α-Amylase variant

Abstract

The present invention provides α-amylase variants, polynucleotides encoding the variants, and methods for producing the variants. The present invention relates to variants of parent α-amylases that have improved cleaning performance compared to the parent α-amylase. The invention also relates to polynucleotides encoding the variants, nucleic acid constructs, vectors and host cells comprising the polynucleotides, and methods for producing the variants of the invention.

Description

Technical Field

[0001] Reference to Sequence Listing This application includes a sequence listing in computer-readable form, which is incorporated herein by reference.

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

Background Art

[0003] α-Amylase (α-1,4-glucan-4-glucanohydrolase, E.C. 3.2.1.1) constitutes 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 the industrial use of α-amylase in numerous known applications such as in detergents, bread making, brewing, for example, starch liquefaction and saccharification in the preparation of isomerized sugars or the production of ethanol from starch. These and other uses of α-amylase are known, and in particular, α-amylases derived from microorganisms such as bacterial α-amylases are utilized.

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

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

[0007] For environmental protection, 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 commonly used in low - temperature washing. α - Amylase is an important enzyme used in detergent compositions, and its 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 washing performance, stain - removing effect 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 increased use of low washing temperatures (e.g., cold water) and short washing cycles. Therefore, it is desirable to obtain starch - degrading enzymes that can function at low temperatures while maintaining or increasing other desirable properties such as specific activity (starch - degrading activity), stability and / or washing performance.

[0008] Therefore, an object of the present invention is to provide an α - amylase mutant that can be used in washing, dishwashing and / or cleaning processes at low temperatures such as temperatures of 5 to 40°C. Further, an object of the present invention is also to provide an α - amylase mutant having improved washing performance at low temperatures as compared with the parent α - amylase or as compared with any one of the α - amylases of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8. Summary of the Invention Means for Solving the Problems

[0009] The present invention relates to mutants of a parent α - amylase, where (i) the mutant contains 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 the amino acid sequence according to SEQ ID NO: 1, (ii) the mutant has a sequence identity of at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, but less than 100% with the amino acid sequence according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8, and (iii) the mutant has α - amylase activity.

[0010] The present invention also relates to a polynucleotide encoding the mutant according to the present invention, a nucleic acid construct containing the polynucleotide encoding the mutant according to the present invention, an expression vector containing the polynucleotide encoding the mutant according to the present invention, and a host cell containing the polynucleotide encoding the mutant according to the present invention.

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

[0012] The present invention further relates to a method for obtaining an α-amylase variant, comprising introducing modifications into a parent α-amylase at one or more positions corresponding to positions 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 the variant has α-amylase activity; and recovering the variant.

Mode for Carrying Out the Invention

[0013] The present invention relates to variants of a parent α-amylase, wherein (i) the variant comprises modifications at one or more positions corresponding to positions 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 a sequence identity of at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, but less than 100% to the amino acid sequence according to SEQ ID NOs: 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 a variant 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 a sequence identity of at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, but less than 100% 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] Definitions Allelic variant: The term "allelic variant" means any of two or more alternative forms of a gene that occupy the same chromosomal locus. Allelic mutations occur naturally through mutation and can also be brought about by polymorphism in a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode a polypeptide with 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, E.C. 3.2.1.1) constitutes a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycoside oligosaccharides and polysaccharides. For the purposes of the present invention, α-amylase activity is determined according to the procedure described in the Examples section. In one aspect, the variant of the present 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 standard 20 genetically encoded amino acids, and their corresponding "D-type" stereoisomers (compared to the natural "L-type"), ω-amino acids, other naturally occurring amino acids, non-conventional 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 functional side groups. Such derivatized molecules include, for example, molecules in which the free amino group is derivatized to form an amine hydrochloride, p-toluenesulfonyl group, carboxybenzoxy group, t-butyloxycarbonyl group, chloroacetyl group or formyl group. The free carboxyl group can be derivatized to form salts, methyl and ethyl esters, or other types of esters and hydrazides. The free hydroxyl group can be derivatized to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are 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 as 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 carboxyl amidation (e.g., by ammonia or methylamine), etc.

[0018] When an amino acid is specifically enumerated (such as "alanine" or "Ala" or "A"), this term refers to both L-alanine and D-alanine, unless otherwise stated. Other non-conventional amino acids can also be suitable components of the polypeptides of the present invention, as long as the desired functional properties are retained by the polypeptide. For the peptides shown, each of the encoded amino acid residues is represented, where appropriate, by a one-letter symbol corresponding to the conventional amino acid nomenclature. In one embodiment, the polypeptides of the present invention comprise, or consist of, L-amino acids.

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

[0020] The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by the 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 combinations thereof.

[0021] The term "control sequence" means a nucleic acid sequence required for the expression of a polynucleotide encoding a variant of the invention. Each control sequence may be native (i.e., derived from the same gene) or foreign (i.e., derived from a different gene) to the polynucleotide encoding the variant, or native or foreign to each other. Such control sequences include, but are not limited to, leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, control sequences include a promoter, as well as transcription and translation stop signals. The control sequences may also comprise a linker for the purpose of introducing specific restriction sites that facilitate the ligation of the control sequences to the coding region of the polynucleotide encoding the variant.

[0022] The term "enhanced cleaning performance" or "improved cleaning performance" means the ability of the polypeptides of the invention to provide an improved cleaning effect (e.g., removal of dirt) in a cleaning process such as laundry or dishwashing, as compared to the parent α-amylase of SEQ ID NO:1. The cleaning performance can be determined using methods well known in the art, such as using an automated mechanical stress assay (AMSA). It will be appreciated by those skilled in the art that enhanced cleaning performance can be achieved under only some, or perhaps all, of the cleaning conditions, such as a cleaning temperature of 20 °C or higher (e.g., 40 °C).

[0023] As used herein, the term "benefit due to enzymatic detergency" refers to the advantageous effects that an enzyme can impart to a detergent, compared to an identical but enzyme-free detergent. Important benefits due to enzymatic detergency that are possible include removal of dirt with little or no visible contaminants after washing and / or cleaning; prevention or reduction of redeposition of contaminants released during the washing process (also called the anti-redeposition effect); complete or partial restoration of the whiteness of fabrics that were originally white but have become grayish or yellowish in appearance after repeated use and washing (also called the whitening effect). Also important for the benefit due to enzymatic detergency, although not directly related to catalytic removal of dirt or prevention of redeposition of contaminants, is the textile care benefit. Examples of such textile care benefits include prevention or reduction of migration (also called anti-migration or anti-reverse soiling effect) from one fabric to another or to other parts of the same fabric; removal of protruding or broken fibers from the fabric surface or of existing pills or fuzz to reduce pilling (also called the anti-pilling effect); improvement of fabric softness; clarification of the color of the fabric; and removal of particulate contaminants trapped in the fibers of the fabric or garment. Enzymatic bleaching is a further benefit due to enzymatic detergency, where the 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 variants, 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 that contains a polynucleotide encoding a variant and is operably linked to control sequences that effect its expression.

[0026] The term "fragment" means a polypeptide lacking one or more (e.g., a plurality of) amino acids at the amino and / or carboxyl termini 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 contiguous amino acid residues, or at least 350 contiguous amino acid residues, or at least 400 contiguous amino acid residues, 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" means any cell type that is susceptible to transformation, transfection, transduction, etc. by a nucleic acid construct or expression vector containing the polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parental cell that is not identical to the parental cell due to a mutation that occurs during replication.

[0028] As used herein, the term "intensity value" refers to a measured value of washing performance. This is measured as luminance, represented as the intensity of light reflected from a sample when irradiated with white light. When the sample is dirty, the intensity of the reflected light is lower than that from a clean sample. Thus, it is possible to measure the washing performance using the intensity of the reflected light, where a higher intensity value correlates with a higher washing performance. Color measurement is performed using a professional flatbed scanner (Kodak iQsmart, Kodak) used to image the washed fabric. To extract the light intensity value from the 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 taking the length of the resulting vector.

Number

[0029] The term "Δ intensity" or "Δ intensity value" is defined herein as the result of measuring the strength of a test material, such as a fabric swatch CS-28 (Center For Testmaterials BV, P.O.Box 120, 3133 KT Vlaardingen, the Netherlands) or a hard surface. The fabric swatch is measured together with a part of the fabric swatch washed under equivalent conditions as a background. Δ intensity is the difference obtained by subtracting the strength value of the test material washed without amylase from the strength value of the test material washed with 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, washing performance, thermal activity, heat resistance, as well as stability under storage conditions and chemical stability. The improved property can be any of those such as stability defined and described herein.

[0031] The term "improved washing performance" is defined herein as indicating a modification of the washing performance of the amylases of the present invention based on the washing performance of the amylases of SEQ ID NO: 2 or 1, and this modification can be shown, for example, as an improvement in the removal of dirt. The improved washing performance is determined according to Example 1. When the α-amylase variant concentration is 0.2 mg / L in Model Detergent A at 20°C, or when the α-amylase variant concentration is 0.05 mg / L in Model Detergent A at 40°C, or when the α-amylase variant concentration is 0.2 mg / L in Model Detergent J at 20°C, or when the α-amylase variant concentration is 0.05 mg / L in Model Detergent J at 30°C, or when the α-amylase variant concentration is 0.2 mg / L in Detergent K at 20°C, in one or more of the conditions listed in Example 1, if the improvement factor (IF) is greater than 1.0, preferably greater than 1.05, the washing performance is improved. The washing conditions are described in the section of the examples.

[0032] The term "washing performance" generally includes cleaning such as hard surface cleaning in, for example, dishwashing, but also includes washing performance in fabrics such as laundry, as well as industrial and institutional cleaning. Improved washing performance can be measured by comparing the Δ intensity as described in the definitions herein.

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

[0034] Isolated polynucleotide: The term "isolated polynucleotide" means a polynucleotide that has been artificially modified. In one aspect, an isolated polynucleotide has a purity of at least 1%, such as at least 5%, at least 10%, at least 20%, at least 40%, at least 60%, at least 80%, at least 90% and at least 95% as measured by agarose electrophoresis. The polynucleotide can be genomic, cDNA, RNA, semi-synthetic, synthetically derived, or any combination thereof.

[0035] Isolated variant: The term "isolated variant" means a variant that has been artificially modified. In one embodiment, this variant has a purity of at least 1% as measured by SDS-PAGE, for example, at least 5% purity, at least 10% purity, at least 20% purity, at least 40% purity, at least 60% purity, at least 80% purity, and at least 90% purity.

[0036] Low temperature: "Low temperature" refers to 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 particularly 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" means a polypeptide in its final form following translation and any post-translational modifications such as N-terminal processing, C-terminal cleavage, glycosylation, phosphorylation, etc. It is known in the art that a host cell can result in a mixture of two different mature polypeptides (i.e., those having different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide.

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

[0039] The term "variant" means a polynucleotide that encodes a variant.

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

[0041] The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule, which is isolated from a natural gene or modified to contain segments of nucleic acid so as not to exist naturally, or is a synthetic compound containing one or more control sequences. The term "nucleic acid construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for the expression of the coding sequence of the present invention.

[0042] The term "operably linked" means a structure in which a control sequence is positioned at an appropriate position relative to the coding sequence of a polynucleotide so that the control sequence causes the coding sequence to be expressed.

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

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

[0045] For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) 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 and later. The parameters used can be a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percentage of identity and is calculated as follows. (Number of 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 the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percentage of identity and is calculated as follows. (Number of equivalent deoxyribonucleotides × 100) / (Length of alignment - Total number of gaps in the 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 where starch is removed from fabric, for example in laundry or fabric cleaning. The starch removal process can also be for hard surface cleaning such as dishwashing, or can be a general cleaning process such as industrial or institutional cleaning. This expression also generally includes other starch removal processes or starch conversion, ethanol production, starch liquefaction, fabric sizing removal, paper and pulp production, beer production, as well as detergents.

[0048] Substantially pure polynucleotide: The term "substantially pure polynucleotide" means a preparation of polynucleotide in a form suitable for use in a genetically engineered polypeptide production system and which does not contain other foreign or unwanted nucleotides. Thus, a substantially pure polynucleotide contains other polynucleotide material that is originally or recombinantly related 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%. However, a substantially pure polynucleotide can include natural 5'- and 3'-untranslated regions such as promoters and terminators. A substantially pure polynucleotide is preferably at least 90% pure, for example 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 by weight. The polynucleotides of the present invention are preferably in substantially pure form.

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

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

[0051] Fabric: The fabric sample CS-28 (rice starch on cotton) is obtained from Center For Testmaterials BV, P.O.Box 120, 3133 KT Vlaardingen, the Netherlands.

[0052] As used herein, the term "benefit by fabric handling" is defined as being important for the benefit by enzymatic detergency, although not directly related to the removal of catalyst-induced soil or the prevention of re-deposition of contaminants. Examples of such benefits by fabric handling include the prevention or reduction of migration (also called the effect of anti-migration or anti-backstaining) from one fabric to another or to other parts of the same fabric; the removal of protruding or broken fibers from the fabric surface to reduce pilling, or the removal of existing pills or fuzz (also called the anti-pilling effect); the improvement of fabric softness; the clarification of the color of the fabric; and the removal of particulate contaminants trapped in the fibers of the fabric. Enzymatic bleaching is a further benefit by enzymatic detergency, where the 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" means 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 associated with the "parent" α-amylase of SEQ ID NO: 1 or SEQ ID NO: 2, 3, 4, 5, 6, 7 or 8. Substitution means replacing an amino acid at a position with a different amino acid; deletion means removing an amino acid at a position; and insertion means adding an amino acid adjacent to and in direct continuity with an amino acid at a position. The variants of the present invention have at least 20% of the α-amylase activity of the mature polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2, 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%.

[0054] The term "wild-type" α-amylase means an α-amylase expressed by a natural microorganism such as bacteria, yeast or filamentous fungi found in nature.

[0055] Determination of Conventional Variants The polypeptide of the present invention having α-amylase activity corresponds to a variant of α-amylase derived from Bacillus shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8.

Chemical formula

[0056] For the purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 1 is used to determine the corresponding amino acid residues in other α-amylase polypeptides. However, those skilled in the art will recognize that the sequence of SEQ ID NO: 2 can also be used to determine the corresponding amino acid residues in other α-amylase polypeptides. The amino acid sequence of another 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 preferably determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) implemented in the Needle program of the EMBOSS package 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 (the EMBOSS version of BLOSUM62) substitution matrix.

[0057] Identification of the corresponding amino acid residues in other α-amylases can be determined by alignment of multiple polypeptide sequences using a number of computer programs including, but not limited to, MUSCLE (multiple sequence comparison by log-expectation; version 3.5 and later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (version 6.857 and 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 (version 1.83 and later; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680) which employs ClustalW, using their respective default parameters.

[0058] When other α-amylases diverge from the mature polypeptide of SEQ ID NO:1 and relationships cannot be detected by comparison based on conventional sequences (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), it is possible to use other pair-wise sequence comparison algorithms. The sensitivity in sequence-based searches can be enhanced by using search programs that utilize the probabilistic representation of polypeptide families (profiles) in database searches. For example, the PSI-BLAST program can generate profiles through an iterative database search process and detect distantly related homologs (Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402). When a family or superfamily of polypeptides has one or more representations in a protein structure database, it is possible to further enhance the sensitivity. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) utilize information from diverse sources (PSI-BLAST, secondary structure prediction, structure alignment profiles, and solvation potential) as input to a neural network that predicts the fold structure of the query sequence. Similarly, the method by Gough et al., 2000, J. Mol. Biol. 313:903-919 can be used for alignment of an unknown structure sequence with a superfamily model present in the SCOP database. Subsequently, it is possible to generate a homology model for the polypeptide using these alignments, and such models 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 superfamilies of proteins are structurally aligned, and these alignments are accessible and downloadable. Two or more protein structures can be aligned using various algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33:88-96) or the combinatorial extension method (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), and these algorithms can be additionally run against a query structure database with the target structure to discover potential structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0060] In the description of the α-amylase variants of the present invention, the following nomenclature is adopted to facilitate reference. The recognized one-letter or three-letter amino acid abbreviations of IUPAC are used.

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

[0062] Deletion. For amino acid deletions, the following nomenclature is used: original amino acid, position, * . Thus, 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 * ”.

[0063] Insertion. For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, for example, the insertion of lysine after glycine at position 195 is denoted “Gly195GlyLys” or “G195GK”. Multiple amino acid insertions are denoted as [original amino acid, position, original amino acid, inserted amino acid 1, inserted amino acid 2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is denoted “Gly195GlyLysAla” or “G195GKA”.

[0064] In such cases, the inserted amino acid residues are numbered by appending a subscript to the amino acid residue preceding the inserted amino acid residue. Therefore, in the above example, the sequence is as follows.

[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 at positions 170 and 195 with tyrosine and glutamate 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, e.g., “Arg170Tyr,Glu” represents substitution at position 170 with tyrosine or glutamate for arginine. Therefore, “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 to the polypeptide of SEQ ID NO: 1 having α-amylase activity, such as at least 85%, at least 90%, for example, 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%. In one embodiment, the amino acid sequence of the parent α-amylase differs from the polypeptide of SEQ ID NO: 1 by no more than 10 amino acids, such as 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0070] The parent α-amylase preferably comprises or consists of the amino acid sequence of SEQ ID NO: 1. In other embodiments, 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 aspect, the parental α-amylase has at least 80% sequence identity with the polypeptide of SEQ ID NO: 2 having α-amylase activity, such as at least 85%, at least 90%, for example, 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%. In one aspect, the amino acid sequence of the parental α-amylase differs from the polypeptide of SEQ ID NO: 2 by no more than 10 amino acids, such as 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0073] The parental α-amylase preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2. In other embodiments, the parental α-amylase is an allelic variant of the polypeptide of SEQ ID NO: 2.

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

[0075] In one aspect, the parental α-amylase has at least 80% sequence identity with the polypeptide of SEQ ID NO: 3 having α-amylase activity, such as at least 85%, at least 90%, for example, 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%. In one aspect, the amino acid sequence of the parental α-amylase differs from the polypeptide of SEQ ID NO: 3 by no more than 10 amino acids, such as 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0076] The parental α-amylase preferably comprises or consists of the amino acid sequence of SEQ ID NO: 3. In other embodiments, the parental α-amylase is an allelic variant of the polypeptide of SEQ ID NO: 3.

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

[0078] In one embodiment, the parental α-amylase has at least 80% sequence identity, such as at least 85%, at least 90%, for example, 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%, with the polypeptide of SEQ ID NO: 4 having α-amylase activity. In one embodiment, the amino acid sequence of the parental α-amylase differs from the polypeptide of SEQ ID NO: 4 by no more than 10 amino acids, such as 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

[0079] The parental α-amylase preferably comprises or consists of the amino acid sequence of SEQ ID NO: 4. In other embodiments, the parental α-amylase is an allelic variant of the polypeptide of SEQ ID NO: 4.

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

[0081] In one embodiment, the parental α-amylase has at least 80% sequence identity, such as at least 85%, at least 90%, for example, 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%, with the polypeptide of SEQ ID NO: 5 having α-amylase activity. In one embodiment, the amino acid sequence of the parental α-amylase differs from the polypeptide of SEQ ID NO: 5 by no more than 10 amino acids, such as 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids, and 1 amino acid.

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

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

[0084] In one aspect, the parent α-amylase has at least 80% sequence identity, such as at least 85%, at least 90%, for example, 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%, with respect to the polypeptide of SEQ ID NO: 6 having α-amylase activity. In one aspect, the amino acid sequence of the parent α-amylase differs from the polypeptide of SEQ ID NO: 6 by no more than 10 amino acids, such as 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids and 1 amino acid.

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

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

[0087] In one aspect, the parental α-amylase has at least 80% sequence identity, such as at least 85%, at least 90%, for example, 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 the polypeptide of SEQ ID NO: 7 having α-amylase activity. In one aspect, the amino acid sequence of the parental α-amylase differs from the polypeptide of SEQ ID NO: 7 by no more than 10 amino acids, such as 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids and 1 amino acid.

[0088] The parental α-amylase preferably comprises or consists of the amino acid sequence of SEQ ID NO: 7. In other embodiments, the parental α-amylase is an allelic variant of the polypeptide of SEQ ID NO: 7.

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

[0090] In one aspect, the parental α-amylase has at least 80% sequence identity, such as at least 85%, at least 90%, for example, 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 the polypeptide of SEQ ID NO: 8 having α-amylase activity. In one aspect, the amino acid sequence of the parental α-amylase differs from the polypeptide of SEQ ID NO: 8 by no more than 10 amino acids, such as 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids and 1 amino acid.

[0091] The parental α-amylase preferably comprises or consists of the amino acid sequence of SEQ ID NO: 8. In other embodiments, the parental 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 in the design of nucleic acid probes to identify and clone DNA encoding parents 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 genomic or cDNA of the genus or species of interest after standard Southern blotting to identify and isolate the corresponding genes contained therein. Such probes can be considerably shorter than the entire sequence, but should be at least 14 nucleotides, such as 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, such as 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 probe is typically labeled (e.g., 32 P, 3 H, 35 S, biotin or avidin) for detecting the corresponding gene. Such probes are encompassed by the present invention.

[0093] Genomic DNA or cDNA libraries prepared from such other organisms can be hybridized with the above probes and screened for DNA encoding parents. 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 the separated DNA can be transferred and immobilized onto nitrocellulose or other suitable carrier materials, which are used in Southern blotting.

[0094] Regarding the object of the present invention, hybridization means that a polynucleotide hybridizes to a labeled nucleotide probe corresponding to 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 sub-sequence thereof under low to extremely high stringency conditions. The molecule to which the probe hybridizes is detectable, for example, using an X-ray film or any other detection means known in the art.

[0095] In one aspect, 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 of at least 100 nucleotides in length, extremely low to extremely high stringency conditions are optimally defined as prehybridization and hybridization at 42°C in 5×SSPE, 0.3% SDS, 200 micrograms / ml fragmented and denatured salmon sperm DNA, following a standard Southern blotting procedure for 12 - 24 hours, and 25% formamide for extremely low and low stringency, 35% formamide for medium and medium-high stringency, or 50% formamide for high and extremely high stringency. The carrier material is finally washed three times for 15 minutes at 2×SSC, 0.2% SDS, 45°C (extremely low stringency), 50°C (low stringency), 55°C (medium stringency), 60°C (medium-high stringency), 65°C (high stringency) or 70°C (extremely high stringency).

[0097] For short probes of about 15 nucleotides to about 70 nucleotides in length, the stringency conditions are calculated using the formula of Bolton and McCarthy (1962, Proc. Natl. Acad. Sci. USA 48: 1390) following a standard Southern blotting procedure for 12 - 24 hours. mDefined as prehybridization and hybridization at a temperature approximately 5°C to about 10°C lower than, 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 of yeast RNA / ml. The carrier material is finally washed once in 6X SCC, and also for 15 minutes in 0.1% SDS, and the calculated T m Washed twice for 15 minutes each with 6× SSC at a temperature 5°C to 10°C lower.

[0098] The parent can be obtained from a microorganism of any genus. For the purposes of the present invention, the term "obtained from" as used herein, in relation to a given source, means that the parent encoded by the polynucleotide is produced by the source, or by a cell into which the 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, the parent can be a gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces α-amylase, 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 aspect, 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 aspects, the parent is Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis or Streptococcus equi subsp. Zooepidemicus α-amylase.

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

[0103] In other embodiments, the parent is, for example, an α-amylase of Bacillus sp., such as the α-amylases 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 foregoing species, it will be understood that the present invention includes both the complete and incomplete generations, as well as other taxonomic equivalents such as, for example, the asexual generation, regardless of the name of the known species. One of ordinary skill in the art will readily recognize the identity of appropriate equivalents.

[0105] The lineages of these species are readily publicly available in a number of microorganism depositories such as the American Type Culture Collection (ATCC), the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), the Centraalbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

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

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

[0108] The parent can also be a fusion polypeptide or a cleavable fusion polypeptide in which one polypeptide is fused to the N-terminus or C-terminus of another polypeptide. The fusion polypeptide is 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 include the step of ligating the coding sequences encoding the polypeptides such that they fit within the frame and the expression of the fusion polypeptide is under the control of the same promoter and terminator. The fusion protein may also be constructed using intein technology in which the fusion is formed after translation (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0109] The fusion polypeptide can further contain a cleavage site between the two polypeptides. When the fusion protein is secreted, this site is cleaved and the two polypeptides are released. Examples of cleavage sites include, but are not limited to, those disclosed 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., 1989, Proteins: Structure, Function, and Genetics 6:240 - 248; and, Stevens, 2003, Drug Discovery World 4:35 - 48.

[0110] Preparation of Variants The present invention relates to a method for obtaining a variant having α - amylase activity, comprising: (a) introducing a modification into the parental 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 according to SEQ ID NO: 1, wherein each modification is independently a substitution or a deletion, and 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, comprising: (a) introducing a modification 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 sequences set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7 and 8, wherein the numbers follow SEQ ID NO: 1, and each modification is independently a substitution or a deletion, and 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 other embodiments, the present invention relates to a method for obtaining a variant having α-amylase activity, comprising: (a) introducing a substitution at one or more positions into a parent α-amylase, wherein the substitution is selected from H1A, G7A, G109A, N280S, W284H, K320A, M323N and E391A of the polypeptide of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 or 8, and the numbers follow SEQ ID NO: 1; and (b) recovering the variant.

[0114] In one embodiment, the method further comprises introducing a deletion at one or more positions into the parent α-amylase, wherein the deletion is selected from H1 * , R181 * , G182 * , D183 * and G184 * of the polypeptide of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 or 8, and the numbers follow SEQ ID NO: 1, and recovering the variant.

[0115] In one embodiment, the method further comprises introducing substitutions at one or more positions in the parental 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 variant.

[0116] The variant 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, etc.

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

[0118] Site-directed mutagenesis can be achieved in vitro by PCR using oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis in which a site in a plasmid containing the polynucleotide encoding the parent is cleaved by a restriction enzyme and then an oligonucleotide containing the mutation is ligated to the polynucleotide. Usually, the restriction enzyme used to digest the plasmid and the oligonucleotide is the same, and the sticky ends of the plasmid and the insert fragment are ligated to each other. 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-specific mutagenesis can also be achieved in vivo by methods known in the art. See, for example, 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-specific mutagenesis method can be used in the present invention. Many commercially available kits that can be used for the preparation of mutants are available.

[0121] Synthetic gene construction involves the in vitro synthesis of a designed polynucleotide molecule for encoding a polypeptide of interest. Gene synthesis can be performed using a number of techniques, such as the multiplex microchip-based technology described by Tian et al. (2004, Nature 432:1050-1054), as well as similar technologies where oligonucleotides are synthesized and assembled into a microfluidic chip 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 methods, recombination methods, 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 relevant 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 site-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0123] The mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of the cloned mutagenized polypeptides expressed by the host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). The mutagenized DNA molecules encoding the active polypeptides can be recovered from the host cells and immediately sequenced using standard methods in the art. With these methods, it is possible to immediately determine the importance of individual amino acid residues in the polypeptide.

[0124] Semi-synthetic gene construction is achieved by combining aspects of synthetic gene construction and / or site-directed mutagenesis and / or random mutagenesis and / or shuffling. Semi-synthetic construction is typified by processes that utilize synthesized polynucleotide fragments, which are combined with PCR technology. Thus, defined regions of a gene can be newly synthesized, while other regions can be amplified using site-directed mutagenic primers, while still other regions can be subjected to error-prone PCR or non-error-prone PCR amplification. Subsequently, polynucleotide sub-sequences can be shuffled.

[0125] Variant The present invention also provides variants of a parent α-amylase that (i) contain 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 the amino acid sequence according to SEQ ID NO: 1, (ii) the variant has a sequence identity of at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, but less than 100% to the amino acid sequence according to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8, and (iii) the variant has α-amylase activity. Thereby, variants with improved washing performance at low temperatures are provided as compared to the parent α-amylase or as compared to the α-amylases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8.

[0126] In embodiments, the variant has a sequence identity of at least 80%, such as 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% to the amino acid sequence of the parent α-amylase.

[0127] In other embodiments, the present invention relates to variants of an isolated parent α-amylase that (i) include 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 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%, such as at least 85%, such as at least 90%, at least 95%, such as 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%, such as at least 85%, such as at least 90%, at least 95%, such as 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: 2.

[0130] In other embodiments, the variant has at least 80%, such as at least 85%, such as at least 90%, at least 95%, such as 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: 3.

[0131] In other embodiments, the variant has a sequence identity of 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% to the mature polypeptide of SEQ ID NO: 4.

[0132] In other embodiments, the variant has a sequence identity of 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% to the mature polypeptide of SEQ ID NO: 5.

[0133] In other embodiments, the variant has a sequence identity of 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% to the mature polypeptide of SEQ ID NO: 6.

[0134] In other embodiments, the variant has a sequence identity of 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% to the mature polypeptide of SEQ ID NO: 7.

[0135] In other embodiments, the variant has a sequence identity of 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% to the mature polypeptide of SEQ ID NO: 8.

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

[0137] In one embodiment, the variant comprises a modification such as a substitution at one or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323, and 391, and optionally a modification at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304, and 476, where the numbers follow SEQ ID NO: 1.

[0138] In other embodiments, the variant comprises a modification such as a substitution at two or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323, and 391, and optionally a modification at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304, and 476, where the numbers follow SEQ ID NO: 1.

[0139] In other embodiments, the variant comprises a modification such as a substitution at three or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323, and 391, and optionally a modification at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304, and 476, where the numbers follow SEQ ID NO: 1.

[0140] In other embodiments, the variant comprises a modification such as a substitution at four or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323, and 391, and optionally a modification at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304, and 476, where the numbers follow SEQ ID NO: 1.

[0141] In other embodiments, the variant comprises a modification such as a substitution at five or more positions corresponding to positions 109, 1, 7, 280, 284, 320, 323, and 391, and optionally a modification at one or more positions corresponding to positions 140, 181, 182, 183, 184, 195, 203, 243, 260, 304, and 476, where the numbers follow SEQ ID NO: 1.

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

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

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

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

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

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

[0153] In a preferred embodiment, the variant comprises modifications 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 variant comprises modifications at positions selected from the group of positions consisting of: 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; X280+X284; X280+X320; X280+X323; X280+X391; X284+X320; X284+X323; X284+X391; X320+X323; X320+X391; and X323+X391, where the numbers follow SEQ ID NO: 1.

[0157] In one embodiment, the variant comprises a modification at a position selected from the group of positions consisting of: 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; X109+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+X391+X284; X1+X391+X320; X1+X391+X323; X1+X280+X284; X1+X280+X320; X1+X280+X323; X1+X284+X320; X1+X284+X323; X1+X320+X323; X391+X280+X284; X391+X280+X320; X391+X280+X323; X391+X284+X320; X391+X284+X323; X391+X320+X323; X280+X284+X320; X280+X284+X323; X280+X320+X323; and X284+X320+X323, where the numbers follow 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+X284+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+X323; X109+X1+X320+X323; X109+X391+X280+X284; X109+X391+X280+X320; X109+X391+X280+X323; X109+X391+X284+X320; X109+X391+X284+X323; X109+X391+X320+X323; X109+X280+X284+X320; X109+X280+X284+X323; X109+X280+X320+X323; X109+X284+X320+X323; X7+X1+X391+X280; X7+X1+X391+X284; 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+X320+X323; X7+X391+X280+X284; X7+X391+X280+X320; X7+X391+X280+X323; X7+X391+X284+X320; X7+X391+X284+X323; X7+X391+X320+X323; X7+X280+X284+X320; X7+X280+X284+X323; X7+X280+X320+X323; X7+X284+X320+X323;A position selected from the group of positions consisting of 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; X1 + X284 + X320 + X323; X391 + X280 + X284 + X320; X391 + X280 + X284 + X323; X391 + X280 + X320 + X323; X391 + X284 + X320 + X323; and X280 + X284 + X320 + X323, wherein the numbers follow 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 numbers follow SEQ ID NO: 1.

[0160] In a particular embodiment, the variant is: X1 * + X1A; X1 * + X7A; X1 * + X109A; X1 * + X280S; X1 * + X284H; X1 * + X320A; X1 * + X323N; X1 *Comprising a modification selected from the group consisting of: 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 numbers follow 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 *Comprising a modification selected from the group consisting of +X323N+X391A; X1A+X7A+X109A; X1A+X7A+X280S; X1A+X7A+X284H; X1A+X7A+X320A; X1A+X7A+X323N; X1A+X7A+X391A; X1A+X109A+X280S; X1A+X109A+X284H; X1A+X109A+X320A; X1A+X109A+X323N; X1A+X109A+X391A; X1A+X280S+X284H; X1A+X280S+X320A; X1A+X280S+X323N; X1A+X280S+X391A; X1A+X284H+X320A; X1A+X284H+X323N; X1A+X284H+X391A; X1A+X320A+X323N; X1A+X320A+X391A; X1A+X323N+X391A; X7A+X109A+X280S; X7A+X109A+X284H; X7A+X109A+X320A; X7A+X109A+X323N; X7A+X109A+X391A; X7A+X280S+X284H; X7A+X280S+X320A; X7A+X280S+X323N; X7A+X280S+X391A; X7A+X284H+X320A; X7A+X284H+X323N; X7A+X284H+X391A; X7A+X320A+X323N; X7A+X320A+X391A; X7A+X323N+X391A; X109A+X280S+X284H; X109A+X280S+X320A; X109A+X280S+X323N; X109A+X280S+X391A; X109A+X284H+X320A; X109A+X284H+X323N; X109A+X284H+X391A; X109A+X320A+X323N; X109A+X320A+X391A; X109A+X323N+X391A; 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 numbers follow 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 * including a modification at a position corresponding to a position selected from the group consisting of +X7A+X109A+X280S+X320A+X323N+X391A, wherein the numbering follows 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 * Selected from the group consisting of +X7A+X109A+X280S+X320A+X323N+X391A, and comprising a modification at a position corresponding to the position of the amino acid sequence according to SEQ ID NO: 1, where the numbering follows SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase described 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 *Comprising a modification at a position corresponding to the 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 follows SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase described 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 * It comprises a modification at a position corresponding to the position of the amino acid sequence according to SEQ ID NO: 2, selected from the group consisting of +G7A+G109A+N280S+K320A+M323N+E391A.

[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 * Selected from the group consisting of +X7A+X109A+X280S+X320A+X323N+X391A, comprising a modification at a position corresponding to the position of the amino acid sequence according to SEQ ID NO: 3, wherein the numbering follows SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase described 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 *Modifications are included at positions corresponding to the positions of the amino acid sequence according to SEQ ID NO: 4, selected from the group consisting of +X7A+X109A+X280S+X320A+X323N+X391A, where the numbering follows SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase described 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 * Comprising a modification at a position corresponding to the 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 follows 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 * Selected from the group consisting of +X7A+X109A+X280S+X320A+X323N+X391A, comprising a modification at a position corresponding to the position of the amino acid sequence according to SEQ ID NO: 6, where the numbering follows SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase according to 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 * Comprising a modification at a position corresponding to the position of the amino acid sequence according to SEQ ID NO: 7, selected from the group consisting of +X7A+X109A+X280S+X320A+X323N+X391A, where the numbering follows SEQ ID NO: 1, and the variant has at least 80% sequence identity to the amylase described 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 selected from the group consisting of, comprising a modification at a position corresponding to the position of the amino acid sequence according to SEQ ID NO: 8, 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 variant according to the present invention is X1 *It is preferably modified at 1, 2, 3, 4 or 5 positions selected from the group of X1A, X7A, X109A, X280S and X391A. In a more preferred embodiment, the modification at the 1, 2, 3, 4 or 5 positions is X1 * , selected from X7A, X109A, X280S and X391A.

[0173] In one aspect, the present invention 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 numbers follow 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 relates to a variant of SEQ ID NO: 1 containing a modification at the positions corresponding to H1 * +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, where the numbers follow SEQ ID NO: 1, and where the variant has at least 80% sequence identity to SEQ ID NO: 1.

[0175] In one embodiment, the present invention relates to a variant of SEQ ID NO: 1 containing a modification at the positions corresponding to H1 * +G109A+N280S+E391A; H1 * +G109A+W284H+E391A; H1 *+G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and, H1 * Regarding the variant of SEQ ID NO: 2 that includes the one corresponding to +G7A+G109A+N280S+W284H+M323N+E391A, where the numbering follows SEQ ID NO: 1, and where the variant has at least 80% sequence identity to SEQ ID NO: 2.

[0176] In one embodiment, the present invention is H1 * +G109A+N280S+E391A;H1 * +G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and, H1 * Regarding the variant of SEQ ID NO: 3 that includes the one corresponding to +G7A+G109A+N280S+W284H+M323N+E391A, where the numbering follows SEQ ID NO: 1, and where the variant has at least 80% sequence identity to SEQ ID NO: 3.

[0177] In one embodiment, the present invention is H1 * +G109A+N280S+E391A;H1 * +G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and, H1 * Regarding the variant of SEQ ID NO: 4 that includes the one corresponding to +G7A+G109A+N280S+W284H+M323N+E391A, where the numbering follows SEQ ID NO: 1, and where the variant has at least 80% sequence identity to SEQ ID NO: 4.

[0178] In one embodiment, the present invention is H1 * +G109A+N280S+E391A;H1 *+G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and, H1 * Regarding the variant of SEQ ID NO: 5 including those corresponding to +G7A+G109A+N280S+W284H+M323N+E391A, where the numbering follows SEQ ID NO: 1, and where the variant has at least 80% sequence identity to SEQ ID NO: 5.

[0179] In one embodiment, the present invention is H1 * +G109A+N280S+E391A;H1 * +G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and, H1 * Regarding the variant of SEQ ID NO: 6 including those corresponding to +G7A+G109A+N280S+W284H+M323N+E391A, where the numbering follows SEQ ID NO: 1, and where the variant has at least 80% sequence identity to SEQ ID NO: 6.

[0180] In one embodiment, the present invention is H1 * +G109A+N280S+E391A;H1 * +G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and, H1 * Regarding the variant of SEQ ID NO: 7 including those corresponding to +G7A+G109A+N280S+W284H+M323N+E391A, where the numbering follows SEQ ID NO: 1, and where the variant has at least 80% sequence identity to SEQ ID NO: 7.

[0181] In one embodiment, the present invention is H1 *+G109A+N280S+E391A;H1 * +G109A+W284H+E391A;H1 * +G109A+N280S+K320A+M323N+E391A;H1 * +G7A+G109A+N280S+E391A; and, H1 * Relates to a variant of SEQ ID NO: 8 including those corresponding to +G7A+G109A+N280S+W284H+M323N+E391A, where the numbering follows SEQ ID NO: 1, and where the variant has at least 80% sequence identity to SEQ ID NO: 8.

[0182] In one embodiment, the variant of the present invention further comprises a modification 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 variant of the present invention is 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 present invention further comprises substitutions at 2, 3 or 4 positions selected from the group consisting of G304R, W140YF, E260GHIKNPRTY and G476EQRK. In a further preferred embodiment, the substitutions at the 2, 3 or 4 positions are selected from the group consisting of G304R, W140Y, E260G and G476K.

[0183] In one embodiment, the variant of the present 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, comprising modifications corresponding thereto, wherein the numbers are according to SEQ ID NO: 1, and the variants have at least 80% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8 and are variants of SEQ ID NO: 1.

[0184] Essential amino acids in the parent can be identified according to methods 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, a single alanine mutation is introduced into all residues in the molecule, and the resulting mutant molecules are tested for α-amylase activity to identify amino acid residues that are important for the activity of the molecule. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site or other biological interactions of α-amylase can also be determined by physical analysis of the structure, which is determined by techniques such as nuclear magnetic resonance, crystal structure analysis, electron diffraction, or photoaffinity labeling, in which mutations of putative contact site amino acids are used in combination. See, for example, 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 identity of the essential amino acids can also be inferred from analysis of the identity by a polypeptide related to the parent.

[0185] Polynucleotide The present invention also relates to an isolated polynucleotide encoding any of the variants of the present invention. Accordingly, the present invention relates to 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 to 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 variant has a sequence identity of less than 100%, such as at least 80%, at least 85%, such as at least 90%, at least 95%, such as at least 97%, to the amino acid sequence according to 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 present invention operably linked to one or more (several) control sequences that express 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 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 the amino acid sequence according to SEQ ID NO: 1, wherein the polynucleotide is operably linked to one or more control sequences that express the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0187] The polynucleotide can be treated in various ways to result in the expression of the variant. Treatment of the polynucleotide before insertion into the vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0188] The control array can be a promoter array recognized by a host cell for the expression of polynucleotides. The promoter array contains a transcriptional control array that mediates the expression of the variant. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in a host cell, including mutant, truncated, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or non-homologous to the host cell.

[0189] Examples of suitable promoters that effect transcription of the nucleic acid constructs of the invention in bacterial host cells are 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 agarase gene (dagA), and the prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), and promoters obtained 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 that effect transcription of the nucleic acid constructs of the present invention in filamentous fungal host cells are 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 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 reeseiEndoglucanase IV from Trichoderma reesei, Endoglucanase V from Trichoderma reesei, Xylanase I from Trichoderma reesei, Xylanase II from Trichoderma reesei, β-Xylosidase from Trichoderma reesei, and a promoter obtained from a gene of the NA2-tpi promoter (a modified promoter containing a gene encoding neutral α-amylase in Aspergilli, wherein the untranslated leader is replaced by an untranslated leader derived from a gene encoding triose phosphate isomerase in Aspergilli; non-limiting examples include a modified promoter containing a gene encoding neutral α-amylase in Aspergillus niger, wherein the untranslated leader is replaced by an untranslated leader derived from a gene encoding triose phosphate isomerase in Aspergillus nidulans or Aspergillus oryzae), and mutants, truncated, and hybrid promoters thereof.

[0191] In an East fungus host, useful promoters are obtained from the genes of 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 can also be a suitable transcription terminator sequence recognized by the 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 can be used.

[0193] Preferred terminators for filamentous fungal host cells are obtained from the genes of 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 of 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 that is an untranslated region of the mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5'-end of the polynucleotide encoding the variant. Any leader sequence that functions in the host cell may be used.

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

[0197] Suitable leaders for yeast host cells are obtained from the genes of 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 can also be a polyadenylation sequence that is operably linked to the 3'-end of the sequence encoding the variant, and when transcribed, is recognized by the host cell as a signal that adds polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that functions in the host cell can be used.

[0199] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes of 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 can also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the variant and sends the variant into the cell's secretory pathway. The 5'-end of the coding sequence of the polynucleotide can essentially contain a signal peptide coding region originally linked in the translation reading frame together with a segment of the coding region encoding the variant. Alternatively, the 5'-end of the coding sequence can contain a signal peptide coding region that is foreign to the coding sequence. When the coding sequence does not originally contain a signal peptide coding region, a foreign signal peptide coding region may be required. Alternatively, the foreign signal peptide coding region may simply replace the native signal peptide coding region to enhance the secretion of the variant. However, any signal peptide coding region that sends the expressed variant into the secretory pathway of the host cell can be used.

[0202] Effective signal peptide coding sequences for bacterial host cells are signal peptide coding sequences obtained from the genes of Bacillus NCIB11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM) and Bacillus subtilis prsA. Further 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 signal peptide coding sequences obtained from the genes of 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 of 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 encoding the propeptide position at the N-terminus of the variant. The resulting polypeptide is known as a zymogen or propolypeptide (or, in some cases, a prothymogen). The propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from the genes of 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] If both a signal peptide and a propeptide region are present at the N-terminus of the variant, the propeptide region is located adjacent to the N-terminus of the variant, and the signal peptide region is located adjacent to the N-terminus of the propeptide region.

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

[0208] Expression vector The present invention also relates to a recombinant expression vector comprising the polynucleotide, promoter, and transcription and translation termination signals of the present invention. Accordingly, the present invention relates to a polynucleotide encoding a variant comprising 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 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, modifications at these positions, a promoter, and transcription and translation termination signals. Various nucleotide and control sequences can be combined to generate a recombinant expression vector that may contain one or more (several) convenient restriction sites to allow insertion or substitution of a polynucleotide encoding a variant at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a vector suitable for expression. In the formation of the expression vector, the coding sequence is positioned in the vector such that the coding sequence is operably linked to a control sequence suitable for expression.

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

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

[0211] The vector preferably has one or more (several) selectable markers that facilitate the selection of cells transformed, transfected, transduced, etc. The selectable marker is a gene whose product confers biocide or virus resistance, resistance to heavy metals, prototrophy for auxotrophs, etc.

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

[0213] The vector preferably has elements that permit integration of the vector into the genome of the host cell or autonomous replication of the vector in a cell independent of the genome.

[0214] With respect to integration into the host cell genome, the vector may depend on either the sequence of the polynucleotide encoding the variant or any other element of the vector involved in 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 by homologous recombination at an exact position in the chromosome. To increase the likelihood of integration at an exact position, the integration element should contain a sufficient number of nucleic acids such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of identity to the corresponding target sequence to increase the probability of homologous recombination. The integration element can be any sequence that is homologous to a target sequence in the host cell genome. Furthermore, the integration element can be a non-coding or coding nucleotide sequence. On the other hand, the vector can be integrated into the host cell genome by non-homologous recombination.

[0215] With respect to autonomous replication, the vector may further include an origin of replication that enables autonomous replication of the vector in the host cell of interest. The origin of replication can be any plasmid replicon that mediates autonomous replication functioning in the cell. The term "origin of replication" or "plasmid replicon" means a nucleotide sequence that enables replication of a plasmid or vector in vivo.

[0216] Two or more copies of the polynucleotide of the present invention may be inserted into the host cell to enhance the production of the variant. The number of copies 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 are selectable by culturing the cells in an appropriate selectable agent.

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

[0218] host cell The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more (several) control sequences that result in the production of a variant of the present invention. Accordingly, the present invention relates to recombinant host cells comprising a polynucleotide encoding a variant 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 the amino acid sequence according to SEQ ID NO: 1, wherein the polynucleotide is operably linked to one or more control sequences that produce a variant 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 the amino acid sequence according to SEQ ID NO: 1. A construct or vector comprising the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrant or as the aforementioned self-replicating episomal-chromosomal vector. The term "host cell" includes 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 depend largely 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 prokaryotic or eukaryotic cell.

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

[0221] The bacterial host cell can be any Bacillus cell including, but 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 subtilis, and Bacillus thuringiensis cells.

[0222] The bacterial host cell can also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

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

[0224] Introduction of DNA into Bacillus cells can be carried out, 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 carried out, 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). Introduction of DNA into Streptomyces cells can be carried out, for example, by 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). Introduction of DNA into Streptococcus cells can be carried out, for example, by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), by protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68:189-2070), by electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65:3800-3804), or by conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45:409-436). However, it is possible to use any of the methods known in the art for introducing DNA into host cells.

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

[0226] Fungal cells can be transformed by a process that includes protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable methods for the transformation of Aspergillus and Trichoderma host cells are described in European Patent No. 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 pamphlet. Yeast can be transformed using the methods 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] Production method The present invention also relates to a method for producing a variant, comprising: (a) culturing a host cell of the present invention under conditions suitable for the expression of the variant; and (b) recovering the variant. Accordingly, the present invention relates to a method for producing a variant, comprising: (a) culturing a host cell comprising 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 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, under conditions suitable for the expression of the variant; and (b) recovering the variant.

[0228] In one aspect, the present invention provides a step of introducing a modification into the 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 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, wherein each modification is independently a substitution or a deletion, and the variant has α-amylase activity; and a step of recovering the variant. The present invention relates to a method for obtaining an α-amylase variant.

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

[0230] The variant can be detected using methods known in the art specific to the variant. These detection methods can include the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, the activity of the variant can be determined using an enzyme assay.

[0231] Variants can be recovered by methods known in the art. For example, variants can 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 to obtain substantially pure variants, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatofocusing and size exclusion), electrophoretic techniques (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

[0233] In an alternative embodiment, the variant is not recovered and the host cell of the invention expressing the variant can be used as a source of the variant.

[0234] Compositions The invention also relates to compositions comprising a variant of the invention. Accordingly, the invention relates to compositions comprising 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. Preferably, the composition is enriched in 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 can be, for example, a single-component composition that contains a variant as the main enzyme component. Alternatively, the composition can contain 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, pectin-degrading enzyme, peptidoglutaminase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase or xylanase. Additional enzymes can be, for example, from the genus Bacillus such as Bacillus licheniformis or Bacillus subtilis; from the genus Aspergillus such as Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae;For example, it can be produced, for example, by microorganisms belonging to the genus Fusarium such as Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides or Fusarium venenatum; the genus Humicola, for example, Humicola insolens or Humicola lanuginosa; or the genus Trichoderma such as Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride, or any other host cell of the present specification.;

[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 granules or fine particles. The variant can be stabilized according to methods known in the art.

[0237] According to the present invention, the above-mentioned α-amylase variant can typically be a component in a cleaning composition such as a detergent composition, for example, a laundry detergent composition or a dishwashing detergent composition. A liquid laundry detergent composition is particularly preferred.

[0238] Such a cleaning composition contains a cleaning / detergent aid, preferably a mixture of components. Typically, the cleaning aid will be present in the composition in an amount of 0.001 to 99.9 wt%, more typically 0.01 to 80 wt%.

[0239] In another preferred embodiment, the composition can contain one or more surfactants that are nonionic and contain semi-polar and / or anionic and / or cationic and / or zwitterionic and / or amphoteric and / or semi-polar nonionic and / or mixtures thereof. The surfactant is typically present at a level of 0.1% to 60 wt% or 0.5 to 50 wt% or 1 to 40 wt% of the composition.

[0240] Use The present invention also relates to a method of using the α-amylase variant.

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

[0242] Method of use The present invention also relates to a method for cleaning and / or treating a location (especially a surface or fabric). In one aspect, such a method is disclosed which, optionally, comprises the steps of washing and / or rinsing the surface or fabric, contacting the surface or fabric with any of the consumer products disclosed herein, and then, optionally, washing and / or rinsing the surface or fabric.

[0243] As used herein, washing is not particularly limited, and examples thereof include rubbing and mechanical agitation. Drying of such a surface or fabric can be achieved by any one of the common means employed in a household or industrial environment. Such means include, but are not limited to, forced air drying or static air drying in the presence or absence of electromagnetic waves including irradiation with ambient temperature or high temperature, pressure from 5 to 0.01 atmospheres, sunlight, infrared rays, ultraviolet rays, and microwaves. In one aspect, the drying may be achieved at a temperature exceeding the ambient temperature by using an iron, where, for example, the fabric may be brought into direct contact with the iron for a relatively short time or even for a long time, and where pressure may be applied exceeding the pressure normally present by gravity. In another aspect, the drying may be achieved at a temperature exceeding the ambient temperature by using a dryer. Devices for drying fabrics are well known and are frequently referred to as clothes dryers. In addition to clothes, such appliances are used for drying many other articles including towels, sheets, pillow covers, diapers, etc., and such appliances are accepted as standard household items that substantially replace the use of drying ropes for drying fabrics in many countries around the world. In most dryers used today, as the fabric is rotated in the dryer, heated air passing over and / or through the fabric is used. The air can be heated, for example, electrically, by a gas flame, or even by microwave radiation. Such air can be heated to from about 15°C to about 400°C, from about 25°C to about 200°C, from about 35°C to about 100°C, or from about 40°C to about 85°C and is used to dry the surface and / or fabric in the dryer. As will be understood by those skilled in the art, the cleaning composition of the present invention is ideally suitable for use in laundry applications. Accordingly, the present invention includes a method for washing fabrics. The method includes the step of contacting the fabric to be washed with the cleaning washing solution comprising at least one embodiment of a cleaning composition, a cleaning additive, or a mixture thereof by the applicant.The fabric may include most fabrics that can be washed under normal consumer use conditions or organizational 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 is typically in the range of about 5°C to about 90°C. The ratio of water to fabric is typically about 1:1 to about 30:1.

[0244] The following are examples of 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] Raw materials and notes for Composition Examples 1 to 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 C having an average ethoxylation degree of 7 12-15 Alcohol ethoxylate AE9 is C having an average ethoxylation degree of 9 12-16 Alcohol ethoxylate HSAS is medium-chain branched primary alkyl sulfate having a carbon chain length of about 16 to 17 disclosed in U.S. Patent No. 6,020,303 and U.S. Patent No. 6,060,443 Polyacrylate MW4500 is manufactured by BASF Carboxymethyl cellulose is Finnfix® V manufactured by CP Kelco, Arnhem, Netherlands CHEC is a cationic modified hydroxyethyl cellulose polymer. Phosphonate chelating agents are, for example, diethylenetetraaminepentaacetic 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 Fluorescent brightener 1 is Tinopal® AMS, fluorescent brightener 2 is Tinopal® CBS-X, Direct Violet 9 is Pergasol® Violet BN-Z, and NOBS is sodium nonanoyloxybenzenesulfonate TAED is tetraacetylethylenediamine S-ACMC is carboxymethyl cellulose conjugated with C.I. Reactive Blue 19 (product name AZO-CM-CELLULOSE) The contaminant release agent 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, and the (S,S) isomer foam inhibitor aggregate is manufactured by Dow Corning, Midland, Michigan, USA HSAS is medium-chain branched alkyl sulfate Liquitint® Violet CT is manufactured by Milliken, Spartanburg, South Carolina, USA 1 The random graft copolymer is a polyvinyl acetate graft polyethylene oxide copolymer having a polyethylene oxide main chain and a plurality of polyvinyl acetate side chains. The molecular weight of the polyethylene oxide main chain is about 6000, the weight ratio of polyethylene oxide to polyvinyl acetate is about 40 - 60, and the number of graft points per 50 ethylene oxide units is 1 or less. 2 Polyethyleneimine (MW = 600) having 20 ethoxylate groups per -NH 3 The amphiphilic alkoxylated polymer is a polyethyleneimine (MW600) prepared from a polymer derivatized to contain 24 ethoxylate groups per -NH and 16 propoxylate groups per -NH. Amylase 4 is any one of a) to k) herein (mg active protein).

[0254]

Table 10

[0255]

Table 11

[0256]

Table 12

[0257] The dimensions and values disclosed in this specification should not be understood to be narrowly limited to the exact numerical values recited. Instead, unless otherwise specified, each of such dimensions 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 "about 40 mm".

Examples

[0258] pNP-G7 assay for α-amylase activity determination α-Amylase activity can be determined by a method using 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, α-glucosidase in the kit further digests the hydrolyzed substrate to release yellow free pNP molecules, enabling measurement by visible spectroscopy at λ = 405 nm (400 - 420 nm). Kits containing the G7-pNP substrate and α-glucosidase are manufactured by Roche / Hitachi (catalog number 11876473).

[0259] Reagents: The G7-pNP substrate of this kit contains 22 mM of 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 of HEPES, 87 mM of NaCl, 12.6 mM of MgCl2, 0.075 mM of CaCl2, and ≥4 kU / L of α-glucosidase).

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

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

[0263] Procedure: The amylase sample to be analyzed was diluted in the dilution buffer to ensure that the pH in the diluted sample was 7. The assay was performed by transferring 20 μl of the diluted enzyme sample to a 96-well microtiter plate and adding 80 μl of the substrate treatment solution. The solution was mixed and pre-incubated at room temperature for 1 minute, and the absorbance was measured every 20 seconds for 5 minutes at OD405nm.

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

[0265] Automatic Mechanical Stress Assay for Laundry (AMSA) To evaluate the washing performance in laundry, a washing experiment is carried out using an Automatic Mechanical Stress Assay (AMSA). In AMSA, it is possible to test the washing performance of a large amount of small - volume enzyme - detergent solution. The AMSA plate has a number of slots for the test solution and a lid that firmly presses the fabric, which is the laundry sample to be washed, against all slot openings. During the washing time, the plate, the test solution, the fabric, and the lid are vigorously shaken to bring the test solution into contact with the fabric and mechanical stress is applied with regular periodic vibrations. For further explanation, refer to the pamphlet of International Publication No. 02 / 42740, especially the paragraphs "Special method embodiments" on pages 23 - 24.

[0266] Description of general washing performance A test solution containing water (10°dH), detergent (for example, 5.1 g / L of a European - made liquid detergent described below), and the enzyme of the present invention (for example, at concentrations of 0, 0.8, and / or 1.2 mg enzyme protein / L) is prepared. A fabric soiled with starch (for example, CS - 28 manufactured by Center For Testmaterials BV, P.O.Box 120, 3133 KT, Vlaardingen, The Netherlands) is added and washed at 20°C for 20 minutes. After thoroughly rinsing with running water from the tap and drying in the dark, the light intensity or reflectance value of the soiled fabric is then measured as a measure of the washing performance. The Δ remission value is obtained using the test at 0 mg enzyme protein / L as a blank. During the washing step, it is preferable that a mechanical action is applied, for example, in the form of shaking, rotating, or stirring the washing solution with the fabric.

[0267] The AMSA washing performance experiment was carried out under the experimental conditions specified below.

[0268] [Table 13]

[0269] Amylase Dilution Buffer: Amylase was diluted with ultrapure water (MilliQ water) containing low-concentration 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 - = 3:1:4.5) were added to the test system to adjust the water hardness to 10 °dH. After washing, the fabric was rinsed with tap water and dried.

[0271] The washing performance was measured as the luminance of the color of the washed fabric. Luminance can also be expressed as the intensity of the reflected light from the sample when irradiated with white light. When the sample is dirty, the intensity of the reflected light is lower than that of a clean sample. Therefore, it is possible to measure the washing performance using the intensity of the reflected light.

[0272] Color measurement was performed using a professional flatbed scanner (Kodak iQsmart, Kodak, Midtager 29, DK-2605 Brondby, Denmark) used to image the washed fabric.

[0273] To extract the light intensity value from the scanned image, the 24-bit pixel values from the image were 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 tests for different mutants are shown in Tables 1 and 2. In the results, the index is 100. The performance result of the parental α-amylase is set to a value of 100, and the results of the mutants are compared to this value.

[0275] TOM washing performance The water hardness was adjusted to the strength described below by adding CaCl2, MgCl2 and NaHCO3. The washing solution was prepared in a bucket with the desired amount of detergent, temperature and water hardness as described below. The detergent was dissolved during 10 minutes of magnetic stirring. (The washing solution was used within 30 - 60 minutes after 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 adjusting the temperature according to the settings (tolerance is + / - 0.5 °C), the washing solution was added to the TOM beaker according to the amounts described below.

[0277] Stirring in the beaker was at 200 rpm. Two hand - made rice starch cloths (HM CS - 28), two hand - made tapioca starch cloths (HM CS - 29) and ballast were added to each of the beakers and washing was carried out according to the times shown below. The cloths were rinsed with cold tap water for 5 minutes, placed in a washing bag and rinsed in a washing machine (AEG OEKO LAVAMAT 86820) in the "STIVN" program. The cloths were sorted, sandwiched between filter papers and left to dry overnight in a dryer without heating.

[0278] Fabric samples HM CS - 28 (rice starch on cotton, 5×5 cm, applied in a circular shape with a starch diameter of 2.5 cm) and HM CS - 29 (tapioca starch on cotton, 5×5 cm, applied in a circular shape with a starch diameter of 2.5 cm) were obtained from Center for Testmaterials BV, P.O.Box 120, 3133 KT Vlaardingen, the Netherlands.

[0279] White cotton jersey 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] The cleaning performance was measured as the brightness of the color of the cleaned fabric represented by the remission value (REM). The remission measurement was performed using a Macbeth 7000 Color Eye spectrophotometer. Each of the dried cloth pieces was measured. Since there was a risk of interference from the background, the cloth pieces were placed on a two-layer fabric during the remission measurement. The remission was measured at 460 nm. A UV filter was not used. The average result of the remission for the cloth pieces was calculated.

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

Equation

[0284] Example 1 Cleaning performance of α-amylase using an automated mechanical stress assay To evaluate the cleaning performance of α-amylase in a detergent-based composition, a cleaning experiment may be conducted using an automated mechanical stress assay (AMSA). In the AMSA test, it is possible to test the cleaning performance of a large number of small-volume enzyme-detergent solutions. The AMSA plate has a number of slots for the test solution and a lid that firmly presses the cloth pieces of the fabric, which is the laundry sample to be cleaned, against all the slot openings. During the cleaning time, the plate, the test solution, the fabric, and the lid are vigorously shaken to bring the test solution into contact with the fabric, and mechanical stress is applied with regular periodic vibrations. For further description, refer to WO 02 / 42740 pamphlet (especially, the paragraph "Embodiments of the special method" on pages 23-24).

[0285] Description of general cleaning performance Prepare a test solution containing water (6°dH or 15°dH), 0.79 g / L of detergent (for example, model detergent J as described below), and the enzyme of the present invention at a concentration of 0 or 0.2 mg enzyme protein / L. Add a cloth soiled with starch (CS-28 manufactured by Center For Test materials BV, P.O.Box 120, 3133 KT, Vlaardingen, the Netherlands), and wash at 20°C and 40°C for 10 minutes, or alternatively, as described in the examples, wash at 20°C and 30°C for 10 minutes. Rinse thoroughly with running water from the tap and dry in the dark. Then, measure the light intensity value of the soiled fabric as a measure of cleaning performance. Use the test at 0 mg enzyme protein / L as a blank and correlate it with the contribution of the detergent. During the cleaning step, mechanical action is preferably applied, for example, in the form of shaking, rotating, or stirring the cleaning solution with the fabric. The AMSA cleaning performance experiment can be carried out under the experimental conditions specified below.

[0286]

Table 16

[0287]

Table 17

[0288] CaCl2, MgCl2 and NaHCO3 (Ca 2+ :Mg 2+ :HCO 3- =2:1:4.5) was added to the test system to adjust the water hardness to 6°dH. After washing, the fabric was rinsed with tap water and dried.

[0289]

Table 18

[0290]

Table 19

[0291] CaCl2, MgCl2 and NaHCO3 (Ca 2+ :Mg 2+ :HCO 3- =4:1:7.5) were added to the test system to adjust the water hardness to 15°dH. After washing, the fabric was rinsed with tap water and dried.

[0292]

Table 20

[0293]

Table 21

[0294] CaCl2, MgCl2 and NaHCO3 (Ca 2+ :Mg 2+ :HCO 3- =4:1:7.5) were added to the test system to adjust the water hardness to 15°dH. After washing, the fabric was rinsed with tap water and dried.

[0295] The washing performance is measured as the luminance represented by the intensity of the light reflected from the sample when irradiated with white light. When the sample is dirty, the intensity of the reflected light is lower than that from a clean sample. Therefore, it is possible to measure the washing performance using the intensity of the reflected light.

[0296] The color measurement is performed with a professional flatbed scanner (EPSON Expression10000XL, EPSON) used to image the washed fabric.

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

Number

[0298] The cleaning performance of the mutants according to the present invention is shown in the following table. Table 3 shows the results obtained from experiments 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 different temperatures (20 °C and 40 °C). Table 4 shows the results obtained from experiments 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 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 the mutants tested have improved cleaning performance compared to the reference (SEQ ID NO: 2) in at least one of the test conditions.

[0304] Example 2 - Detergency Performance of α-Amylase in Liquid Detergent K in TOM The detergency performance of the tested variants and the corresponding parental α-amylase (SEQ ID NO: 2) was tested for TOM-scale washing as described above. The detergent used was Detergent K. The results are shown as (variant performance - blank performance) divided by (parental 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 as illustrations of several 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 case of conflict, the present disclosure, including definitions, will control.

Claims

1. A variant of a parent α - amylase consisting of the amino acid sequence of SEQ ID NO: 1 or 2, wherein said variant comprises deletions and / or substitutions, (i) includes a deletion at the position corresponding to position 1 of the amino acid sequence of SEQ ID NO: 1 or 2, includes serine at the position corresponding to position 280, includes the amino acid substitution of G109A / S, and includes the amino acid substitution of E391A / V, with the numbering according to SEQ ID NO: 1, (ii) has at least 90% but less than 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, and (iii) has α - amylase activity.

2. The variant according to claim 1, wherein said variant further comprises one or more deletions and / or substitutions at one or more positions corresponding to positions 7, 140, 181, 182, 183, 184, 195, 206, 243, 260, 284, 304, 320, 323, and 476 of the amino acid sequence of SEQ ID NO: 1 or 2.

3. wherein said deletion and / or substitution is selected from the group consisting of X7A, X7K, X7E, X7N, X7Q, X7L, X7D, X109S, X140Y, X181 ＊ , X182 ＊ , X183 ＊ , X184 ＊ , X195F, X206Y, X243F, X260G, X284H, X284R, X284F, X304R, X320A, X320M, X320T, X320V, X320S, X323N, X323R, X323S, X323K, X391V and X476K, a variant according to claim 2

4. The variant according to any one of claims 1 to 3, wherein said variant has improved washing performance compared to the parent α - amylase consisting of the amino acid sequence of SEQ ID NO: 1 or 2.

5. The variant according to any one of claims 1 to 4, wherein said variant has a sequence identity of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% to the amino acid sequence of SEQ ID NO: 1 or 2.

6. The variant according to claim 1, wherein the number of said deletions and / or substitutions is 2 - 30.

7. 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 + 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 + N280S + K320A + M323N + E391A; H1* + G109A + N280S + W284H + E391A; and H1* + G109A + N280S + M323S + E391A A variant according to any one of claims 1 to 6, comprising deletions and / or substitutions at positions corresponding to the positions of the amino acid sequence of SEQ ID NO: 2, numbered according to SEQ ID NO:

1.

8. The variant according to claim 1, wherein the variant is an α-amylase having at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 or 2.

9. A polynucleotide encoding the variant according to any one of claims 1 to 8.

10. A nucleic acid construct comprising the polynucleotide according to claim 9.

11. An expression vector comprising the polynucleotide according to claim 9.

12. A host cell comprising the polynucleotide according to claim 9.

13. A method for producing an α-amylase variant, comprising: a. culturing the host cell according to claim 12 under conditions suitable for the expression of the variant; and b. recovering the variant A method comprising.

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