, can be used to design nucleic acid probes to identify and clone parent-encoding DNA from lineages of different genera or species, according to methods well known in the art. In particular, such probes can be used for hybridization with the genome or cDNA of the genus or species of interest, followed by standard Southern blotting techniques, to identify and isolate the corresponding gene contained therein. Such probes can be much shorter than the entire sequence, but should be at least 14 nucleotides, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled (e.g., RNA probes) to detect the corresponding gene. 32 P, 3 H, 35 S, biotin or avidin). Such probes are encompassed by the present invention.

[0093] Genomic DNA or cDNA libraries prepared from such other organisms can be screened for DNA that hybridizes with the above-described probes and encodes the parent. Genomic or other DNA from such other organisms can be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. DNA from these libraries or separated DNA can be transferred and immobilized on nitrocellulose or other suitable carrier material, which can be used in Southern blotting.

[0094] For purposes of this invention, hybridization refers to the hybridization of a polynucleotide under low to very high stringency conditions to a labeled nucleotide probe corresponding to a polynucleotide encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or a subsequence thereof. Molecules to which the probe hybridizes can be detected, for example, using x-ray film or any other detection means known in the art.

[0095] In one embodiment, the nucleic acid probe is a polynucleotide encoding the polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or a fragment thereof.

[0096] For long probes at least 100 nucleotides in length, very low to very high stringency conditions are optimally defined as 12 to 24 hours of standard Southern blotting followed by prehybridization and hybridization in 5x SSPE, 0.3% SDS, 200 micrograms / ml fragment-treated and denatured salmon sperm DNA at 42°C, and 25% formamide for very low and low stringency, 35% formamide for medium and medium-high stringency, or 50% formamide for high and very high stringency. The carrier material is finally washed three times for 15 minutes in 2xSSC, 0.2% SDS at 45°C (very low stringency), 50°C (low stringency), 55°C (moderate stringency), 60°C (moderate-high stringency), 65°C (high stringency) or 70°C (very high stringency).

[0097] For short probes, from about 15 nucleotides to about 70 nucleotides in length, stringent conditions are optimally determined using calculations according to Bolton and McCarthy (1962, Proc. Natl. Acad. Sci. USA 48:1390) following standard Southern blotting for 12 to 24 hours. mThe hybridization time is defined as prehybridization and hybridization in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg yeast RNA / ml at a temperature approximately 5°C to approximately 10°C lower than the calculated T. The carrier material was finally hybridized once in 6× SCC and once in 0.1% SDS for 15 minutes and then at a temperature approximately 5°C to approximately 10°C lower than the calculated T. m The tubes are washed twice for 15 minutes each in 6x SSC at a temperature 5-10°C lower than the standard temperature.

[0098] The parent may be obtained from a microorganism of any genus. For purposes of the present invention, the term "obtained from," as used herein in reference to a given source, shall mean that the parent encoded by the polynucleotide is produced by the source or by a cell into which a polynucleotide from the source has been inserted. In one embodiment, the parent is secreted extracellularly.

[0099] The parent can be a bacterial α-amylase, for example, a Gram-positive α-amylase such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces α-amylase. It may be a bacterial polypeptide or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma α-amylase.

[0100] In one embodiment, the parent is Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis or Bacillus thuringiensis α-amylase.

[0101] In other embodiments, the parent is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis or Streptococcus zooepidemicus α-amylase.

[0102] In other embodiments, the parent is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus or Streptomyces lividans α-amylase.

[0103] In other embodiments, the parent is a Bacillus sp. α-amylase, such as the α-amylase of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8.

[0104] For the aforementioned species, it will be understood that the invention includes both the perfect and imperfect forms, regardless of the species name by which they are known, as well as other taxonomic equivalents, such as asexual forms. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0105] Strains of these species are readily available to the public at numerous microbial collections, such as the American Type Culture Collection (ATCC), the German Collection of Microbial Cell Cultures (DSM), the Netherlands Center for Microbial Strains (CBS), and the Agricultural Research Service Patent Culture Collection and the Northern Regional Research Center (NRRL).

[0106] The parent can be identified and obtained using the above-described probes from microorganisms isolated from nature (e.g., pollutants, compost, water, etc.) or from other sources, including DNA samples obtained directly from natural materials (e.g., pollutants, compost, water, etc.). Techniques for isolating microorganisms and DNA directly from natural environments are well known in the art. Polynucleotides encoding the parent can then be obtained by similarly screening genomic or cDNA libraries of other microorganisms or mixed DNA samples. Once a polynucleotide encoding the parent is detected with a probe, the polynucleotide can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, supra).

[0107] The parent may be a hybrid polypeptide in which a portion of one polypeptide is fused at the N-terminus or C-terminus of a portion of another polypeptide.

[0108] The parent may also be a fusion polypeptide or a cleavable fusion polypeptide in which one polypeptide is fused at the N-terminus or C-terminus of another polypeptide. Fusion polypeptides are produced by fusing a polynucleotide encoding one polypeptide to a polynucleotide encoding another polypeptide. Techniques for producing fusion polypeptides are known in the art and involve ligating coding sequences encoding the polypeptides in frame and such that expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion proteins may also be constructed using intein technology, in which the fusion is formed post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0109] The fusion polypeptide may further comprise a cleavage site between the two polypeptides that is cleaved to release the two polypeptides when the fusion protein is secreted. Examples of cleavage sites include, but are not limited to, those described in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al. al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0110] Preparation of mutants The present invention relates to a method for obtaining a variant having α-amylase activity, comprising the steps of: (a) introducing modifications into a parent α-amylase at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence of SEQ ID NO: 1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence of SEQ ID NO: 1, wherein each modification is independently a substitution or a deletion, and wherein the variant has α-amylase activity; and (b) recovering the variant.

[0111] In one aspect, the present invention relates to a method for obtaining a variant having α-amylase activity, the method comprising the steps of: (a) introducing modifications into a parent α-amylase at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence according to SEQ ID NO: 1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7 and 8, numbered according to SEQ ID NO: 1, and wherein each modification is independently a substitution or a deletion, and wherein the variant has α-amylase activity; and (b) recovering the variant.

[0112] In one embodiment, the modification is a substitution. In one embodiment, the modification is a deletion.

[0113] In another embodiment, the present invention relates to a method for obtaining a variant having α-amylase activity, comprising the steps of: (a) introducing substitutions into a parent α-amylase at one or more positions, wherein the substitutions are selected from H1A, G7A, G109A, N280S, W284H, K320A, M323N and E391A of the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8, numbered according to SEQ ID NO: 1; and (b) recovering the variant.

[0114] In one embodiment, the method further comprises the step of introducing deletions into the parent α-amylase at one or more positions, the deletions being: H1 of the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8 * , R181 * , G182 * , D183 * and G184 * and numbered according to SEQ ID NO: 1, and recovering the mutant.

[0115] In one embodiment, the method further comprises introducing substitutions at one or more positions in the parent α-amylase, wherein the substitutions are selected from: W140Y, N195F, V206Y, Y243F, E260G, G304R and G476K of the polypeptide of SEQ ID NO: 1, 3, 4, 5, 6, 7 or 8, and recovering the variants.

[0116] Variants can be prepared using any mutagenesis method known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, and the like.

[0117] Site-directed mutagenesis is a technique for making one or more mutations at one or more defined sites in a parent coding polynucleotide.

[0118] Site-specific mutagenesis can be achieved in vitro by PCR, using oligonucleotide primers containing the desired mutation. Site-specific mutagenesis can also be performed in vitro by cassette mutagenesis, in which a site in a plasmid containing a parent encoding polynucleotide is cleaved with a restriction enzyme, and then an oligonucleotide containing the mutation is ligated to the polynucleotide. Usually, the restriction enzymes used to digest the plasmid and the oligonucleotide are the same, allowing the cohesive ends of the plasmid and the insert to be ligated together. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

[0119] Site-directed mutagenesis can also be achieved in vivo by methods known in the art (see, e.g., U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16).

[0120] Any site-directed mutagenesis method can be used in the present invention. Many commercial kits are available that can be used to prepare mutants.

[0121] Synthetic gene construction involves the in vitro synthesis of designed polynucleotide molecules to encode a polypeptide of interest. Gene synthesis can be performed using a number of techniques, including the multiplexed microchip-based technology described by Tian et al. (2004, Nature 432:1050-1054), as well as similar technologies in which oligonucleotides are synthesized and assembled into microfluidic chips that can be controlled by light.

[0122] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625, followed by associated screening methods. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0123] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from the host cells and readily sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0124] Semisynthetic gene construction is achieved by combining aspects of synthetic gene construction and / or site-directed mutagenesis and / or random mutagenesis and / or shuffling. Semisynthetic construction is typified by a process that utilizes synthesized polynucleotide fragments combined with PCR technology. Thus, defined regions of a gene can be synthesized de novo, while other regions can be amplified using site-directed mutagenic primers, while still other regions can be subjected to error-prone or non-error-prone PCR amplification. The polynucleotide subsequences can then be shuffled.

[0125] Mutants The present invention also provides variants of a parent α-amylase, wherein (i) the variant comprises a modification at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323, and 391 of the amino acid sequence of SEQ ID NO: 1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304, and 476 of the amino acid sequence of SEQ ID NO: 1, (ii) the variant has at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8, and (iii) the variant has α-amylase activity. This provides variants that have improved cleaning performance at low temperatures compared to the parent α-amylase or compared to the α-amylase of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.

[0126] In embodiments, the variant has at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, but less than 100% sequence identity to the amino acid sequence of the parent α-amylase.

[0127] In other embodiments, the invention relates to an isolated variant of a parent α-amylase, wherein (i) the variant comprises a modification at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence according to SEQ ID NO: 1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence according to SEQ ID NO: 1; (ii) the variant has at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, but less than 100% sequence identity to the amino acid sequence according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8; and (iii) the variant has α-amylase activity.

[0128] In other embodiments, the variant has at least 80%, at least 85%, such as at least 90%, at least 95%, at least 96%, such as at least 97%, at least 98%, and at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO:1.

[0129] In other embodiments, the variant has at least 80%, at least 85%, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO:2.

[0130] In other embodiments, the variant has at least 80%, at least 85%, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO:3.

[0131] In other embodiments, the variant has at least 80%, at least 85%, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO:4.

[0132] In other embodiments, the variant has at least 80%, at least 85%, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO:5.

[0133] In other embodiments, the variant has at least 80%, at least 85%, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO:6.

[0134] In other embodiments, the variant has at least 80%, at least 85%, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO:7.

[0135] In other embodiments, the variant has at least 80%, at least 85%, such as at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%, but less than 100% sequence identity to the mature polypeptide of SEQ ID NO:8.

[0136] In one embodiment, the number of modifications in the variants of the invention is 1-20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, for example 1-10 and 1-5 modifications.

[0137] In one embodiment, the variant includes modifications such as substitutions at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and optionally modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0138] In other embodiments, the variant includes modifications such as substitutions at two or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and optionally modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0139] In other embodiments, the variant includes modifications such as substitutions at three or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and optionally modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0140] In other embodiments, the variant includes modifications such as substitutions at four or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and optionally modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0141] In other embodiments, the variant includes modifications such as substitutions at five or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and optionally modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0142] In other embodiments, the variant includes modifications such as substitutions at six or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and optionally modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0143] In other embodiments, the variant includes modifications such as substitutions at seven or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and optionally modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0144] In other embodiments, the variant includes modifications, such as substitutions, at eight positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and optionally modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0145] In one embodiment, the variant includes modifications such as substitutions at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0146] In other embodiments, the variant includes modifications such as substitutions at two or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0147] In other embodiments, the variant includes modifications such as substitutions at three or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0148] In other embodiments, the variant includes modifications such as substitutions at four or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0149] In other embodiments, the variant includes modifications such as substitutions at five or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0150] In other embodiments, the variant includes modifications such as substitutions at six or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0151] In other embodiments, the variant includes modifications such as substitutions at seven or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0152] In other embodiments, the variants include modifications, such as substitutions, at eight positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391, and modifications at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304 and 476, where the numbering is according to SEQ ID NO: 1.

[0153] In a preferred embodiment, the variant comprises a modification at 1, 2, 3, 4 or 5 positions selected from the group consisting of 1, 7, 109, 280 and 391.

[0154] In one embodiment, the variant comprises at least one deletion and at least one substitution at 2, 3, 4 or 5 positions selected from the group consisting of 1, 7, 109, 280 and 391.

[0155] In one embodiment, the variant comprises substitutions at 1, 2, 3 or 4 positions selected from 7, 109, 280 and 391.

[0156] In one embodiment, the variants are: X1 + X7; X1 + X109; X1 + X280; X1 + X284; X1 + X320; X1 + X323; X1 + X391; X109 + X280; X109 + X284; X109 + X320; X109 + X323; X109 + X391; X7 + X109; X7 + X280; X7 + X284; X7 + X320; X7 + X X280+X323; X280+X391; X284+X320; X284+X323; X284+X391; X320+X323; X320+X391; and X323+X391, wherein the numbering is according to SEQ ID NO: 1.

[0157] In one embodiment, the variants are: X109 + X7 + X1; X109 + X7 + X391; X109 + X7 + X280; X109 + X7 + X284; X109 + X7 + X320; X109 + X7 + X323; X109 + X1 + X391; X109 + X1 + X280; X109 + X1 + X284; X109 + X1 + X320; X109 + X1 + X323; X109 + X391 + X280; X109 + X391 + X284; X109 + X391 + X320; X1 09+X391+X323;X109+X280+X284;X109+X280+X320;X109+X280+X323;X109+X284+X320;X109+X284+X323;X109+X320+X323 ;X7+X1+X391;X7+X1+X280;X7+X1+X284;X7+X1+X320;X7+X1+X323;X7+X391+X280;X7+X391+X284;X7+X391+X320;X7+X391 +X323;X7+X280+X284;X7+X280+X320;X7+X280+X323;X7+X284+X320;X7+X284+X323;X7+X320+X323;X1+X391+X280;X1+X 391+X284;X1+X391+X320;X1+X391+X323;X1+X280+X284;X1+X280+X320;X1 +X280+X323; X280+X284; X391+X280+X320; X391+X280+X323; X391+X284+X320; X391+X284+X323; X391+X320+X323; X280+X284+X320; X280+X284+X320; X280+X284+X323; X280+X320+X323; and X284+X320+X323, wherein the numbering is according to SEQ ID NO: 1.

[0158] In one embodiment, the variants are: X109 + X7 + X1 + X391; X109 + X7 + X1 + X280; X109 + X7 + X1 + X284; X109 + X7 + X1 + X320; X109 + X7 + X1 + X323; X109 + X7 + X391 + X280; X109 + X7 + X391 + X284; X109 + X7 + X391 + X320; X109 + X7 + X391 + X323; X109 + X7 + X280 + X284; X109 + X7 + X280 + X320; X109 + X7 + X280 + X323; X109 + X7 + X284 + X320; X109 + X7 + X 284+X323;X109+X7+X320+X323;X109+X1+X391+X280;X109+X1+X391+X284 ;X109+X1+X391+X320;X109+X1+X391+X323;X109+X1+X280+X284;X109+X1 +X280+X320;X109+X1+X280+X323;X109+X1+X284+X320;X109+X1+X284+X3 23;X109+X1+X320+X323;X109+X391+X280+X284;X109+X391+X280+X320;X1 09+X391+X280+X323;X109+X391+X284+X320;X109+X391+X284+X323;X109 +X391+X320+X323;X109+X280+X284+X320;X109+X280+X284+X323;X109+X 280+X320+X323;X109+X284+X320+X323;X7+X1+X391+X280;X7+X1+X391+X 284;X7+X1+X391+X320;X7+X1+X391+X323;X7+X1+X280+X284;X7+X1+X280+ X320;X7+X1+X280+X323;X7+X1+X284+X320;X7+X1+X284+X323;X7+X1+X32 0+X323;X7+X391+X280+X284;X7+X391+X280+X320;X7+X391+X280+X323;X 7+X391+X284+X320;X7+X391+X284+X323;X7+X391+X320+X323;X7+X280+X 284+X320;X7+X280+X284+X323;X7+X280+X320+X323;X7+X284+X320+X323;X1+X391+X280+X284;X1+X391+X280+X320;X1+X391+X280+X323;X1+X391+X284+X320;X1+X391+ X284+X323;X1+X391+X320+X323;X1+X280+X284+X320;X1+X280+X284+X323;X1+X280+X320+X323 and X280 + X284 + X320 + X323, wherein the numbering is according to SEQ ID NO: 1.

[0159] In one embodiment, the variant is X1 * ,X1A,X7A,X7K,X7E,X7N,X7Q,X7L,X7D,X109A,X109S,X140Y,X181 * , X182 * , X183 * , X184 * , X195F, X206Y, X243F, X260G, X280S, X284H, X284R, X284F, X304R, X320A, X320M, X320T, X320V, X320S, X323N, X323R, X323S, X323K, X391A, X391V and X476K, wherein the numbering is according to SEQ ID NO: 1.

[0160] In one particular embodiment, the variant is: X1 * +X1A;X1 * +X7A;X1 * +X109A;X1 * +X280S;X1 * +X284H;X1 * +X320A;X1 * +X323N;X1 *+X391A;X1A+X7A;X1A+X109A;X1A+X280S;X1A+X284H;X1A+X320A;X1A+X323N;X1A+X391A;X7A+X109A;X7A +X280S;X7A+X284H;X7A+X320A;X7A+X323N;X7A+X391A;X109A+X280S;X109A+X284H;X109A+X320A;X109A +X323N; X109A+X391A; X280S+X284H; X280S+X320A; X280S+X323N; X280S+X391A; X284H+X320A; X284H+X323N; X284H+X391A; X320A+X323N; X320A+X391A; and X323N+X391A, wherein the numbering is according to SEQ ID NO: 1.

[0161] In one embodiment, the variant is: X1 * +X7A+X109A;X1 * +X7A+X280S;X1 * +X7A+X284H;X1 * +X7A+X320A;X1 * +X7A+X323N;X1 * +X7A+X391A;X1 * +X109A+X280S;X1 * +X109A+X284H;X1 * +X109A+X320A;X1 * +X109A+X323N;X1 * +X109A+X391A;X1 * +X280S+X284H;X1 * +X280S+X320A;X1 * +X280S+X323N;X1 * +X280S+X391A;X1 * +X284H+X320A;X1 * +X284H+X323N;X1 * +X284H+X391A;X1 * +X320A+X323N;X1 * +X320A+X391A;X1 *+X323N+X391A;X1A+X7A+X109A;X1A+X7A+X280S;X1A+X7A+X284H;X1A+X 7A+X320A;X1A+X7A+X323N;X1A+X7A+X391A;X1A+X109A+X280S;X1A+X109 A+X284H;X1A+X109A+X320A;X1A+X109A+X323N;X1A+X109A+X391A;X1A+X 280S+X284H;X1A+X280S+X320A;X1A+X280S+X323N;X1A+X280S+X391A;X1 A+X284H+X320A;X1A+X284H+X323N;X1A+X284H+X391A;X1A+X320A+X323N ;X1A+X320A+X391A;X1A+X323N+X391A;X7A+X109A+X280S;X7A+X109A+X2 84H;X7A+X109A+X320A;X7A+X109A+X323N;X7A+X109A+X391A;X7A+X280S +X284H;X7A+X280S+X320A;X7A+X280S+X323N;X7A+X280S+X391A;X7A+X2 84H+X320A;X7A+X284H+X323N;X7A+X284H+X391A;X7A+X320A+X323N;X7 A+X320A+X391A;X7A+X323N+X391A;X109A+X280S+X284H;X109A+X280S+X 320A;X109A+X280S+X323N;X109A+X280S+X391A;X109A+X284H+X320A;X1 09A+X284H+X323N;X109A+X284H+X391A;X109A+X320A+X323N;X109A+X32 X280S+X284H+X320A; X280S+X284H+X323N; X280S+X284H+X391A; X280S+X320A+X323N; X280S+X320A+X391A; X280S+X323N+X391A; X284H+X320A+X323N; X284H+X320A+X391A; X284H+X323N+X391A; and X320A+X323N+X391A, wherein the numbering is according to SEQ ID NO: 1.

[0162] In a preferred embodiment, the variant is: X1 * +X109A+X280S+X391A;X1 * +X7K+X109A+X280S+X391A;X1 * +X7E+X109A+X280S+X391A;X1 * +X7N+X109A+X280S+X391A;X1 * +X7Q+X109A+X280S+X391A;X1 * +X7L+X109A+X280S+X391A;X1 * +X7D+X109A+X280S+X391A;X1 * +X109A+X280S+X320A+X391A;X1 * +X109A+X280S+X320M+X391A;X1 * +X109A+X280S+X320T+X391A;X1 * +X109A+X280S+X320V+X391A;X1 * +X109A+X280S+X323R+X391A;X1 * +X109A+X280S+X320S+X391A;X1 * +X109A+X280S+X391V;X1 * +X109A+X284R+X391A;X1 * +X109A+X284F+X391A;X1 * +X109A+X280S+X320A+X323S+X391A;X1 * +X109A+X280S+X284F+X391A;X1 * +X109A+X280S+X323N+X391A;X1 * +X109A+X280S+X323K+X391A;X1 * +X109S+X280S+X391A;X1 * +X109A+X284H+X391A;X1 * +X109A+X280S+X320A+X323N+X391A;X1 * +X7A+X109A+X280S+X391A;X1 *+X7A+X109A+X280S+X284H+X320A+X323N+X391A;X7A+X284H+X320A+X323N;X7A+X320A+X323N;X320A;X7A+X320A;X1 * +X7A+X109A+X280S+X391A;X1 * +X109A+X280S+X284H+X391A;X1 * +X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X320A+X391A;X1 * +X7A+X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X323N+X391A;X1 * +X7A+X109A+X280S+X284F+X391A;X1 * +X7A+X109A+X280S+X284R+X391A;X1 * +X7A+X109A+X280S+X320A+X323S+X391A;X1 * +X7A+X109A+X284R+X391A; and, X1 * +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant has at least 80% sequence identity to any one of the amylases set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 or 8.

[0163] In one embodiment, the variant is: X1 * +X109A+X280S+X391A;X1 * +X7K+X109A+X280S+X391A;X1 * +X7E+X109A+X280S+X391A;X1 * +X7N+X109A+X280S+X391A;X1 * +X7Q+X109A+X280S+X391A;X1 * +X7L+X109A+X280S+X391A;X1 *+X7D+X109A+X280S+X391A;X1 * +X109A+X280S+X320A+X391A;X1 * +X109A+X280S+X320M+X391A;X1 * +X109A+X280S+X320T+X391A;X1 * +X109A+X280S+X320V+X391A;X1 * +X109A+X280S+X323R+X391A;X1 * +X109A+X280S+X320S+X391A;X1 * +X109A+X280S+X391V;X1 * +X109A+X284R+X391A;X1 * +X109A+X284F+X391A;X1 * +X109A+X280S+X320A+X323S+X391A;X1 * +X109A+X280S+X284F+X391A;X1 * +X109A+X280S+X323N+X391A;X1 * +X109A+X280S+X323K+X391A;X1 * +X109S+X280S+X391A;X1 * +X109A+X284H+X391A;X1 * +X109A+X280S+X320A+X323N+X391A;X1 * +X7A+X109A+X280S+X391A;X1 * +X7A+X109A+X280S+X284H+X320A+X323N+X391A;X7A+X284H+X320A+X323N;X7A+X320A+X323N;X320A;X7A+X320A;X1 * +X7A+X109A+X280S+X391A;X1 * +X109A+X280S+X284H+X391A;X1 * +X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X320A+X391A;X1 * +X7A+X109A+X280S+X323S+X391A;X1* +X7A+X109A+X280S+X323N+X391A;X1 * +X7A+X109A+X280S+X284F+X391A;X1 * +X7A+X109A+X280S+X284R+X391A;X1 * +X7A+X109A+X280S+X320A+X323S+X391A;X1 * +X7A+X109A+X284R+X391A; and, X1 * +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant comprises a modification at a position corresponding to a position of the amino acid sequence according to SEQ ID NO: 1 selected from the group consisting of: +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase set forth in SEQ ID NO: 1.

[0164] In one embodiment, the variant is: X1 * +X109A+X280S+X391A;X1 * +X7K+X109A+X280S+X391A;X1 * +X7E+X109A+X280S+X391A;X1 * +X7N+X109A+X280S+X391A;X1 * +X7Q+X109A+X280S+X391A;X1 * +X7L+X109A+X280S+X391A;X1 * +X7D+X109A+X280S+X391A;X1 * +X109A+X280S+X320A+X391A;X1 * +X109A+X280S+X320M+X391A;X1 * +X109A+X280S+X320T+X391A;X1 * +X109A+X280S+X320V+X391A;X1 * +X109A+X280S+X323R+X391A;X1 * +X109A+X280S+X320S+X391A;X1 * +X109A+X280S+X391V;X1 *+X109A+X284R+X391A;X1 * +X109A+X284F+X391A;X1 * +X109A+X280S+X320A+X323S+X391A;X1 * +X109A+X280S+X284F+X391A;X1 * +X109A+X280S+X323N+X391A;X1 * +X109A+X280S+X323K+X391A;X1 * +X109S+X280S+X391A;X1 * +X109A+X284H+X391A;X1 * +X109A+X280S+X320A+X323N+X391A;X1 * +X7A+X109A+X280S+X391A;X1 * +X7A+X109A+X280S+X284H+X320A+X323N+X391A;X7A+X284H+X320A+X323N;X7A+X320A+X323N;X320A;X7A+X320A;X1 * +X7A+X109A+X280S+X391A;X1 * +X109A+X280S+X284H+X391A;X1 * +X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X320A+X391A;X1 * +X7A+X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X323N+X391A;X1 * +X7A+X109A+X280S+X284F+X391A;X1 * +X7A+X109A+X280S+X284R+X391A;X1 * +X7A+X109A+X280S+X320A+X323S+X391A;X1 * +X7A+X109A+X284R+X391A; and, X1 *+X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant comprises a modification at a position corresponding to a position of the amino acid sequence according to SEQ ID NO: 2, selected from the group consisting of: +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase set forth in SEQ ID NO: 2.

[0165] In a preferred embodiment, the variant is: H1 * +G109A+N280S+E391A;H1 * +G7K+G109A+N280S+E391A;H1 * +G7E+G109A+N280S+E391A;H1 * +G7N+G109A+N280S+E391A;H1 * +G7Q+G109A+N280S+E391A;H1 * +G7L+G109A+N280S+E391A;H1 * +G7D+G109A+N280S+E391A;H1 * +G109A+N280S+K320A+E391A;H1 * +G109A+N280S+K320M+E391A;H1 * +G109A+N280S+K320T+E391A;H1 * +G109A+N280S+K320V+E391A;H1 * +G109A+N280S+M323R+E391A;H1 * +G109A+N280S+K320S+E391A;H1 * +G109A+N280S+E391V;H1 * +G109A+W284R+E391A;H1 * +G109A+W284F+E391A;H1 * +G109A+N280S+K320A+M323S+E391A;H1 * +G109A+N280S+W284F+E391A;H1 * +G109A+N280S+M323N+E391A;H1 * +G109A+N280S+M323K+E391A;H1 *+G109S+N280S+E391A;H1 * +G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A;H1 * +G7A+G109A+N280S+W284H+K320A+M323N+E391A;G7A+W284H+K320A+M323N;G7A+K320A+M323N;K320A;G7A+K320A;H1 * +G7A+G109A+N280S+E391A;H1 * +G109A+N280S+W284H+E391A;H1 * +G109A+N280S+M323S+E391A;H1 * +G7A+G109A+N280S+K320A+E391A;H1 * +G7A+G109A+N280S+M323S+E391A;H1 * +G7A+G109A+N280S+M323N+E391A;H1 * +G7A+G109A+N280S+W284F+E391A;H1 * +G7A+G109A+N280S+W284R+E391A;H1 * +G7A+G109A+N280S+K320A+M323S+E391A;H1 * +G7A+G109A+W284R+E391A; and H1 * +G7A +G109A +N280S +K320A +M323N +E391A, at positions corresponding to positions in the amino acid sequence according to SEQ ID NO: 2.

[0166] In one embodiment, the variant is: X1 * +X109A+X280S+X391A;X1 * +X7K+X109A+X280S+X391A;X1 * +X7E+X109A+X280S+X391A;X1 * +X7N+X109A+X280S+X391A;X1 *+X7Q+X109A+X280S+X391A;X1 * +X7L+X109A+X280S+X391A;X1 * +X7D+X109A+X280S+X391A;X1 * +X109A+X280S+X320A+X391A;X1 * +X109A+X280S+X320M+X391A;X1 * +X109A+X280S+X320T+X391A;X1 * +X109A+X280S+X320V+X391A;X1 * +X109A+X280S+X323R+X391A;X1 * +X109A+X280S+X320S+X391A;X1 * +X109A+X280S+X391V;X1 * +X109A+X284R+X391A;X1 * +X109A+X284F+X391A;X1 * +X109A+X280S+X320A+X323S+X391A;X1 * +X109A+X280S+X284F+X391A;X1 * +X109A+X280S+X323N+X391A;X1 * +X109A+X280S+X323K+X391A;X1 * +X109S+X280S+X391A;X1 * +X109A+X284H+X391A;X1 * +X109A+X280S+X320A+X323N+X391A;X1 * +X7A+X109A+X280S+X391A;X1 * +X7A+X109A+X280S+X284H+X320A+X323N+X391A;X7A+X284H+X320A+X323N;X7A+X320A+X323N;X320A;X7A+X320A;X1 * +X7A+X109A+X280S+X391A;X1 * +X109A+X280S+X284H+X391A;X1 * +X109A+X280S+X323S+X391A;X1 *+X7A+X109A+X280S+X320A+X391A;X1 * +X7A+X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X323N+X391A;X1 * +X7A+X109A+X280S+X284F+X391A;X1 * +X7A+X109A+X280S+X284R+X391A;X1 * +X7A+X109A+X280S+X320A+X323S+X391A;X1 * +X7A+X109A+X284R+X391A; and, X1 * +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant comprises a modification at a position corresponding to a position of the amino acid sequence according to SEQ ID NO: 3, selected from the group consisting of: +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase set forth in SEQ ID NO: 3.

[0167] In one embodiment, the variant is: X1 * +X109A+X280S+X391A;X1 * +X7K+X109A+X280S+X391A;X1 * +X7E+X109A+X280S+X391A;X1 * +X7N+X109A+X280S+X391A;X1 * +X7Q+X109A+X280S+X391A;X1 * +X7L+X109A+X280S+X391A;X1 * +X7D+X109A+X280S+X391A;X1 * +X109A+X280S+X320A+X391A;X1 * +X109A+X280S+X320M+X391A;X1 * +X109A+X280S+X320T+X391A;X1 * +X109A+X280S+X320V+X391A;X1 * +X109A+X280S+X323R+X391A;X1 *+X109A+X280S+X320S+X391A;X1 * +X109A+X280S+X391V;X1 * +X109A+X284R+X391A;X1 * +X109A+X284F+X391A;X1 * +X109A+X280S+X320A+X323S+X391A;X1 * +X109A+X280S+X284F+X391A;X1 * +X109A+X280S+X323N+X391A;X1 * +X109A+X280S+X323K+X391A;X1 * +X109S+X280S+X391A;X1 * +X109A+X284H+X391A;X1 * +X109A+X280S+X320A+X323N+X391A;X1 * +X7A+X109A+X280S+X391A;X1 * +X7A+X109A+X280S+X284H+X320A+X323N+X391A;X7A+X284H+X320A+X323N;X7A+X320A+X323N;X320A;X7A+X320A;X1 * +X7A+X109A+X280S+X391A;X1 * +X109A+X280S+X284H+X391A;X1 * +X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X320A+X391A;X1 * +X7A+X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X323N+X391A;X1 * +X7A+X109A+X280S+X284F+X391A;X1 * +X7A+X109A+X280S+X284R+X391A;X1 * +X7A+X109A+X280S+X320A+X323S+X391A;X1 * +X7A+X109A+X284R+X391A; and, X1 *+X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant comprises a modification at a position corresponding to a position of the amino acid sequence according to SEQ ID NO: 4 selected from the group consisting of: +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase set forth in SEQ ID NO: 4.

[0168] In one embodiment, the variant is: X1 * +X109A+X280S+X391A;X1 * +X7K+X109A+X280S+X391A;X1 * +X7E+X109A+X280S+X391A;X1 * +X7N+X109A+X280S+X391A;X1 * +X7Q+X109A+X280S+X391A;X1 * +X7L+X109A+X280S+X391A;X1 * +X7D+X109A+X280S+X391A;X1 * +X109A+X280S+X320A+X391A;X1 * +X109A+X280S+X320M+X391A;X1 * +X109A+X280S+X320T+X391A;X1 * +X109A+X280S+X320V+X391A;X1 * +X109A+X280S+X323R+X391A;X1 * +X109A+X280S+X320S+X391A;X1 * +X109A+X280S+X391V;X1 * +X109A+X284R+X391A;X1 * +X109A+X284F+X391A;X1 * +X109A+X280S+X320A+X323S+X391A;X1 * +X109A+X280S+X284F+X391A;X1 * +X109A+X280S+X323N+X391A;X1 * +X109A+X280S+X323K+X391A;X1 *+X109S+X280S+X391A;X1 * +X109A+X284H+X391A;X1 * +X109A+X280S+X320A+X323N+X391A;X1 * +X7A+X109A+X280S+X391A;X1 * +X7A+X109A+X280S+X284H+X320A+X323N+X391A;X7A+X284H+X320A+X323N;X7A+X320A+X323N;X320A;X7A+X320A;X1 * +X7A+X109A+X280S+X391A;X1 * +X109A+X280S+X284H+X391A;X1 * +X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X320A+X391A;X1 * +X7A+X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X323N+X391A;X1 * +X7A+X109A+X280S+X284F+X391A;X1 * +X7A+X109A+X280S+X284R+X391A;X1 * +X7A+X109A+X280S+X320A+X323S+X391A;X1 * +X7A+X109A+X284R+X391A; and, X1 * +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant comprises a modification at a position corresponding to a position of the amino acid sequence according to SEQ ID NO: 5 selected from the group consisting of: +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase set forth in SEQ ID NO: 5.

[0169] In one embodiment, the variant is: X1 * +X109A+X280S+X391A;X1 * +X7K+X109A+X280S+X391A;X1 *+X7E+X109A+X280S+X391A;X1 * +X7N+X109A+X280S+X391A;X1 * +X7Q+X109A+X280S+X391A;X1 * +X7L+X109A+X280S+X391A;X1 * +X7D+X109A+X280S+X391A;X1 * +X109A+X280S+X320A+X391A;X1 * +X109A+X280S+X320M+X391A;X1 * +X109A+X280S+X320T+X391A;X1 * +X109A+X280S+X320V+X391A;X1 * +X109A+X280S+X323R+X391A;X1 * +X109A+X280S+X320S+X391A;X1 * +X109A+X280S+X391V;X1 * +X109A+X284R+X391A;X1 * +X109A+X284F+X391A;X1 * +X109A+X280S+X320A+X323S+X391A;X1 * +X109A+X280S+X284F+X391A;X1 * +X109A+X280S+X323N+X391A;X1 * +X109A+X280S+X323K+X391A;X1 * +X109S+X280S+X391A;X1 * +X109A+X284H+X391A;X1 * +X109A+X280S+X320A+X323N+X391A;X1 * +X7A+X109A+X280S+X391A;X1 * +X7A+X109A+X280S+X284H+X320A+X323N+X391A;X7A+X284H+X320A+X323N;X7A+X320A+X323N;X320A;X7A+X320A;X1 * +X7A+X109A+X280S+X391A;X1 *+X109A+X280S+X284H+X391A;X1 * +X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X320A+X391A;X1 * +X7A+X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X323N+X391A;X1 * +X7A+X109A+X280S+X284F+X391A;X1 * +X7A+X109A+X280S+X284R+X391A;X1 * +X7A+X109A+X280S+X320A+X323S+X391A;X1 * +X7A+X109A+X284R+X391A; and, X1 * +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant comprises a modification at a position corresponding to a position of the amino acid sequence according to SEQ ID NO: 6 selected from the group consisting of: +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase set forth in SEQ ID NO: 6.

[0170] In one embodiment, the variant is: X1 * +X109A+X280S+X391A;X1 * +X7K+X109A+X280S+X391A;X1 * +X7E+X109A+X280S+X391A;X1 * +X7N+X109A+X280S+X391A;X1 * +X7Q+X109A+X280S+X391A;X1 * +X7L+X109A+X280S+X391A;X1 * +X7D+X109A+X280S+X391A;X1 * +X109A+X280S+X320A+X391A;X1 * +X109A+X280S+X320M+X391A;X1 * +X109A+X280S+X320T+X391A;X1 *+X109A+X280S+X320V+X391A;X1 * +X109A+X280S+X323R+X391A;X1 * +X109A+X280S+X320S+X391A;X1 * +X109A+X280S+X391V;X1 * +X109A+X284R+X391A;X1 * +X109A+X284F+X391A;X1 * +X109A+X280S+X320A+X323S+X391A;X1 * +X109A+X280S+X284F+X391A;X1 * +X109A+X280S+X323N+X391A;X1 * +X109A+X280S+X323K+X391A;X1 * +X109S+X280S+X391A;X1 * +X109A+X284H+X391A;X1 * +X109A+X280S+X320A+X323N+X391A;X1 * +X7A+X109A+X280S+X391A;X1 * +X7A+X109A+X280S+X284H+X320A+X323N+X391A;X7A+X284H+X320A+X323N;X7A+X320A+X323N;X320A;X7A+X320A;X1 * +X7A+X109A+X280S+X391A;X1 * +X109A+X280S+X284H+X391A;X1 * +X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X320A+X391A;X1 * +X7A+X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X323N+X391A;X1 * +X7A+X109A+X280S+X284F+X391A;X1 * +X7A+X109A+X280S+X284R+X391A;X1 *+X7A+X109A+X280S+X320A+X323S+X391A;X1 * +X7A+X109A+X284R+X391A; and, X1 * +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant comprises a modification at a position corresponding to a position of the amino acid sequence according to SEQ ID NO: 7 selected from the group consisting of: +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase set forth in SEQ ID NO: 7.

[0171] In one embodiment, the variant is: X1 * +X109A+X280S+X391A;X1 * +X7K+X109A+X280S+X391A;X1 * +X7E+X109A+X280S+X391A;X1 * +X7N+X109A+X280S+X391A;X1 * +X7Q+X109A+X280S+X391A;X1 * +X7L+X109A+X280S+X391A;X1 * +X7D+X109A+X280S+X391A;X1 * +X109A+X280S+X320A+X391A;X1 * +X109A+X280S+X320M+X391A;X1 * +X109A+X280S+X320T+X391A;X1 * +X109A+X280S+X320V+X391A;X1 * +X109A+X280S+X323R+X391A;X1 * +X109A+X280S+X320S+X391A;X1 * +X109A+X280S+X391V;X1 * +X109A+X284R+X391A;X1 * +X109A+X284F+X391A;X1 * +X109A+X280S+X320A+X323S+X391A;X1 * +X109A+X280S+X284F+X391A;X1* +X109A+X280S+X323N+X391A;X1 * +X109A+X280S+X323K+X391A;X1 * +X109S+X280S+X391A;X1 * +X109A+X284H+X391A;X1 * +X109A+X280S+X320A+X323N+X391A;X1 * +X7A+X109A+X280S+X391A;X1 * +X7A+X109A+X280S+X284H+X320A+X323N+X391A;X7A+X284H+X320A+X323N;X7A+X320A+X323N;X320A;X7A+X320A;X1 * +X7A+X109A+X280S+X391A;X1 * +X109A+X280S+X284H+X391A;X1 * +X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X320A+X391A;X1 * +X7A+X109A+X280S+X323S+X391A;X1 * +X7A+X109A+X280S+X323N+X391A;X1 * +X7A+X109A+X280S+X284F+X391A;X1 * +X7A+X109A+X280S+X284R+X391A;X1 * +X7A+X109A+X280S+X320A+X323S+X391A;X1 * +X7A+X109A+X284R+X391A; and, X1 * +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant comprises a modification at a position corresponding to a position of the amino acid sequence according to SEQ ID NO: 8, selected from the group consisting of: +X7A +X109A +X280S +X320A +X323N +X391A, wherein the numbering is according to SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase set forth in SEQ ID NO: 8.

[0172] The mutant of the present invention is X1 *In a further preferred embodiment, the modifications at positions 1, 2, 3, 4 or 5 are selected from the group consisting of X1, X1A, X7A, X109A, X280S and X391A. * , X7A, X109A, X280S and X391A.

[0173] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: X1 * +X109A+X280S+X391A, X1 * +X109A+X284H+X391A, X1 * +X109A+X280S+X320A+X323N+X391A, X1 * +X7A+X109A+X280S+X391A and X1 * +X7A+X109A+X280S+X284H+X323N+X391A, where the numbering is according to SEQ ID NO: 1, and where the variant has at least 80% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8.

[0174] In one embodiment, the present invention provides a method for producing a compound comprising: * +G109A+N280S+E391A;H1 * +G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and H1 * +G7A+G109A+N280S+W284H+M323N+E391A, wherein the numbering is according to SEQ ID NO: 1, and wherein the variant has at least 80% sequence identity to SEQ ID NO: 1.

[0175] In one embodiment, the present invention provides a method for producing a compound comprising: * +G109A+N280S+E391A;H1 * +G109A+W284H+E391A;H1 *+G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and H1 * +G7A+G109A+N280S+W284H+M323N+E391A, wherein the numbering is according to SEQ ID NO: 1, and wherein the variant has at least 80% sequence identity to SEQ ID NO: 2.

[0176] In one embodiment, the present invention provides a method for producing a compound comprising: * +G109A+N280S+E391A;H1 * +G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and H1 * +G7A+G109A+N280S+W284H+M323N+E391A, wherein the numbering is according to SEQ ID NO: 1, and wherein the variant has at least 80% sequence identity to SEQ ID NO: 3.

[0177] In one embodiment, the present invention provides a method for producing a compound comprising: * +G109A+N280S+E391A;H1 * +G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and H1 * +G7A+G109A+N280S+W284H+M323N+E391A, wherein the numbering is according to SEQ ID NO: 1, and wherein the variant has at least 80% sequence identity to SEQ ID NO: 4.

[0178] In one embodiment, the present invention provides a method for producing a compound comprising: * +G109A+N280S+E391A;H1 *+G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and H1 * +G7A+G109A+N280S+W284H+M323N+E391A, wherein the numbering is according to SEQ ID NO: 1, and wherein the variant has at least 80% sequence identity to SEQ ID NO: 5.

[0179] In one embodiment, the present invention provides a method for producing a compound comprising: * +G109A+N280S+E391A;H1 * +G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and H1 * +G7A+G109A+N280S+W284H+M323N+E391A, wherein the numbering is according to SEQ ID NO: 1, and wherein the variant has at least 80% sequence identity to SEQ ID NO: 6.

[0180] In one embodiment, the present invention provides a method for producing a compound comprising: * +G109A+N280S+E391A;H1 * +G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and H1 * +G7A+G109A+N280S+W284H+M323N+E391A, wherein the numbering is according to SEQ ID NO: 1, and wherein the variant has at least 80% sequence identity to SEQ ID NO: 7.

[0181] In one embodiment, the present invention provides a method for producing a compound comprising: *+G109A+N280S+E391A;H1 * +G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and H1 * +G7A+G109A+N280S+W284H+M323N+E391A, wherein the numbering is according to SEQ ID NO: 1, and wherein the variant has at least 80% sequence identity to SEQ ID NO: 8.

[0182] In one embodiment, the variants of the invention further comprise modifications at one or more positions selected from the group of 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476. In a particular embodiment, the variants of the invention comprise modifications at one or more positions selected from the group of W140Y / F, R181 * , G182 * , D183 * , G184 * , N195F / Y, I206Y / F, Y243F, E260A / D / C / Q / L / M / F / P / S / W / V / G / H / I / K / N / R / T / Y, G304R / K / E / Q and G476E / Q / R / K. In a preferred embodiment, the variant according to the invention further comprises substitutions at two, three or four positions selected from the group consisting of G304R, W140YF, E260GHIKNPRTY and G476EQRK. In a further preferred embodiment, the substitutions at two, three or four positions are selected from the group consisting of G304R, W140Y, E260G and G476K.

[0183] In one embodiment, the variant of the invention is H1 * +G109A+W140Y+D183 * +G184 * +N195F+I206Y+Y243F+E260G+N280S+G304R+E391A+G476K, H1 *+G109A+W140Y+D183 * +G184 * +N195F+I206Y+Y243F+E260G+W284H+G304R+E391A+G476K, H1 * +G109A+W140Y+D183 * +G184 * +N195F+I206Y+Y243F+E260G+N280S+G304R+K320A+M323N+E391A+G476K, H1 * +G7A+G109A+W140Y+D183 * +G184 * +N195F+I206Y+Y243F+E260G+N280S+G304R+E391A+G476K, and H1 * +G7A+G109A+W140Y+D183 * +G184 * +N195F+I206Y+Y243F+E260G+N280S+W284H+G304R+M323N+E391A+G476K, wherein the numbering is according to SEQ ID NO: 1, and the variant has at least 80% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8 and is a variant of SEQ ID NO: 1.

[0184] Essential amino acids in the parent can be identified according to techniques known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting mutant molecules are tested for α-amylase activity to identify amino acid residues important for the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site or other biological interactions of α-amylases can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, coupled with mutations of putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred from analysis of identities with parent-related polypeptides.

[0185] Polynucleotides The present invention also relates to isolated polynucleotides encoding any of the variants of the invention. Accordingly, the present invention relates to isolated polynucleotides encoding variants comprising modifications at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence of SEQ ID NO: 1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence of SEQ ID NO: 1, wherein the variant has at least 80%, such as at least 85%, at least 90%, such as at least 95%, such as at least 97%, but less than 100%, sequence identity to the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8, and wherein the variant has α-amylase activity.

[0186] nucleic acid construct The present invention also relates to a nucleic acid construct comprising a polynucleotide encoding a variant of the invention operably linked to one or more control sequences that provide for the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Accordingly, the present invention relates to a nucleic acid construct comprising a polynucleotide encoding a variant comprising a modification at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence according to SEQ ID NO: 1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence according to SEQ ID NO: 1, wherein the polynucleotide is operably linked to one or more control sequences that provide for the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0187] Polynucleotides can be manipulated in a variety of ways to result in expression of variants. Manipulation of the polynucleotide prior to insertion into a vector may be desirable or necessary, depending on the expression vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0188] The control sequence may be a promoter sequence recognized by a host cell for expression of the polynucleotide. The promoter sequence contains transcriptional control sequences that mediate expression of the variant. The promoter may be any nucleic acid sequence that exhibits transcriptional activity in the host cell, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0189] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the invention in bacterial host cells include the Bacillus amyloliquefaciens α-amylase gene (amyQ), the Bacillus licheniformis α-amylase gene (amyL), the Bacillus licheniformis penicillinase gene (penP), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis xylA and xylB genes, the E. coli lac operon, the Streptomyces coelicolor operon, the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis xylA and xylB genes, the Bacillus subtilis levansucrase gene (sacB), ... coelicolor agarase gene (dagA) and the prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), as well as a promoter derived from the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Additional promoters are described in "Useful proteins from recombinant bacteria," Gilbert et al., 1980, Scientific American 242:74-94; and Sambrook et al., 1989, supra.

[0190] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the invention in filamentous fungal host cells include those encoding Aspergillus nidulans acetamidase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum L. oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 00 / 56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma

[0039] Examples of promoters that may be used include: Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei β-xylosidase, and the NA2-tpi promoter (a modified promoter comprising a gene encoding neutral α-amylase in Aspergilli in which the non-translated leader has been replaced by a non-translated leader derived from a gene encoding triose phosphate isomerase in Aspergilli; a non-limiting example is the Aspergillus niger promoter in which the non-translated leader has been replaced by a non-translated leader derived from a gene encoding triose phosphate isomerase in Aspergillus nidulans or Aspergillus oryzae). Modified promoters include promoters derived from genes encoding neutral α-amylase in Escherichia coli (L. niger); and mutant, truncated and hybrid promoters thereof.

[0191] In yeast hosts, useful promoters can be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0192] The control sequence may also be a suitable transcription terminator sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3'-end of the polynucleotide encoding the variant. Any terminator that functions in the host cell may be used.

[0193] Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0194] 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 in Romanos et al., 1992, supra.

[0195] The control sequence may also be a suitable leader sequence, a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5'-terminus of the polynucleotide encoding the variant. Any leader sequence that is functional in the host cell may be used.

[0196] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0197] Preferred leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0198] The control sequence may also be a polyadenylation sequence, which is a sequence operably linked to the 3'-end of the sequence encoding the variant, which, when transcribed, is recognized by the host cell as a signal for the addition of polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that functions in the host cell may be used.

[0199] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0200] Useful polyadenylation sequences for yeast host cells are described in Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0201] The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the variant and directs the variant into the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding region originally linked in translation reading frame with the segment of the coding region encoding the variant. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding region foreign to the coding sequence. A foreign signal peptide coding region may be required if the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the native signal peptide coding region to enhance secretion of the variant. However, any signal peptide coding region that directs the expressed variant into the secretory pathway of the host cell may be used.

[0202] Effective signal peptide coding sequences for bacterial host cells are those obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0203] Effective signal peptide coding sequences for filamentous fungal host cells are those obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

[0204] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0205] The control sequence may also be a propeptide coding region that encodes a propeptide position at the N-terminus of the variant. The resulting polypeptide is known as a zymogen or propolypeptide (or, in some cases, a zymogen). Propolypeptides are generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding regions may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae α-factor.

[0206] When both the signal peptide and propeptide regions are present at the N-terminus of the variant, the propeptide region is positioned adjacent to the N-terminus of the variant, and the signal peptide region is positioned adjacent to the N-terminus of the propeptide region.

[0207] It may also be desirable to add regulatory sequences that allow for regulation of expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA α-amylase promoter, and the Aspergillus oryzae glucoamylase promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein gene, which is amplified with heavy metals. In these cases, the polynucleotide encoding the variant would be operably linked to the regulatory sequence.

[0208] Expression vector The present invention also relates to recombinant expression vectors comprising the polynucleotides of the present invention, promoters, and transcriptional and translational stop signals. Thus, the present invention relates to recombinant vectors comprising polynucleotides encoding variants containing modifications, promoters, and transcriptional and translational stop signals at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323, and 391 of the amino acid sequence set forth in SEQ ID NO:1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304, and 476 of the amino acid sequence set forth in SEQ ID NO:1. The various nucleotides and control sequences combine to produce recombinant expression vectors that may contain one or more convenient restriction sites to allow for the insertion or substitution of polynucleotides encoding the variants at such sites. Alternatively, polynucleotides can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In forming an expression vector, a coding sequence is placed in a vector so that it is operably linked to appropriate control sequences for expression.

[0209] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA techniques and can result in expression of a polynucleotide. The choice of vector will typically depend on the affinity of the vector for the host cell into which it will be introduced. The vector can be a linear or closed circular plasmid.

[0210] The vector may be a self-replicating vector (i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication), such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may contain some means for ensuring self-replication. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Moreover, a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0211] Vectors preferably carry one or more selectable markers that facilitate selection of cells that have been transformed, transfected, transduced, etc. A selectable marker is a gene the product of which confers biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0212] Examples of bacterial selectable markers are the dal genes from Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin or tetracycline resistance. Preferred markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.

[0213] Preferably, the vector contains elements that allow for integration of the vector into the genome of the host cell or for autonomous replication of the vector in the cell independent of the genome.

[0214] For integration into the host cell genome, the vector may rely on the sequence of the polynucleotide encoding the variant or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may have additional nucleotide sequences that result in integration into the host cell genome at a precise location in the chromosome by homologous recombination. To increase the likelihood of integration at a precise location, the integrative elements should contain a sufficient number of nucleic acids, such as 100-10,000 base pairs, 400-10,000 base pairs, or 800-10,000 base pairs, with a high degree of identity to the corresponding target sequence to increase the probability of homologous recombination. The integrative elements can be any sequence that is homologous to the target sequence in the host cell genome. Furthermore, the integrative elements can be non-coding or coding nucleotide sequences. Alternatively, the vector can be integrated into the host cell genome by non-homologous recombination.

[0215] With regard to autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously in a host cell of interest. The origin of replication may be any plasmid replicator that mediates autonomous replication that functions in the cell. The term "origin of replication" or "plasmid replicator" refers to a nucleotide sequence that allows a plasmid or vector to replicate in vivo.

[0216] More than one copy of a polynucleotide of the invention may be inserted into a host cell to enhance the production of variants. The copy number of the polynucleotide can be increased by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene in the polynucleotide, where cells containing amplified copies of the selectable marker gene, and thus additional copies of the polynucleotide, can be selected by culturing the cells in an appropriate selectable agent.

[0217] The techniques used to ligate the above elements to construct the recombinant expression vectors of the invention to obtain substantially pure variants are well known to those skilled in the art (see, e.g., Sambrook et al., 1989, supra).

[0218] host cell The present invention also relates to recombinant host cells containing a polynucleotide of the present invention operably linked to one or more control sequences that provide for the production of the variants of the present invention. Accordingly, the present invention relates to a recombinant host cell comprising a polynucleotide encoding a variant comprising a modification at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence according to SEQ ID NO:1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence according to SEQ ID NO:1, wherein the polynucleotide is operably linked to one or more control sequences that cause the production of the variant comprising a modification at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence according to SEQ ID NO:1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence according to SEQ ID NO:1. A construct or vector containing a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as previously described. 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 host cell will largely depend on the gene encoding the variant and its source.

[0219] The host cell can be any cell useful in the recombinant production of the variant, for example, a prokaryote or a eukaryote.

[0220] Prokaryotic host cells can be either Gram-positive or Gram-negative bacteria, including, but not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0221] Bacterial host cells include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus The cell may be any Bacillus cell, including Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0222] A bacterial host cell may also be any Streptococcus cell, including, but not limited to, any of the following Streptococcus cells: Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus zooepidemicus cells.

[0223] A bacterial host cell can also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0224] DNA can be introduced into Bacillus cells, for example, by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168:111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81:823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). Introduction of DNA into E. coli cells can be accomplished, for example, by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:557-580) or by electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). DNA can be introduced into Streptomyces cells by, for example, protoplast transformation and electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), by conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294).Introduction of DNA into Pseudomonas cells can be carried out, for example, by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64:391-397) or by conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57). DNA can be introduced into Streptococcus cells, for example, by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68:189-2070), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65:3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0225] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant or fungal cell.

[0226] Fungal cells can 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 techniques for the transformation of Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474. Suitable methods for the transformation of Fusarium species are described in Malardier et al., 1989, Gene 78:147-156 and WO 96 / 00787. Yeast can be transformed using the techniques described in 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, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1920.

[0227] Generation method The present invention also relates to a method of producing a variant, comprising the steps of: (a) culturing a host cell of the invention under conditions suitable for expression of the variant; and (b) recovering the variant. Accordingly, the present invention relates to a method of producing a variant, comprising the steps of: (a) culturing a host cell containing an expression vector or polynucleotide encoding a variant comprising a modification at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence of SEQ ID NO: 1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence of SEQ ID NO: 1, under conditions suitable for expression of the variant; and (b) recovering the variant.

[0228] In one aspect, the invention relates to a method for obtaining an α-amylase variant, comprising the steps of: introducing modifications into a parent α-amylase at one or more positions corresponding to positions selected from the group consisting of 109, 7, 1, 391, 280, 284, 320 and 323 of the amino acid sequence of SEQ ID NO: 1, and optionally at one or more positions corresponding to positions selected from the group consisting of 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence of SEQ ID NO: 1, wherein each modification is independently a substitution or a deletion, and wherein the variant has α-amylase activity; and recovering the variant.

[0229] The host cells are cultured in a nutrient medium suitable for producing the variant using methods known in the art. For example, the cells can be cultured by shake flask culture or by small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a suitable medium and under conditions that allow for expression and / or isolation of the polypeptide. Cultivation is carried out in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts using techniques known in the art. Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., catalogs of the American Type Culture Collection). If the variant is secreted into the nutrient medium, it can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0230] Mutants can be detected using methods known in the art that are specific for the variant. These detection methods can include the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, an enzyme assay can be used to determine the activity of the variant.

[0231] The variant may be recovered by methods known in the art. For example, the variant may be recovered from the nutrient medium by conventional techniques including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0232] Variants can be purified by a variety of techniques known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic techniques (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) to obtain substantially pure variants.

[0233] In an alternative embodiment, the variants are not recovered, and the host cells of the invention expressing the variants may be used as a source of the variants.

[0234] composition The present invention also relates to compositions comprising the variants of the invention. Accordingly, the present invention relates to compositions comprising variants comprising modifications at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323, and 391 of the amino acid sequence of SEQ ID NO: 1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304, and 476 of the amino acid sequence of SEQ ID NO: 1. Preferably, the compositions are enriched for such variants. The term "enriched" means that the α-amylase activity of the composition is increased, for example by an enrichment factor of 1.1.

[0235] The composition may be, for example, a mono-component composition, which may contain the variant as the main enzyme component, or the composition may comprise multiple enzyme activities such as aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase. The additional enzyme may be derived from a Bacillus species, such as Bacillus licheniformis or Bacillus subtilis; an Aspergillus species, such as Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae;For example, Fusarium bacterioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcocroum. Fusarium species, such as Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides or Fusarium venenatum; Humicola species, such as Humicola insolens or Humicola lanuginosa; or species such as Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride. It can be produced, for example, by a microorganism belonging to the genus Trichoderma, such as Trichoderma viride, or any other host cell herein;

[0236] The composition can be prepared according to methods known in the art and can be in the form of a liquid or dry composition. For example, the composition can be in the form of a granule or microgranule. The variant can be stabilized according to methods known in the art.

[0237] According to the present invention, the above α-amylase variants may typically be an ingredient in cleaning compositions, such as detergent compositions, for example laundry detergent compositions or dishwashing detergent compositions, with liquid laundry detergent compositions being particularly preferred.

[0238] Such cleaning compositions include a cleaning / detergent adjunct, preferably a mixture of ingredients. Typically, the cleaning adjunct will be present in the composition in an amount of from 0.001 to 99.9 wt %, more typically from 0.01 to 80 wt % of the cleaning adjunct.

[0239] In other preferred embodiments, the composition comprises one or more surfactants which may be semi-polar and / or anionic and / or cationic and / or zwitterionic and / or amphoteric and / or semi-polar nonionic and / or nonionic, including mixtures thereof. The surfactants are typically present at a level of 0.1% to 60% by weight, or 0.5 to 50% by weight, or 1 to 40% by weight of the composition.

[0240] use The present invention also relates to methods of using the α-amylase variants.

[0241] The α-amylase variants of the present invention are useful in detergent compositions, laundry washing, dishwashing and / or cleaning processes at low temperatures.

[0242] How to use The present invention also relates to a method for cleaning and / or treating a locus, particularly a surface or fabric, hi one aspect, such a method is disclosed, comprising the steps of optionally washing and / or rinsing said surface or fabric, contacting said surface or fabric with any of the consumer products disclosed herein, and then, optionally washing and / or rinsing said surface or fabric.

[0243] As used herein, cleaning includes, but is not limited to, scrubbing and mechanical agitation. Drying of such surfaces or fabrics can be accomplished by any one of the common means employed in domestic or industrial environments. Such means include, but are not limited to, forced air drying or still air drying at ambient or elevated temperatures, under pressures of 5 to 0.01 atmospheres, and in the presence or absence of electromagnetic radiation, including sunlight, infrared, ultraviolet, and microwave radiation. In one embodiment, the drying can be achieved at temperatures above ambient temperature by utilizing an iron, where, for example, the fabric can be placed in direct contact with the iron for a relatively short period of time or even for a long period of time, and where pressures in excess of those normally present due to gravity can be applied. In another embodiment, the drying can be achieved at temperatures above ambient temperature by utilizing a dryer. Apparatus for drying fabrics is well known and is often referred to as a clothes dryer. In addition to clothing, such appliances are used to dry many other items, including towels, sheets, pillowcases, diapers, etc., and such appliances have become standard commodity in many countries around the world, essentially replacing the use of clotheslines for drying fabrics. Most dryers in use today use heated air that passes over and / or through fabrics as they are tumbled in the dryer. The air can be heated, for example, electrically, by a gas flame, or even by microwave radiation. Such air can be heated to temperatures of about 15°C to about 400°C, about 25°C to about 200°C, about 35°C to about 100°C, or about 40°C to about 85°C and used to dry surfaces and / or fabrics in the dryer. As will be appreciated by those skilled in the art, the cleaning compositions of the present invention are ideally suited for use in laundry applications. Accordingly, the present invention includes a method for laundering fabrics. The method comprises the step of contacting a fabric to be laundered with said cleaning laundry solution comprising at least one embodiment of Applicant's cleaning composition, cleaning additive or mixtures thereof.The fabric may include most fabrics that can be laundered under normal consumer or institutional use conditions. The solution preferably has a pH of about 8 to about 10.5. The composition may be utilized at a concentration of about 500 ppm to about 15,000 ppm in the solution. The water temperature typically ranges from about 5°C to about 90°C. The water-to-fabric ratio is typically about 1:1 to about 30:1.

[0244] The following are exemplary detergent compositions:

[0245] [Table 2]

[0246] [Table 3]

[0247] [Table 4]

[0248] [Table 5]

[0249] [Table 6]

[0250] [Table 7]

[0251] [Table 8]

[0252] [Table 9]

[0253] Ingredients and Notes for Composition Examples 1-21 C 11 ~C 18 Linear alkylbenzene sulfonate having an average aliphatic carbon chain length of C 12-18 Dimethylhydroxyethylammonium chloride AE3S is C 12-15 Alkyl ethoxy (3) sulfate AE7 is a C ethoxylate with an average degree of ethoxylation of 7 12-15 Alcohol Ethoxylate AE9 is a C ethoxylate with an average degree of ethoxylation of 9 12-16 Alcohol Ethoxylate HSAS is a mid-chain branched primary alkyl sulfate having a carbon chain length of about 16-17 as disclosed in U.S. Pat. Nos. 6,020,303 and 6,060,443. Polyacrylate MW4500 is manufactured by BASF Carboxymethylcellulose was Finnfix® V from CP Kelco, Arnhem, Netherlands. CHEC is a cationic modified hydroxyethyl cellulose polymer. Phosphonate chelating agents include, for example, diethylenetetraminepentaacetic acid (DTPA) hydroxyethanediphosphonate (HEDP), Savinase®, Natalase®, Stainzyme®, Lipex®, Celluclean™, Mannaway® and Whitezyme® are all products of Novozymes, Bagsvaerd, Denmark. Purafect® and Purafect Prime® are products of Genencor International, Palo Alto, California, USA. Optical Brightener 1 is Tinopal® AMS, Optical Brightener 2 is Tinopal® CBS-X, Direct Violet 9 is Pergasol® Violet BN-Z, and NOBS is sodium nonanoyloxybenzene sulfonate TAED is tetraacetylethylenediamine S-ACMC is carboxymethylcellulose conjugated with CI Reactive Blue 19 (product name AZO-CM-CELLULOSE). The stain remover is Repel-o-tex® PF The acrylic acid / maleic acid copolymer has a molecular weight of 70,000 and an acrylate:maleate ratio of 70:30. EDDS is the sodium salt of ethylenediamine-N,N'-disuccinic acid, (S,S) isomer. Foam suppressor aggregates are available from Dow Corning, Midland, Michigan, USA. HSAS is medium-chain branched alkyl sulfate Liquitint® Violet CT is manufactured by Milliken, Spartanburg, South Carolina, USA 1 Random graft copolymers are polyvinyl acetate-grafted polyethylene oxide copolymers with a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is approximately 6000, the weight ratio of polyethylene oxide to polyvinyl acetate is approximately 40-60, and there is no more than one grafting point per 50 ethylene oxide units. 2 Polyethyleneimine (MW=600) with 20 ethoxylate groups per —NH 3 The amphiphilic alkoxylated polymer was prepared from polyethyleneimine (MW 600) polymer derivatized to contain 24 ethoxylate groups per —NH and 16 propoxylate groups per —NH. amylase 4 means any of a) to k) herein (mg active protein).

[0254] [Table 10]

[0255] [Table 11]

[0256] [Table 12]

[0257] The dimensions and values ​​disclosed herein should not be understood to be narrowly limited to the exact numerical values ​​recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range above and below that value. For example, a dimension disclosed as "40 mm" is intended to mean "approximately 40 mm." [Example]

[0258] pNP-G7 assay for determining α-amylase activity Alpha-amylase activity can be determined by a method utilizing the G7-pNP substrate. G7-pNP is an abbreviation for 4,6-ethylidene (G7)-p-nitrophenyl (G1)-α,D-maltoheptaoside, a blocked oligosaccharide that can be cleaved by endo-amylases such as α-amylase. After cleavage, the α-glucosidase in the kit further digests the hydrolyzed substrate, liberating yellow free pNP molecules, which can be measured by visible spectroscopy at λ = 405 nm (400-420 nm). A kit containing the G7-pNP substrate and α-glucosidase is manufactured by Roche / Hitachi (catalog number 11876473).

[0259] reagent: The G7-pNP substrate in this kit contains 22 mM 4,6-ethylidene-G7-pNP and 52.4 mM HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid), pH 7.0).

[0260] The α-glucosidase reagent contains 52.4 mM HEPES, 87 mM NaCl, 12.6 mM MgCl2, 0.075 mM CaCl2, ≥ 4 kU / L α-glucosidase).

[0261] The substrate treatment solution is formed by mixing 1 mL of α-glucosidase reagent with 0.2 mL of G7-pNP substrate immediately before use.

[0262] Dilution buffer: 50 mM MOPS, 0.05% (w / v) Triton X100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (C 14 H 22 O(C2H4O) n (n=9–10)), 1 mM CaCl2, pH 8.0.

[0263] Method: Amylase samples to be analyzed were diluted in dilution buffer to ensure a pH of 7 in the diluted samples. Assays were performed by transferring 20 μl of diluted enzyme sample to a 96-well microtiter plate and adding 80 μl of substrate treatment solution. The solution was mixed and pre-incubated at room temperature for 1 minute, and absorbance was measured at OD 405 nm every 20 seconds for 5 minutes.

[0264] The slope of the time-dependent absorbance curve (absorbance / min) is directly proportional to the specific activity (activity / mg enzyme) of the α-amylase of interest under a given set of conditions. Amylase samples should be diluted to a level where the slope is less than 0.4 absorbance units / min.

[0265] Automated Mechanical Stress Assay (AMSA) for Laundry To evaluate washing performance in laundries, washing experiments are performed using an automated mechanical stress assay (AMSA). The AMSA allows for testing the washing performance of large volumes of small-volume enzyme-detergent solutions. The AMSA plate has multiple slots for test solutions and a lid into which fabrics representing laundry samples to be washed are firmly pressed against all slot openings. During the wash time, the plate, test solutions, fabrics, and lid are vigorously shaken to bring the test solutions into contact with the fabrics and apply mechanical stress with regular, periodic vibrations. For further explanation, see WO 02 / 42740, especially pages 23-24, paragraph "Special method description."

[0266] General cleaning performance description A test solution containing water (10° dH), detergent (e.g., 5.1 g / L of a European liquid detergent as described below), and an enzyme of the present invention (e.g., at concentrations of 0, 0.8, and / or 1.2 mg enzyme protein / L) is prepared. Fabrics soiled with starch (e.g., CS-28 from Center for Test Materials BV, PO Box 120, 3133 KT, Vlaardingen, The Netherlands) are added and washed for 20 minutes at 20°C. After thorough rinsing with running tap water and drying in the dark, the light intensity or reflectance values ​​of the soiled fabrics are then measured as a measure of cleaning performance. A Δ remission value was obtained using the test with 0 mg enzyme protein / L as a blank. Mechanical action is preferably applied during the washing step, for example, in the form of shaking, rotating, or stirring the washing solution with the fabrics.

[0267] AMSA cleaning performance experiments were carried out under the experimental conditions specified below.

[0268] [Table 13]

[0269] Amylase dilution buffer: Amylase was diluted in ultrapure water (MilliQ water) containing a low concentration of calcium (0.1 mM) to stabilize the amylase during storage and 0.01% Triton X-100 to reduce the risk of enzyme protein adsorption to containers and pipettes.

[0270] CaCl2, MgCl2 and NaHCO3(Ca 2+ :Mg 2+ :HCO3 - The water hardness was adjusted to 10°dH by adding 10% ethanol (=3:1:4.5) to the test system. After washing, the fabrics were rinsed with tap water and dried.

[0271] Washing performance is measured as the brightness of the color of the washed fabric. Brightness can also be expressed as the intensity of light reflected from the sample when illuminated with white light. If the sample is dirty, the intensity of the reflected light will be lower than that of a clean sample. Therefore, the intensity of the reflected light can be used to measure washing performance.

[0272] The color measurements are made with a professional flatbed scanner (Kodak iQsmart, Kodak, Midtager 29, DK-2605 Brondby, Denmark) which is used to take an image of the washed fabric.

[0273] To extract light intensity values ​​from a scanned image, the 24-bit pixel values ​​from the image are converted to red, green, and blue (RGB) values. The intensity value (Int) is calculated by adding the RGB values ​​together as a vector and then calculating the length of the resulting vector.

number

[0274] The results of the AMSA laundry test for the different variants are shown in Tables 1 and 2. In the results, the index is 100. The performance result of the parent α-amylase is given a value of 100, and the results of the variants are compared to this value.

[0275] TOM cleaning performance Water hardness was adjusted to the strengths listed below by adding CaCl2, MgCl2, and NaHCO3. Washing solutions were prepared in buckets with the desired amount of detergent, temperature, and water hardness, as listed below. The detergent was dissolved during 10 minutes of magnetic stirring. (Washing solutions were used within 30-60 minutes of preparation.)

[0276] The temperature and rotation (rpm) in the water bath in the Terg-O-tometer were set according to the settings in Table 2 below. When the temperature was adjusted according to the settings (tolerance + / - 0.5°C), the cleaning solution was added to the TOM beaker according to the amounts listed below.

[0277] The stirring in the beakers was 200 rpm. Two homemade rice starch rags (HM CS-28), two homemade tapioca starch rags (HM CS-29), and ballast were added to each beaker and washed according to the times indicated below. The rags were rinsed in cold tap water for 5 minutes, placed in a wash bag, and rinsed in a washing machine (AEG OEKO LAVAMAT 86820) using the "STIVN" program. The rags were sorted, placed between filter paper, and placed in a dryer to dry overnight without heat.

[0278] Fabric samples HM CS-28 (rice starch on cotton, 5 × 5 cm, starch applied to a 2.5 cm diameter circle) and HM CS-29 (tapioca starch on cotton, 5 × 5 cm, starch applied to a 2.5 cm diameter circle) were obtained from Center for Test Materials BV, PO Box 120, 3133 KT Vlaardingen, the Netherlands.

[0279] White cotton stockinette was used as ballast and was obtained from Warwick Equest Ltd, Unit 55, Consett Business Park, Consett, County Durham, DH8 6BN UK.

[0280] [Table 14]

[0281] [Table 15]

[0282] Wash performance was measured as the brightness of the color of the washed fabric expressed as a remission value (REM). Remission measurements were performed using a Macbeth 7000 Color Eye spectrophotometer. Each dried swatch was measured. Due to the possibility of interference from the background, the swatches were placed on two layers of fabric during remission measurements. Remission was measured at 460 nm. No UV filter was used. The average remission result for the swatches was calculated.

[0283] The cleaning performance of the different variants is shown in Table 5 as the improvement factor (IF), which is calculated as shown below.

number

[0284] Example 1 Cleaning performance of α-amylase using an automated mechanical stress assay To evaluate the cleaning performance of α-amylase in detergent-based compositions, wash experiments can be performed using an automated mechanical stress assay (AMSA). The AMSA test allows for testing the cleaning performance of large volumes of small-volume enzyme-detergent solutions. The AMSA plate has multiple slots for test solutions and a lid into which fabric swatches, representing the laundry samples to be washed, are firmly pressed against all slot openings. During the wash time, the plate, test solutions, fabrics, and lid are vigorously shaken to bring the test solutions into contact with the fabrics and apply mechanical stress with regular, periodic vibrations. For further description, see WO 02 / 42740 (especially the paragraph "Special Method Embodiments" on pages 23-24).

[0285] General cleaning performance description A test solution containing water (6° dH or 15° dH), 0.79 g / L detergent (e.g., Model Detergent J, as described below), and an enzyme of the present invention at a concentration of 0 or 0.2 mg enzyme protein / L is prepared. Starch-soiled fabrics (CS-28, manufactured by Center for Test Materials BV, PO Box 120, 3133 KT, Vlaardingen, the Netherlands) are added and washed for 10 minutes at 20°C and 40°C, or alternatively, as described in the Examples, for 10 minutes at 20°C and 30°C. After thorough rinsing with running tap water and drying in the dark, the light intensity value of the soiled fabric is then measured as a measure of washing performance. A test with 0 mg enzyme protein / L is used as a blank to correlate with the contribution of the detergent. Mechanical action is preferably applied during the washing step, for example, in the form of shaking, rotating, or stirring the washing solution with the fabric. AMSA washing performance experiments can be performed under the experimental conditions specified below.

[0286] [Table 16]

[0287] [Table 17]

[0288] CaCl2, MgCl2 and NaHCO3(Ca 2+ :Mg 2+ :HCO 3- The water hardness was adjusted to 6°dH by adding 100% ethanol (2:1:4.5) to the test system. After washing, the fabrics were rinsed with tap water and dried.

[0289] [Table 18]

[0290] [Table 19]

[0291] CaCl2, MgCl2 and NaHCO3(Ca 2+ :Mg 2+ :HCO 3- The water hardness was adjusted to 15°dH by adding 10% ethanol (4:1:7.5) to the test system. After washing, the fabrics were rinsed with tap water and dried.

[0292] [Table 20]

[0293] [Table 21]

[0294] CaCl2, MgCl2 and NaHCO3(Ca 2+ :Mg 2+ :HCO 3- The water hardness was adjusted to 15°dH by adding 10% ethanol (4:1:7.5) to the test system. After washing, the fabrics were rinsed with tap water and dried.

[0295] Cleaning performance is measured as brightness, which is the intensity of light reflected from a sample when illuminated with white light. If the sample is dirty, the intensity of the reflected light is lower than that from a clean sample. Therefore, the intensity of the reflected light can be used to measure cleaning performance.

[0296] The color measurements are taken with a professional flatbed scanner (EPSON Expression10000XL, EPSON) which is used to capture images of the washed fabrics.

[0297] To extract light intensity values ​​from a scanned image, the 48->24-bit color pixel values ​​from the image are converted to red, green, and blue (RGB) values. The intensity value (Int) is calculated by adding the RGB values ​​together as a vector and then calculating the length of the resulting vector.

number

[0298] The cleaning performance of the variants of the present invention is shown in the following tables: Table 3 shows the results obtained from an experiment to evaluate the cleaning performance of model detergents A (Table D) and J (Table B) at different concentrations (0.05 mg enzyme / L detergent and 0.2 mg enzyme / L detergent) and at different temperatures (20°C and 40°C). Table 4 shows the results obtained from an experiment to evaluate the cleaning performance of detergent K (Table F) at different concentrations (0.05 mg enzyme / L detergent and 0.2 mg enzyme / L detergent) and at different temperatures (20°C and 40°C).

[0299] [Table 22]

[0300] [Table 23]

[0301] [Table 24]

[0302] [Table 25]

[0303] As can be seen from Tables 10 and 11, all of the variants tested have improved cleaning performance compared to the benchmark (SEQ ID NO: 2) in at least one of the conditions tested.

[0304] Example 2 - Cleaning performance of α-amylase in liquid detergent K in TOM The cleaning performance of the tested variants and the corresponding parent α-amylase (SEQ ID NO: 2) was tested on the TOM scale cleaning as described above. The detergent used was detergent K. Results are presented as (variant performance - blank performance) divided by (parent performance - blank performance).

[0305] [Table 26]

[0306] [Table 27]

[0307] [Table 28]

[0308] The invention described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, since these embodiments are intended to be illustrative of the various aspects of the invention. Any equivalent embodiments are intended to be within the scope of the 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.

Claims

1. A variant of a parent α-amylase, (i) comprising modifications at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence according to SEQ ID NO: 1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of said amino acid sequence according to SEQ ID NO: 1; (ii) has at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8; and (iii) has α-amylase activity; Variants of the parent α-amylase.

2. The mutant of claim 1 , wherein the modification is a deletion or substitution.

3. 3. The variant of claim 1 or 2, wherein the variant comprises a modification at at least one position corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence according to SEQ ID NO:

1.

4. 4. The variant of any one of claims 1 to 3, wherein the variant comprises a modification at at least one position corresponding to positions 109, 1, 7, 140, 181, 182, 183, 184, 195, 206, 243, 260, 280, 284, 304, 320, 323, 391 and 476 of the amino acid sequence according to SEQ ID NO:

1.

5. The variant according to any one of claims 1 to 4, wherein the one or more modifications are substitutions.

6. The variant according to any one of claims 1 to 4, wherein at least one of the one or more modifications is a deletion.

7. The variant of any one of claims 1 to 6, wherein the variant comprises modifications at at least two positions selected from the group consisting of 1, 109 and 391.

8. The modification is: X1 ＊ , X1A, X7A, X7K, X7E, X7N, X7Q, X7L, X7D, X109A, X109S, X140Y, X181 ＊ , X182 ＊ , X183 ＊ , X184 ＊ 8. The mutant of any one of claims 1 to 7, selected from the group consisting of: X195F, X206Y, X243F, X260G, X280S, X284H, X284R, X284F, X304R, X320A, X320M, X320T, X320V, X320S, X323N, X323R, X323S, X323K, X391A, X391V and X476K.

9. 9. The variant of any one of claims 1 to 8, wherein the variant has improved performance, such as improved cleaning performance.

10. 10. The variant of any one of claims 1 to 9, wherein the variant has at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to the amino acid sequence of the parent α-amylase.

11. 11. The variant according to any one of claims 1 to 10, wherein the number of modifications is 1 to 30, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifications, for example 1 to 20, such as 1 to 10 and 1 to 5 modifications.

12. The variants are: X1 + X7; X1 + X109; X1 + X280; X1 + X284; X1 + X320; X1 + X323; X1 + X391; X109 + X280; X109 + X284; X109 + X320; X109 + X323; X109 + X391; X7 + X109; X7 + X280; X7 + X284; X7 + X320; X7 + X323; X7 + X391; X28 12. The variant of any one of claims 1 to 11, comprising a modification at a position selected from the group of positions consisting of: X280 + X284; X280 + X320; X280 + X323; X280 + X391; X284 + X320; X284 + X323; X284 + X391; X320 + X323; X320 + X391; and X323 + X391, wherein the numbering is according to SEQ ID NO:

1.

13. The mutant is: H1 ＊ +G109A+N280S+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G7K+G109A+N280S+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G7E+G109A+N280S+E391A; H1 ＊ +G7N+G109A+N280S+E391A; H1 ＊ +G7Q+G109A+N280S+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G7L+G109A+N280S+E391A; H1 ＊ +G7D+G109A+N280S+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G109A+N280S+K320A+E391A; H1 ＊ +G109A+N280S+K320M+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G109A+N280S+K320T+E391A; H1 ＊ +G109A+N280S+K320V+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G109A+N280S+M323R+E391A; H1 ＊ +G109A+N280S+K320S+E391A; H1 ＊ +G109A+N280S+E391V; H1 ＊ +G109A+W284R+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G109A+W284F+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G109A+N280S+K320A+M323S+E391A; H1 ＊ +G109A+N280S+W284F+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G109A+N280S+M323N+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G109A+N280S+M323K+E391A; H1 ＊ +G109S+N280S+E391A; H1 ＊ +G109A+W284H+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G109A+N280S+K320A+M323N+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G7A+G109A+N280S+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G7A+G109A+N280S+W284H+K320A+M323N+E391A; G7A+W284H+K320A+M323N; G7A+K320A+M323N; K320A; G7A+K320A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G7A+G109A+N280S+E391A; H1 ＊ +G109A+N280S+W284H+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G109A+N280S+M323S+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G7A+G109A+N280S+K320A+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G7A+G109A+N280S+M323S+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G7A+G109A+N280S+M323N+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G7A+G109A+N280S+W284F+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G7A+G109A+N280S+W284R+E391A; <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G7A+G109A+N280S+K320A+M323S+E391A; H1 ＊ +G7A+G109A+W284R+E391A; and <h2 style=";text-align:left;direction:ltr">H1<h2 style=";text-align:left;direction:ltr"> ＊ <h2 style=";text-align:left;direction:ltr"> +G7A+G109A+N280S+K320A+M323N+E391A 13. The variant of any one of claims 1 to 12, comprising a modification at a position corresponding to a position of the amino acid sequence according to SEQ ID NO: 2, selected from the group consisting of:

14. 14. The variant of any one of claims 1 to 13, wherein the parent α-amylase is selected from the amino acid sequences according to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 and 8, or any α-amylase having at least 90%, such as at least 92%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99%, or 100% sequence identity to any of the amino acid sequences according to SEQ ID NOs: 1 and 2.

15. The variant according to any one of claims 1 to 14, wherein the parent α-amylase comprises or consists of the amino acid sequence according to SEQ ID NOs: 1 and 2.

16. A polynucleotide encoding the variant of any one of claims 1 to 15.

17. A nucleic acid construct comprising the polynucleotide of claim 16.

18. An expression vector comprising the polynucleotide of claim 16.

19. A host cell comprising the polynucleotide of claim 16.

20. 1. A method for producing an α-amylase variant, comprising: a. culturing the host cell of claim 19 under conditions suitable for expression of the variant; and b. Recovering the mutant A method comprising:

21. 1. A method for obtaining an α-amylase variant, the method comprising the steps of: introducing modifications into a parent α-amylase at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323 and 391 of the amino acid sequence of SEQ ID NO:1, and optionally at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 206, 243, 260, 304 and 476 of the amino acid sequence of SEQ ID NO:1, wherein each modification is independently a substitution or a deletion, and wherein the variant has α-amylase activity; and recovering the variant.

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