Amylase variants

Abstract

In the present invention, a novel amylase is provided. More specifically, genetically engineered amylases, compositions comprising these enzymes, and methods of making and using these enzymes or compositions comprising these enzymes are provided.

Description

Technical Field

[0001] In the present invention, novel amylases are provided. More specifically, genetically engineered amylases, compositions comprising these enzymes, and methods of making and using these enzymes or compositions comprising these enzymes are provided. Background Art

[0002] Enzymes are increasingly used in various applications as a sustainable alternative to petrochemicals. Enzymes are biodegradable and can perform catalytic activity at lower temperatures, thereby reducing energy consumption. Specifically, in the detergent industry, enzymes are applied in washing formulations to improve cleaning efficiency and reduce energy consumption in the washing step.

[0003] Amylases are enzymes that can hydrolyze starch. Therefore, amylases have been used to remove starch stains and have been added to detergent compositions for this purpose. In detergent applications, amylases should be stable at high temperatures and / or within the denaturing conditions of detergents and washing liquids. Therefore, there is a need for new amylases that meet these requirements. Summary of the invention

[0004] The present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0005] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3,

[0006] (ii) amino acid substitutions at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 176, 181, 186, 195, 206, 225, 320 and 482 according to the numbering of SEQ ID NO: 3, and

[0007] (iii) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1, 3, 4 or any one of SEQ ID NO: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1.

[0008] In one embodiment, the amylase variant of the invention comprises

[0009] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3,

[0010] (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3, and

[0011] (iii) with SEQ ID NO: 1, 3, 4 or with any one of SEQ ID NO: 15-41, preferably with SEQ ID NO: 1 or SEQ ID NO: 3, preferably with SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity.

[0012] In another embodiment, the invention relates to polynucleotides encoding amylase variants.

[0013] Furthermore, the present invention relates to a composition comprising the amylase variant.Preferably, the composition comprising the amylase variant is a detergent composition.

[0014] The present invention further relates to a method for preparing the amylase variants and methods of using the amylase variants, particularly for cleaning objects. DETAILED DESCRIPTION

[0015] The present invention may be understood more readily by reference to the following detailed description of embodiments of the invention and the Examples included therein.

[0016] Although the invention will be described with respect to specific embodiments, this description should not be construed in a limiting sense.

[0017] definition

[0018] Unless otherwise noted, terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0019] Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given. Unless otherwise stated or obvious from the nature of the definitions, the definitions apply to all compounds, methods and uses described herein.

[0020] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include the corresponding plural forms as well, unless the context clearly dictates otherwise.

[0021] In the context of the present invention, the terms "about" and "approximately" represent an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the discussed feature. The term typically indicates a deviation from the indicated numerical value of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5%.

[0022] In addition, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" and the like in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequential order or a chronological order. It should be understood that the terms so used are interchangeable where appropriate, and that the embodiments of the invention described herein are capable of operating in other sequences than those described or illustrated herein. Where the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", "i", "ii" and the like refer to steps of a method or use or an assay, there is no temporal or temporal time interval coherence between these steps, i.e., these steps may be performed simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in this application as described above or below.

[0023] Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0024] It should be understood that the term "comprising" is not limiting. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising at least a certain number of members, this means that a group consisting only of these members is also encompassed.

[0025] A "variant" enzyme differs from a "parent" enzyme by certain amino acid alternations, preferably by amino acid substitutions at one or more amino acid positions.

[0026] In describing the variants of the invention, abbreviations for the individual amino acids are used according to the generally accepted IUPAC single-letter or three-letter amino acid abbreviations.

[0027] As used herein, "amino acid change" refers to an amino acid substitution, deletion or insertion.

[0028] "Substitutions" are described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the amino acid that replaces the original amino acid. For example, a histidine at position 120 replaced by an alanine is represented as "His120Ala" or "H120A". Substitutions can also be described by naming only the resulting amino acid in the variant, e.g., "X120A" or "120A" or "Xaa120Ala" or "120Ala", without specifying the parent amino acid at that position.

[0029] "Deletions" are described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by *. Accordingly, the deletion of glycine at position 150 is indicated as "Gly150*" or "G150*". Alternatively, a deletion is indicated by, for example, "deletion of D183 and G184".

[0030] "Insertion" is described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the original amino acid and the newly added amino acid. For example, the insertion of lysine at position 180 near glycine is represented as "Gly180GlyLys" or "G180GK". When more than one amino acid residue is inserted, such as, for example, Lys and Ala are inserted after Gly180, such insertion may be represented as "Gly180GlyLysAla" or "G195GKA".

[0031] In the case where the substitution and insertion occur at the same position, this can be expressed as "S99SD+S99A" or simply "S99AD". Variants containing multiple changes are separated by a plus sign "+", for example, "Arg170Tyr+Gly195Glu", "R170Y+G195E" or "X170Y+X195E" represent that the arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively. Alternatively, multiple changes can be separated by spaces or commas, for example, "R170Y G195E" or "R170Y, G195E". When different substitution changes can be introduced at a certain position, the different changes are separated by commas, for example, "Arg170Tyr, Glu" and "R170T, E" represent that the arginine at position 170 is replaced by tyrosine or glutamic acid, respectively. The substitution of a specific position can also be represented as "X120A, G, H", "120A, G, H", "X120A / G / H" or "120A / G / H". Alternatively, different changes or optional substitutions can be represented by brackets, such as "Arg170[Tyr, Gly]" or "Arg170{Tyr, Gly}" or abbreviated as "R170[Y, G]" or "R170{Y, G}".

[0032] The term "native" (or naturally or wild-type or endogenous) cell or organism or polynucleotide or polypeptide refers to a cell or organism or polynucleotide or polypeptide as found in nature (ie, without any human intervention).

[0033] The term "heterologous" (or exogenous or foreign or recombinant or non-original or non-naturally) polypeptide is defined herein as a polypeptide that is not native to a host cell; a polypeptide that is native to a host cell, wherein structural modifications (e.g., deletions, substitutions, and / or insertions) have been made by recombinant DNA techniques to alter the native polypeptide; or a polypeptide that is native to a host cell, the expression of which is quantitatively altered or the expression of which is from a genomic location different from that of the native host cell due to manipulation of the host cell DNA by recombinant DNA techniques (e.g., a stronger promoter). Similarly, the term "heterologous" (or exogenous or foreign or recombinant or non-original or non-naturally) polynucleotide refers to a polynucleotide that is not native to a host cell; a polynucleotide that is native to a host cell, wherein structural modifications (e.g., deletions, substitutions, and / or insertions) have been made by recombinant DNA techniques to alter the native polynucleotide; or a polynucleotide that is native to a host cell, whose expression is quantitatively altered due to manipulation of the regulatory elements of the polynucleotide by recombinant DNA techniques (e.g., a stronger promoter); or a polynucleotide that is native to a host cell, but has not been integrated into its natural genetic environment due to genetic manipulation by recombinant DNA techniques. With respect to the relationship between two or more polynucleotide sequences or the relationship between two or more amino acid sequences, the term "heterologous" is used to characterize that the two or more polynucleotide sequences or the two or more amino acid sequences do not naturally occur in a specific combination with each other.

[0034] For the purposes of this invention, "recombinant" (or transgenic) with respect to a cell or organism means that the cell or organism contains a heterologous polynucleotide, which has been introduced by a human using genetic techniques. With respect to polynucleotides, "recombinant" includes all constructs produced using genetic techniques / recombinant DNA technology, wherein

[0035] (a) a polynucleotide sequence or a portion thereof, or

[0036] (b) one or more genetic control sequences, including but not limited to a promoter, operably linked to the polynucleotide, or

[0037] (c) Both a) and b)

[0038] Not in its wild-type genetic context or has been artificially modified.

[0039] "Synthetic" compounds are obtained by in vitro chemical and / or enzymatic synthesis.

[0040] Variant polynucleotides and variant polypeptide sequences can be defined by their sequence identity when compared to another sequence. Sequence identity is usually provided in the form of "% sequence identity" or "% identity". In order to calculate sequence identity, the first step is to generate a sequence alignment. According to the present invention, a paired global alignment is generated, which means that the two sequences are aligned over their entire length, which is usually generated by using a mathematical method known as an alignment algorithm.

[0041] According to the present invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J.Mol.Biol. [Journal of Molecular Biology] (1979) 48, pp. 443-453). Preferably, the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the present invention, wherein the program default parameters are used (polynucleotides: gap opening = 10.0, gap extension = 0.5 and matrix = EDNAFULL; polypeptides: gap opening = 10.0, gap extension = 0.5, matrix = EBLOSUM62). After the two sequences are aligned, in a second step, the identity value is determined based on the alignment generated. For this purpose, % identity is calculated by dividing the number of identical residues by the length of the alignment region showing the corresponding sequence of the present invention in its full length multiplied by 100: % identity = (identical residues / length of the alignment region showing the corresponding sequence of the present invention in its full length) * 100.

[0042] For calculating the percentage identity of two nucleic acid sequences, it is also applicable to calculating the percentage identity of two amino acid sequences with certain specifications. For the nucleic acid sequence of coding protein, pairwise comparison should be carried out on the complete length (from start codon to stop codon, excluding intron) of the coding region of the sequence of the present invention. Introns present in other sequences compared with the sequence of the present invention should also be removed for pairwise comparison. After comparing the two sequences, in the second step, the identity value is determined according to the comparison produced. Percent identity is calculated by: %-identity=(same residue / show the length of the comparison area of ​​the sequence of the present invention from start codon to stop codon and excluding intron on its complete length)*100.

[0043] In addition, a preferred alignment program for nucleic acid sequences implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, pp. 443-453) is "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)), using the program default parameters (gap opening = 10.0, gap extension = 0.5, and matrix = EDNAFULL).

[0044] Variant polypeptides can also be defined by their sequence similarity when compared to another sequence. Sequence similarity is usually provided as "% sequence similarity" or "%-similarity". The % sequence similarity takes into account that the defined amino acid sets have similar properties, for example by their size, by their hydrophobicity, by their charge or by other features. Here, the exchange of one amino acid with another can be referred to as a "conservative mutation". The definition of similar amino acids according to the present invention is as follows, which should also apply to the determination of %-similarity according to the present invention, which is also consistent with the BLOSUM62 matrix used by the program "NEEDLE", which is one of the most commonly used amino acid similarity matrices for database searches and sequence alignments:

[0045] Amino acid A is similar to amino acid S

[0046] Amino acid D is similar to amino acid E; N

[0047] Amino acid E is similar to amino acid D; K; Q

[0048] Amino acid F is similar to amino acid W; Y

[0049] Amino acid H is similar to amino acid N; Y

[0050] Amino acid I is similar to amino acid L; M; V

[0051] Amino acid K is similar to amino acid E; Q; R

[0052] Amino acid L is similar to amino acid I; M; V

[0053] Amino acid M is similar to amino acid I; L; V

[0054] Amino acid N is similar to amino acid D; H; S

[0055] Amino acid Q is similar to amino acid E; K; R

[0056] Amino acid R is similar to amino acid K; Q

[0057] Amino acid S is similar to amino acid A; N; T

[0058] Amino acid T is similar to amino acid S

[0059] Amino acid V is similar to amino acid I; L; M

[0060] Amino acid W is similar to amino acid F; Y

[0061] Amino acid Y is similar to amino acid F; H; W

[0062] To calculate sequence similarity, in a first step, a sequence alignment is generated as described above. After the two sequences have been aligned, in a second step, a similarity value is determined based on the alignment generated. For this purpose, %-similarity is calculated by adding the number of identical residues to the number of similar residues divided by the length of the alignment region showing the sequence of the invention over its full length multiplied by 100: %-similarity=[(identical residues+similar residues) / length of the alignment region showing the sequence of the invention over its full length]*100.

[0063] For nucleic acids, similar sequences can also be determined by hybridization using corresponding stringent conditions. The term "high stringency conditions" means probes of at least 100 nucleotides in length, prehybridized and hybridized in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA and 50% formamide at 42°C, according to standard protein blotting procedures for 12 to 24 hours. Finally, the carrier material was washed three times at 65°C using 2X SSC, 0.2% SDS for 15 minutes each time. The term "very high stringency conditions" means probes of at least 100 nucleotides in length, prehybridized and hybridized in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA and 50% formamide at 42°C, according to standard protein blotting procedures for 12 to 24 hours. Finally, the carrier material was washed three times at 70°C using 2X SSC, 0.2% SDS for 15 minutes each time.

[0064] As used herein, "fragment" or "subsequence" is a part of a polynucleotide or amino acid sequence. The term "functional fragment" refers to any nucleic acid or amino acid sequence that only comprises a part of a full-length amino acid sequence, but still has the same or similar activity and / or function. Preferably, the functional fragment is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99% or at least 99.5% identical to the original full-length amino acid sequence. The functional fragment comprises consecutive nucleotides or amino acids compared to the original nucleic acid or original amino acid sequence, respectively.

[0065] As used herein, a "gene construct" or "expression cassette" is a nucleic acid molecule consisting of at least one sequence of interest to be expressed, which is operably linked to one or more control sequences as described herein (at least to a promoter).

[0066] As used herein, the term "vector" includes any kind of construct suitable for carrying an exogenous polynucleotide sequence to transfer to another cell or to stably or transiently express in a given cell. As used herein, the term "vector" encompasses any kind of cloning vehicle, such as, but not limited to, plasmids, phagemids, viral vectors (e.g., phage), phages, baculoviruses, cosmids, forsmids, artificial chromosomes, and any other vectors specific to a particular host of interest. Exogenous polynucleotide sequences typically include a coding sequence that may be referred to herein as a "target gene". The target gene may include introns and exons, depending on the source or destination of the host cell.

[0067] The term "introduction of polynucleotides" or "transformation of polynucleotides" referred to herein encompasses the transfer of exogenous polynucleotides into host cells, regardless of the method used for transfer. That is, as used herein, the term "transformation of polynucleotides" is independent of vectors, shuttle systems or host cells, and it not only relates to polynucleotide transfer methods of transformation known in the art (see, e.g., Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), but it also encompasses any other kind of polynucleotide transfer method, such as, but not limited to, transduction or transfection.

[0068] A polynucleotide encoding a polypeptide can be "expressed". The term "expression" or "gene expression" means that one or more genes or gene constructs are transcribed into structural RNA (e.g., rRNA, tRNA) or mRNA, which is then translated into protein or not. The process includes DNA transcription and processing of the resulting mRNA product.

[0069] The term "purification" or "purifying" refers to a process in which at least one component (e.g., a protein of interest) is separated from at least another component (e.g., particulate matter of a fermentation broth) and transferred to different compartments or phases, wherein the different compartments or phases do not necessarily need to be separated by a physical barrier. Examples of such different compartments are two compartments separated by a filter membrane or cloth, namely a filtrate and a retentate; examples of such different phases are precipitate and supernatant or filter cake and filtrate, respectively. The solution obtained after purifying the enzyme of interest from the fermentation broth is referred to herein as a "purified enzyme solution."

[0070] By "protein formulation" (or "enzyme preparation"), e.g., "protein variant formulation", it is meant any non-complex formulation comprising minor ingredients, wherein these ingredients are used for the purpose of stabilizing the protein contained in the protein formulation and / or the stability of the protein formulation itself. Preferably, the non-complex protein formulation comprises a higher concentration of protein than a complex formulation (e.g., than a detergent composition). Thus, preferably, the non-complex protein formulation is a concentrated protein variant formulation. Preferably, the non-complex protein formulation comprises 2 mg / g to 120 mg / g active enzyme, wherein a complex formulation such as a detergent composition comprises 0.002 mg / g to 6 mg / g active enzyme.

[0071] "Enzyme properties" include, but are not limited to, catalytic activity, substrate / cofactor specificity, product specificity, stability over time, thermal stability, pH stability, and chemical stability. "Enzyme activity" or "catalytic activity" means the catalytic effect exerted by an enzyme, expressed in units / mg of enzyme (specific activity) or substrate molecules converted / minute / enzyme molecule (molecular activity). Enzyme activity can be specified by the actual function of the enzyme, for example, proteases exert proteolytic activity by catalyzing the hydrolytic cleavage of peptide bonds, lipases exert lipolytic activity by hydrolytic cleavage of ester bonds, amylase activity involves the hydrolysis of glycosidic linkages in polysaccharides, etc.

[0072] The term "enzyme stability" according to the present invention relates to the retention of enzyme activity as a function of time during storage or handling. The retention of enzyme activity as a function of time during storage is called "storage stability" and is preferred in the context of the present invention.

[0073] In order to determine and quantify the catalytic activity of an enzyme stored or used under certain conditions over time, the "initial enzyme activity" is measured at time zero (100%) and at a later time point (x%) under the specified conditions. By comparing the measured values, the potential loss of enzyme activity can be determined. The extent of enzyme activity loss determines the stability or instability of the enzyme.

[0074] As used herein, "enzyme inhibitors" are compounds that slow down or stop the activity of an enzyme. Enzyme inhibitors can also typically stabilize the three-dimensional structure of an enzyme. Therefore, enzyme inhibitors also typically act as "enzyme stabilizers."

[0075] "pH stability" refers to the ability of an enzyme to exert enzymatic activity after exposure to a certain pH value.

[0076] The terms "thermal stability", "thermostability" or "temperature-dependent activity" refer to the ability of an enzyme to exert catalytic activity or washing performance after exposure to elevated temperatures, preferably at a temperature of 40°C for 28 days, preferably 56 days, preferably in a detergent composition (preferably, in a model ES1-C detergent), or at 92°C for at least 10 min.

[0077] The term "detergent stability" or "stability under storage in a detergent composition" refers to the ability of an enzyme to exert catalytic activity or washing performance after storage in a detergent composition, preferably in a detergent composition (preferably, in a model ES1-C detergent) at a temperature of 40°C or 50°C for 28 days, preferably 56 days.

[0078] "Improvement Factor" (IF) is the degree of improvement of an enzyme variant in some property, preferably relative to the corresponding parent enzyme. The improvement of an enzyme variant (preferably relative to the corresponding parent enzyme) is characterized by an improvement factor (IF) of >1.0. The improvement factor can alternatively be expressed as a percentage, e.g., an IF of 1.1 is equal to 110%.

[0079] As used herein, the "washing performance" (also referred to herein as "cleaning performance") of an enzyme refers to the contribution of the enzyme to the cleaning performance of a detergent composition, i.e., the cleaning performance added to the detergent composition by the performance of the enzyme. The term "washing performance" is similarly used herein for laundry and hard surface cleaning. Washing performance is compared under relevant washing conditions. The term "relevant washing conditions" is used herein to indicate the conditions actually used by households in the detergent market segment, particularly washing temperature, time, washing machinery, foam concentration, detergent type, and water hardness. The term "improved washing performance" is used to indicate that a better final result is obtained in stain removal under relevant washing conditions, or that less enzyme is required to obtain the same final result on a weight basis relative to corresponding control conditions.

[0080] In this article, the term "specific performance" refers to the ability of each unit of active enzyme to clean and remove specific stains or dirt. In some embodiments, specific performance is determined using stains or dirt (e.g., eggs, egg yolks, milk, grass, minced meat, blood, chocolate sauce, baby food, sebum, etc.).

[0081] "Detergent composition", "detergent formulation" or "detergent" refers to a composition designated for cleaning soiled materials. Detergent compositions according to the present invention include detergent compositions for different applications such as laundry and hard surface cleaning. The term "detergent component" is defined herein to mean a class of chemicals that can be used in detergent compositions. Typical detergent components are surfactants. "Surfactant" (herein used synonymously with "surfactant (surfaceactive agent)") means an organic chemical that changes the properties of a liquid at an interface when added to a liquid. Depending on its ionic charge, surfactants are referred to as nonionic surfactants, anionic surfactants, cationic surfactants or amphoteric surfactants. The term "effective amount of a detergent component" includes the amount of certain components that provide effective stain removal and / or effective cleaning conditions (e.g., pH, temperature, water hardness), the amount that effectively provides optical benefits (e.g., optical brightening, dye transfer inhibition, color care), and the amount of certain components that effectively assist processing (maintaining physical properties during processing, storage and use; e.g., rheology modifiers, hydrotropes, desiccants).

[0082] The term "laundry" or "laundering" refers to both domestic and industrial laundry and means the process of treating textiles and / or fabrics with a solution containing the detergent composition of the present invention. The laundering process can be carried out using technical equipment such as domestic or industrial washing machines. Alternatively, the laundering process can be done manually.

[0083] The term "textile" means any textile material, including yarn (thread made of natural or synthetic fibers for knitting or weaving), yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and fabrics such as clothing, cloth and other articles made from these materials. As used herein, the term "fabric" (a textile made from woven, knitted or felted fibers) or "garment" (any clothing made from textiles) is also meant to include the broader term textiles.

[0084] The term "fiber" includes natural fibers, synthetic fibers and mixtures thereof. Examples of natural fibers are plant fibers (e.g. flax, jute and cotton) or fibers of animal origin, containing proteins such as collagen, keratin and fibroin (e.g. silk, sheep wool, angora wool, mohair, cashmere). Examples of fibers of synthetic origin are polyurethane fibers such as or Polyester fibers, polyolefins such as elastofin, or polyamide fibers such as nylon. The fibers may be single fibers or part of a textile, such as a knitted, woven or nonwoven fabric.

[0085] The term "hard surface cleaning" refers to both domestic hard surface cleaning and industrial hard surface cleaning and means the process of treating hard surfaces with a solution containing the detergent composition of the present invention. Hard surfaces can include any hard surface in the home or industry, such as floors, furniture, walls, sanitary ceramics, glass, metal surfaces (including medical equipment), knives and tableware. A special form of hard surface cleaning is dishwashing, manual dishwashing (MDW) or automatic dishwashing (ADW).

[0086] The term "warewashing" refers to all forms of dishwashing, such as manual dishwashing or automatic dishwashing. Dishwashing includes, but is not limited to, cleaning all forms of crockery, such as plates, cups, glasses, bowls, all forms of cutlery, such as spoons, knives, forks and serving utensils, as well as ceramics, plastics, such as melamine, metal, porcelain, glass and acrylic.

[0087] The cleaning performance is evaluated under relevant cleaning conditions. In the present context, the term "relevant cleaning conditions" refers to the conditions actually used in washing machines, automatic dishwashers or in manual cleaning processes, in particular cleaning temperature, time, cleaning regime, foam concentration, detergent type and water hardness.

[0088] The term "medical device cleaning" refers to the cleaning steps involved in reprocessing reusable medical devices. Medical device cleaning methods can be divided into two categories: manual cleaning methods and mechanical / automatic cleaning methods. Manual cleaning is used when no mechanical device is available or the medical device to be cleaned is too fragile or difficult to clean with mechanical devices. Mechanical / automatic cleaning methods remove dirt and microorganisms through an automated cleaning and rinsing process that includes ultrasonic cleaning and washing.

[0089] In the field of soil removal, the term "stain" is generally used to refer to laundry, such as the cleaning of textiles, fabrics or fibers, while the term "soil" is generally used to refer to hard surface cleaning, such as the cleaning of dishes and cutlery. However, the terms "stain" and "soil" should be used interchangeably herein.

[0090] As used herein, "chelating builders" are distinguished from precipitating builders because no substantial precipitate is formed when the builder is used in an amount sufficient to combine with all calcium ions in an aqueous solution initially having a hardness of 7° dH (German hardness) at neutral pH. "Strong builders" are classified as highly efficient chelating agents that are capable of strongly binding divalent cations (e.g., Ca2+), with a logarithmic stability constant (Log K of the cation / chelating agent complex) of 1.17kcal / cm2. Ca ) is greater than 4, especially greater than 5, greater than 6 or greater than 7. The stability constant is determined at an ionic strength of 0.1 M and a temperature of 25° C. "Strong chelating builders" combine the above two properties.

[0091] As used herein, the term "low temperature" refers to a temperature range equal to or lower than 40°C, preferably 10-40°C, more preferably 20-40°C, more preferably 20-35°C.

[0092] Detailed Description

[0093] In the present invention, novel amylases are provided. More particularly, amylase variants, methods for preparing these amylase variants, compositions comprising these amylase variants, and methods of using these amylase variants or compositions comprising these amylase variants are provided.

[0094] Amylase variants

[0095] The present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0096] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3,

[0097] (ii) amino acid substitutions at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 176, 181, 186, 195, 206, 225, 320 and 482 according to the numbering of SEQ ID NO: 3, and

[0098] (iii) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1, 3, 4 or any one of SEQ ID NO: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1.

[0099] The amylase variants of the present invention are non-naturally occurring amylases. Preferably, the amylase variants are alpha-amylases. Preferably, the amylase variants of the present invention are purified, isolated, synthesized and / or recombinant amylase variants. Preferably, the amylase variants of the present invention are purified and recombinant amylase variants.

[0100] Amylases and amylase variants according to the invention have "amylolytic activity" or "amylase activity". "Amylolytic activity" or "amylase activity" describes the ability to hydrolyze glycosidic linkages in polysaccharides. Amylase activity can be determined by assays known to those skilled in the art for measuring amylase activity. Examples of assays for measuring amylase activity are the Phadebas assay or the EPS assay ("Infinity reagent"). In the Phadebas assay, amylase activity is determined by using Phadebas tablets as substrate (Phadebas amylase test, provided by Magle LifeScience). Starch is hydrolyzed by amylase, producing soluble blue fragments. The absorbance of the resulting blue solution (measured spectrophotometrically at 620 nm) is a function of amylase activity. The measured absorbance is directly proportional to the specific activity (activity / mg pure amylase protein) of the amylase in question under given conditions.

[0101] Alternatively, amylase activity can also be determined by the method using ethylidene-4-nitrophenyl-α-D-maltoheptaglycoside (EPS). D-maltoheptaglycoside is a closed oligosaccharide that can be cut by endoamylase. After cutting, the α-glucosidase digestion substrate included in the test kit releases yellow free PNP molecules, and therefore can be measured at 405nm by visible spectrophotometry. The test kit containing EPS substrate and α-glucosidase is manufactured by, for example, Roche Costum Biotech (Cat. No. 10880078103). The slope of the time-dependent absorption curve is directly proportional to the specific activity (activity / mg enzyme) of the amylase discussed under given conditions.

[0102] In one embodiment, the amylase variants of the invention exhibit at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200% of the amylolytic activity of the parent amylase. In one embodiment, the amylase variants of the invention exhibit the same or increased amylolytic activity compared to the parent amylase. Preferably, the amylase variants of the invention exhibit increased amylolytic activity compared to the parent amylase.

[0103] Preferably, the parent amylase of the amylase variant of the present invention is an amylase having at least 60% sequence identity with any one of SEQ ID NO: 1, 3, 4 or any one of SEQ ID NO: 15-41, preferably the parent amylase of the amylase variant of the present invention is an amylase according to any one of SEQ ID NO: 1, 3, 4 or any one of SEQ ID NO: 15-41. Most preferably, the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1.

[0104] The present invention relates to an amylase variant comprising an amino acid change, preferably an insertion, a deletion, a substitution or a combination thereof, most preferably a substitution, at two or more positions corresponding to the positions selected from the group consisting of: 25, 116, 176, 181, 186, 195, 206, 225, 320 and 482 according to the numbering of the amino acid sequence shown in SEQ ID NO:3.

[0105] In an alternative manner, amino acid positions 25, 116, 176, 181, 186, 195, 206, 225, 320 and 482 (numbering according to SEQ ID NO: 3) may be described with reference to the numbering of SEQ ID NO: 1 (i.e. numbering according to SEQ ID NO: 1). In this alternative manner of describing the amino acid positions of the invention, amino acid positions 25, 116, 176, 181, 186, 195, 206, 225, 320 and 482 numbered according to SEQ ID NO: 3 correspond to amino acid positions 25, 116, 176, 181, 184, 193, 204, 223, 318 and 480 numbered according to SEQ ID NO: 1.

[0106] Preferably, the parent amylase of the amylase variant of the present invention is an amylase having at least 60% sequence identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 or with any one of SEQ ID NO: 15-41, preferably the parent amylase of the amylase variant of the present invention is an amylase according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 or according to any one of SEQ ID NO: 15-41. Most preferably, the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1.

[0107] Preferably, the present invention relates to an amylase variant comprising one or more amino acid substitutions selected from the group consisting of X25H, X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K and X482W according to the numbering of SEQ ID NO:3.

[0108] Preferably, the present invention relates to an amylase variant comprising two or more amino acid substitutions selected from the group consisting of: X25H, X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K and X482W as compared to the parent sequence and amino acid substitutions at these positions to amino acids similar to the indicated amino acid substitutions (i.e. amino acids similar to the indicated target amino acids at these positions according to the numbering of SEQ ID NO: 3), and wherein the variant has amylase activity, preferably wherein the parent amylase of the amylase variant of the invention is an amylase according to SEQ ID NO: 1 or any amylase having at least 60% sequence identity with SEQ ID NO: 1, most preferably the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1, wherein

[0109] Amino acid A is similar to amino acid S

[0110] Amino acid D is similar to amino acid E; N

[0111] Amino acid E is similar to amino acid D; K; Q

[0112] Amino acid F is similar to amino acid W; Y

[0113] Amino acid H is similar to amino acid N; Y

[0114] Amino acid I is similar to amino acid L; M; V

[0115] Amino acid K is similar to amino acid E; Q; R

[0116] Amino acid L is similar to amino acid I; M; V

[0117] Amino acid M is similar to amino acid I; L; V

[0118] Amino acid N is similar to amino acid D; H; S

[0119] Amino acid Q is similar to amino acid E; K; R

[0120] Amino acid R is similar to amino acid K; Q

[0121] Amino acid S is similar to amino acid A; N; T

[0122] Amino acid T is similar to amino acid S

[0123] Amino acid V is similar to amino acid I; L; M

[0124] Amino acid W is similar to amino acid F; Y

[0125] Amino acid Y is similar to amino acid F; H; W.

[0126] Preferably, the present invention relates to an amylase variant comprising two or more amino acid substitutions selected from the group consisting of: X25H, X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K and X482W as compared to the parent sequence, and wherein the variant has amylase activity, preferably wherein the parent amylase of the amylase variant of the present invention is an amylase according to SEQ ID NO: 1 or any amylase having at least 60% sequence identity with SEQ ID NO: 1, most preferably, the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1.

[0127] Preferably, in this embodiment, the amino acid residue in the parent amylase at the above cited position (ie, X) corresponds to the amino acid residue shown in SEQ ID NO: 1 at the corresponding position (according to the numbering of SEQ ID NO: 3).

[0128] Thus, preferably, the present invention relates to an amylase variant comprising amino acid substitutions at two or more positions corresponding to positions selected from the group consisting of: N25, W116, R176, R181, G186, N195, 1206, T225, R320 and Y482 according to the numbering of SEQ ID NO:3.

[0129] In a preferred embodiment, the present invention relates to an amylase variant comprising amino acid substitutions at two or more positions corresponding to positions selected from the group consisting of: N25H, W116K, R176K, R181T, G186E, N195F, I206Y, T225A, R320K and Y482W according to the numbering of SEQ ID NO:3.

[0130] The present invention relates to an amylase variant comprising an amino acid substitution at position 25 and at one or more positions corresponding to positions selected from the group consisting of: 116, 176, 181, 186, 195, 206, 225, 320 and 482 according to the numbering of SEQ ID NO: 3.

[0131] Preferably, the present invention relates to an amylase variant comprising the amino acid substitution X25H and one or more amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K and X482W according to the numbering of SEQ ID NO:3.

[0132] Preferably, the present invention relates to an amylase variant comprising the amino acid substitution X25H and one or more amino acid substitutions selected from the group consisting of: X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K and X482W as compared to the parent sequence, and wherein the variant has amylase activity, preferably wherein the parent amylase of the amylase variant of the invention is an amylase according to SEQ ID NO: 1 or any amylase having at least 60% sequence identity with SEQ ID NO: 1, most preferably, the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1.

[0133] Preferably, in this embodiment, the amino acid residue in the parent amylase at the above cited position (ie, X) corresponds to the amino acid residue shown in SEQ ID NO: 1 at the corresponding position (according to the numbering of SEQ ID NO: 3).

[0134] Thus, preferably, the present invention relates to an amylase variant comprising an amino acid substitution at amino acid position N25 and at one or more positions corresponding to positions selected from the group consisting of: W116, R176, R181, G186, N195, 1206, T225, R320 and Y482 according to the numbering of SEQ ID NO:3.

[0135] In a preferred embodiment, the present invention relates to an amylase variant comprising an amino acid substitution N25H and an amino acid substitution at one or more positions corresponding to positions selected from the group consisting of: W116K, R176K, R181T, G186E, N195F, I206Y, T225A, R320K and Y482W according to the numbering of SEQ ID NO:3.

[0136] The present invention preferably relates to amylase variants comprising amino acid substitutions at positions 25 and 195 and at one or more positions corresponding to positions selected from the group consisting of: 116, 176, 181, 186, 206, 225, 320 and 482 according to the numbering of SEQ ID NO:3.

[0137] Preferably, the present invention relates to an amylase variant comprising amino acid substitutions X25H and X195F and one or more amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X206Y, X225A, X320K and X482W according to the numbering of SEQ ID NO:3.

[0138] Preferably, in this embodiment, the amino acid residue in the parent amylase at the above cited position (ie, X) corresponds to the amino acid residue shown in SEQ ID NO: 1 at the corresponding position (according to the numbering of SEQ ID NO: 3).

[0139] Thus, preferably, the present invention relates to an amylase variant comprising an amino acid substitution at amino acid positions N25 and N195 and at one or more positions corresponding to positions selected from the group consisting of: W116, R176, R181, G186, 1206, T225, R320 and Y482 according to the numbering of SEQ ID NO: 3.

[0140] In a preferred embodiment, the present invention relates to an amylase variant comprising amino acid substitutions N25H and N195F and amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of: W116K, R176K, R181T, G186E, I206Y, T225A, R320K and Y482W according to the numbering of SEQ ID NO:3.

[0141] The present invention preferably relates to amylase variants comprising amino acid substitutions at positions 25 and 206 and at one or more positions corresponding to positions selected from the group consisting of: 116, 176, 181, 186, 195, 225, 320 and 482 according to the numbering of SEQ ID NO:3.

[0142] Preferably, the present invention relates to an amylase variant comprising amino acid substitutions X25H and X206Y and one or more amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X225A, X320K and X482W according to the numbering of SEQ ID NO:3.

[0143] Preferably, in this embodiment, the amino acid residue in the parent amylase at the above cited position (ie, X) corresponds to the amino acid residue shown in SEQ ID NO: 1 at the corresponding position (according to the numbering of SEQ ID NO: 3).

[0144] Thus, preferably, the present invention relates to an amylase variant comprising an amino acid substitution at amino acid positions N25 and 1206 and at one or more positions corresponding to positions selected from the group consisting of: W116, R176, R181, G186, N195, T225, R320 and Y482 according to the numbering of SEQ ID NO: 3.

[0145] In a preferred embodiment, the present invention relates to an amylase variant comprising amino acid substitutions N25H and I206Y and amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of: W116K, R176K, R181T, G186E, N195F, T225A, R320K and Y482W according to the numbering of SEQ ID NO:3.

[0146] Preferably, the amylase variant comprises an amino acid substitution at one or more of the amino acid positions (according to the numbering of SEQ ID NO: 3) described below compared to the parent amylase. Preferably, the parent amylase of the amylase variant is an amylase according to any one of SEQ ID NOs: 1, 3, 4 or any one of SEQ ID NOs: 15-41, most preferably the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1. Preferably, the amino acid residue at the referenced position (i.e., X) in the parent amylase corresponds to the amino acid residue shown in SEQ ID NO: 1 at the corresponding position (according to the numbering of SEQ ID NO: 3).

[0147] The amylase variant comprises an amino acid substitution at position 25 (according to the numbering of SEQ ID NO: 3), preferably the substitution X25H. Alternatively, the acid substitution at position 25 is X25Y or X25D.

[0148] Preferably, the amylase variant comprises an amino acid substitution at position 116 (according to the numbering of SEQ ID NO: 3), preferably the substitution X116K.

[0149] Preferably, the amylase variant comprises an amino acid substitution at position 176 (according to the numbering of SEQ ID NO: 3), preferably the substitution X176K.

[0150] Preferably, the amylase variant comprises an amino acid substitution at position 181 (according to the numbering of SEQ ID NO: 3), preferably the substitution X181T.

[0151] Preferably, the amylase variant comprises an amino acid substitution at position 186 (according to the numbering of SEQ ID NO: 3), preferably the substitution X186E.

[0152] Preferably, the amylase variant comprises an amino acid substitution at position 195 (according to the numbering of SEQ ID NO: 3), preferably the substitution X195F.

[0153] Preferably, the amylase variant comprises an amino acid substitution at position 206 (according to the numbering of SEQ ID NO: 3), preferably the substitution X206Y.

[0154] Particularly preferably, the amylase variant comprises an amino acid substitution at position 195 or 206 (according to the numbering of SEQ ID NO: 3), preferably the substitution X195F or X206Y.

[0155] Preferably, the amylase variant comprises an amino acid substitution at position 225 (according to the numbering of SEQ ID NO: 3), preferably the substitution X225A.

[0156] Preferably, the amylase variant comprises an amino acid substitution at position 320 (according to the numbering of SEQ ID NO: 3), preferably the substitution X320K.

[0157] Preferably, the amylase variant comprises an amino acid substitution at position 482 (according to the numbering of SEQ ID NO: 3), preferably the substitution X482W.

[0158] The amylase variants of the present invention comprise

[0159] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3, and

[0160] (ii) amino acid substitutions at one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 176, 181, 186, 195, 206, 225, 320, and 482, according to the numbering of SEQ ID NO: 3.

[0161] In a further preferred embodiment, the amylase variant of the present invention comprises

[0162] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3, and

[0163] (ii) amino acid substitutions at one or more, two or more, three or more, or all of the amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 181, 225 and 320 according to the numbering of SEQ ID NO:3.

[0164] In a further preferred embodiment, the amylase variant of the present invention comprises

[0165] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3, and

[0166] (ii) amino acid substitutions at one or more, two or more, three or more, or all of the amino acid positions corresponding to the amino acid positions selected from the group consisting of: 176 and 186 according to the numbering of SEQ ID NO:3.

[0167] In a preferred embodiment, the amylase variant of the present invention comprises

[0168] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3, and

[0169] (ii) an amino acid substitution at position 195 or 206, preferably at either position 195 or 206, according to the numbering of SEQ ID NO: 3.

[0170] In a preferred embodiment, the amylase variant of the present invention comprises

[0171] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3,

[0172] (ii) an amino acid substitution at position 195 or 206, preferably at either position 195 or 206, and

[0173] (iii) an amino acid substitution at one or more, two or more, three or more, four or more, five or more, six or more, or all of the amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 176, 181, 186, 225, 320 and 482, according to the numbering of SEQ ID NO:3.

[0174] In a further preferred embodiment, the amylase variant of the present invention comprises

[0175] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3, and

[0176] (ii) amino acid substitutions at one or more, two or more, three or more, or all of the amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 181, 225 and 320 according to the numbering of SEQ ID NO:3.

[0177] In a further preferred embodiment, the amylase variant of the present invention comprises

[0178] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3,

[0179] (ii) an amino acid substitution at position 195 or 206, preferably at either position 195 or 206, and

[0180] (iii) amino acid substitutions at one or more, two or more, three or more, or all of the amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 181, 225, and 320 according to the numbering of SEQ ID NO:3.

[0181] In a further preferred embodiment, the amylase variant of the present invention comprises

[0182] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3,

[0183] (ii) an amino acid substitution at position 195 or 206, preferably at either position 195 or 206, and

[0184] (iii) amino acid substitution at one or more, or all, of the amino acid positions corresponding to the amino acid positions selected from the group consisting of: 176 and 186 according to the numbering of SEQ ID NO:3.

[0185] In a further preferred embodiment, the amylase variant of the present invention comprises

[0186] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3,

[0187] (ii) an amino acid substitution at position 195 or 206, preferably at either position 195 or 206,

[0188] (iii) an amino acid substitution at one or more, or all, of the amino acid positions corresponding to the amino acid positions selected from the group consisting of: 176 and 186 according to the numbering of SEQ ID NO: 3, and

[0189] (iv) amino acid substitutions at one or more, two or more, three or more, or all of the amino acid positions corresponding to the amino acid positions selected from the group consisting of 116, 181, 225, and 320 according to the numbering of SEQ ID NO:3.

[0190] In a particularly preferred embodiment, the amylase variants of the present invention comprise

[0191] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3,

[0192] (ii) an amino acid substitution at position 195 or 206, preferably at either position 195 or 206,

[0193] (iii) an amino acid substitution at one or more, or all, of the amino acid positions corresponding to the amino acid positions selected from the group consisting of: 176 and 186 according to the numbering of SEQ ID NO: 3,

[0194] (iv) amino acid substitutions at one or more, two or more, three or more, or all of the amino acid positions corresponding to the amino acid positions selected from the group consisting of 116, 181, 225, and 320 according to the numbering of SEQ ID NO: 3, and

[0195] (v) an amino acid substitution at the amino acid position corresponding to amino acid position 482 according to the numbering of SEQ ID NO: 3

[0196] Preferably, the amylase variants of the invention comprise an amino acid substitution at one of the following combinations of amino acid positions (according to the numbering of SEQ ID NO: 3):

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] From the above table, combinations of amino acid substitutions at amino acid positions containing either position 195 or position 206 are particularly preferred.

[0204] Also from the above table, combinations of amino acid substitutions at amino acid positions containing at least one of the amino acid positions selected from the group consisting of: 116, 181, 225 and 320 are particularly preferred.

[0205] Even more preferred from the above table are combinations of amino acid substitutions at amino acid positions containing either position 195 or position 206 and at least one of the amino acid positions selected from the group consisting of: 116, 181, 225 and 320.

[0206] From the above table, further preferred are combinations of amino acid substitutions at amino acid positions containing either position 195 or position 206, and at least one of positions 176 and 186.

[0207] From the above table, the most preferred is a combination of amino acid substitutions at amino acid positions containing either position 195 or position 206, at least one of positions 176 and 186, and at least one of the amino acid positions selected from the group consisting of: 116, 181, 225 and 320.

[0208] The amylase variants of the present invention comprise

[0209] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3, and

[0210] (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K and X482W according to the numbering of SEQ ID NO:3.

[0211] In a preferred embodiment, the amylase variant of the present invention comprises

[0212] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3,

[0213] (ii) amino acid substitution X195F or X206Y according to the numbering of SEQ ID NO: 3, preferably either X195F or X206Y, and

[0214] (iii) one or more, two or more, three or more, four or more, five or more, six or more, or all amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K and X482W according to the numbering of SEQ ID NO:3.

[0215] In a preferred embodiment, the amylase variant of the present invention comprises

[0216] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3,

[0217] (ii) one or more or all amino acid substitutions selected from the group consisting of: X176K and X186E according to the numbering of SEQ ID NO: 3, or

[0218] (iii) one or more, two or more, three or more, or all amino acid substitutions selected from the group consisting of X116K, X181T, X225A and X320K according to the numbering of SEQ ID NO:3.

[0219] In a preferred embodiment, the amylase variant of the present invention comprises

[0220] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3,

[0221] (ii) amino acid substitution X195F or X206Y, preferably either X195F or X206Y,

[0222] (iii) one or more or all amino acid substitutions selected from the group consisting of: X176K and X186E according to the numbering of SEQ ID NO: 3, and

[0223] (iv) one or more, two or more, three or more, or all amino acid substitutions selected from the group consisting of X116K, X181T, X225A and X320K according to the numbering of SEQ ID NO:3.

[0224] In a preferred embodiment, the amylase variant of the present invention comprises

[0225] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3,

[0226] (ii) amino acid substitution X195F or X206Y according to the numbering of SEQ ID NO: 3, preferably either X195F or X206Y,

[0227] (iii) one or more or all amino acid substitutions selected from the group consisting of: X176K and X186E according to the numbering of SEQ ID NO: 3,

[0228] (iv) one or more, two or more, three or more, or all amino acid substitutions selected from the group consisting of X116K, X181T, X225A, and X320K according to the numbering of SEQ ID NO: 3, and

[0229] (v) Amino acid substitution X482W.

[0230] Preferably, the amylase variant of the present invention comprises one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 3):

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239] Combinations of amino acid substitutions derived from the above table containing one or more, two or more, three or more or all substitutions selected from the group consisting of: X116K, X181T, X225A and X320K are particularly preferred.

[0240] Preferred are amylase variants comprising a combination of mutations selected from the group consisting of:

[0241] X25H+X176K+X186E,

[0242] X25H+X116K+X181T+X225A+X320K,

[0243] X25H+X116K+X176K+X181T+X225A+X320K,

[0244] X25H+X116K+X181T+X186E+X225A+X320K,

[0245] X25H+X116K+X181T+X225A+X320K+X482W,

[0246] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K,

[0247] X25H+X116K+X176K+X181T+X225A+X320K+X482W,

[0248] X25H+X116K+X181T+X186E+X225A+X320K+X482W, and

[0249] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W, preferably further comprising any of substitution X195F or substitution X206Y, all according to the numbering of SEQ ID NO: 3.

[0250] Furthermore, from the above table, a combination of amino acid substitutions containing either substitution X195F or substitution X206Y is particularly preferred.

[0251] Combinations of amino acid substitutions obtained from the above table containing at least one, preferably two, more preferably all, selected from the group consisting of: X176K and X186E are particularly preferred.

[0252] Combinations of amino acid substitutions obtained from the above table, in particular containing one or more, two or more, three or more or all substitutions selected from the group consisting of X116K, X181T, X225A and X320K and either X195F or X206Y, are particularly preferred.

[0253] Preferably, the amylase variant of the present invention comprises one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 3):

[0254]

[0255]

[0256]

[0257] Preferably, the amylase variant of the present invention comprises one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 3):

[0258]

[0259]

[0260] In particular, a combination of amino acid substitutions obtained from the above table containing at least one, at least two, at least three or all amino acid substitutions selected from the group consisting of X116K, X181T, X225A and X320K is particularly preferred.

[0261] Preferably, the amylase variant of the present invention comprises one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 3):

[0262] X25H+X176K+X186E+X195F X25H+X116K+X181T+X195F+X225A+X320K X25H+X116K+X176K+X181T+X195F+X225A+X320K X25H+X116K+X181T+X186E+X195F+X225A+X320K X25H+X116K+X181T+X195F+X225A+X320K+X482W X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W

[0263] Also from the above table, combinations of amino acid substitutions containing at least one amino acid substitution selected from the group consisting of: X176K and X186E are particularly preferred.

[0264] Therefore, preferred, in particular from the above table, are combinations of amino acid substitutions containing the substitution X195F, at least one of the substitutions X176K and X186E and at least one, at least two, at least three or all of the amino acid substitutions selected from the group consisting of: X116K, X181T, X225A and X320K.

[0265] Most preferably, the amylase variant of the present invention comprises the following combination of amino acid substitutions X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W (according to the numbering of SEQ ID NO: 3).

[0266] Preferably, the amylase variant of the present invention comprises one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 3):

[0267] X25H+X176K+X186E+X206Y X25H+X116K+X181T+X206Y+X225A+X320K X25H+X116K+X176K+X181T+X206Y+X225A+X320K X25H+X116K+X181T+X186E+X206Y+X225A+X320K X25H+X116K+X181T+X206Y+X225A+X320K+X482W X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W

[0268] Also from the above table, combinations of amino acid substitutions containing at least one amino acid substitution selected from the group consisting of: X176K and X186E are particularly preferred.

[0269] Therefore, preferred, in particular from the above table, are combinations of amino acid substitutions containing the substitution X206Y, at least one of the substitutions X176K and X186E and at least one, at least two, at least three or all of the amino acid substitutions selected from the group consisting of: X116K, X181T, X225A and X320K.

[0270] Particularly preferably, the amylase variant of the invention comprises the following combination of amino acid substitutions X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W (according to the numbering of SEQ ID NO: 3).

[0271] Preferably, the amylase variant further comprises a deletion at one or more, preferably two or more amino acids corresponding to positions selected from the group consisting of: 181, 182, 183 and 184. Preferably, the amylase variant of the invention having one or more amino acid substitutions as described herein comprises a deletion of one or more amino acids corresponding to positions 183 and 184, preferably a deletion of two amino acids corresponding to positions 183 and 184 (according to the numbering of SEQ ID NO: 3). Preferably, the amylase variant of the invention having one or more amino acid substitutions as described herein comprises a deletion of one or more, preferably two or more, most preferably two amino acids corresponding to positions selected from the group consisting of: R181, G182, D183 and G184, preferably D183* and G184*, wherein the numbering is according to the amino acid sequence shown in SEQ ID NO: 3.

[0272] Preferably, the amylase variant comprises a deletion at two amino acids corresponding to positions selected from the group consisting of: 181, 182, 183 and 184, preferably a deletion corresponding to the amino acids at positions 181 and 182, 182 and 183, or 183 and 184, preferably deletion D183* and G184*, wherein the numbering is according to the amino acid sequence shown in SEQ ID NO:3.

[0273] The amylase variants according to the invention having one or more amino acid substitutions as described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, %, at least 98%, at least 98.5%, at least 99% or at least 99.5% but less than 100% sequence identity.

[0274] Preferably, the amylase variants of the present invention having one or more amino acid substitutions as described herein are preferably SEQ ID NO: 1, 3, 4, or any one of SEQ ID NOs 15-41, preferably SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4, more preferably SEQ ID NO: 1 or 3, most preferably SEQ ID NO: The amino acid sequence shown in NO:1 has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100% sequence identity.

[0275] Preferably, the amylase variants according to the invention having one or more amino acid substitutions as described herein preferably have at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5% but less than 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, 3, 4, or any one of SEQ ID NOs 15-41, preferably SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4, more preferably SEQ ID NO: 1 or 3, most preferably SEQ ID NO: 1.

[0276] In one embodiment, the amylase variants according to the present invention having one or more amino acid substitutions as described herein are preferably identical to SEQ ID The amino acid sequence shown in NO:1 has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity.

[0277] In another embodiment, the amylase variants according to the present invention having one or more amino acid substitutions as described herein are preferably identical to SEQ ID The amino acid sequence shown in NO:3 has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity.

[0278] In another embodiment, the amylase variants according to the present invention having one or more amino acid substitutions as described herein are preferably identical to SEQ ID The amino acid sequence shown in NO:4 has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity.

[0279] In another embodiment, the amylase variants according to the invention having one or more amino acid substitutions described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:15.

[0280] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0281] In another embodiment, the amylase variants according to the invention having one or more amino acid substitutions described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 17.

[0282] In another embodiment, the amylase variants according to the invention having one or more amino acid substitutions described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:18.

[0283] In another embodiment, the amylase variants according to the invention having one or more amino acid substitutions described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 19.

[0284] In another embodiment, the amylase variants according to the invention having one or more amino acid substitutions described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:20.

[0285] In another embodiment, the amylase variants according to the invention having one or more amino acid substitutions described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:21.

[0286] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:22.

[0287] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:23.

[0288] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:24.

[0289] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:25.

[0290] In another embodiment, the amylase variants according to the invention having one or more amino acid substitutions described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:26.

[0291] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:27.

[0292] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:28.

[0293] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:29.

[0294] In another embodiment, the amylase variants according to the invention having one or more amino acid substitutions described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:30.

[0295] In another embodiment, the amylase variants according to the invention having one or more amino acid substitutions described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:31.

[0296] In another embodiment, the amylase variants according to the invention having one or more amino acid substitutions described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:32.

[0297] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:33.

[0298] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:34.

[0299] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:35.

[0300] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:36.

[0301] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:37.

[0302] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:38.

[0303] In another embodiment, an amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:39.

[0304] In another embodiment, the amylase variants according to the invention having one or more amino acid substitutions described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:40.

[0305] In another embodiment, the amylase variants according to the invention having one or more amino acid substitutions described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:41.

[0306] Preferably, the amylase variants according to the invention having one or more amino acid substitutions as described herein are preferably identical to SEQ ID The amino acid sequence shown in NO:4 has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity.

[0307] Preferably, the amylase variants according to the invention having one or more amino acid substitutions as described herein are preferably identical to SEQ ID The amino acid sequence shown in NO:3 has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity.

[0308] More preferably, the amylase variants according to the invention having one or more amino acid substitutions as described herein are preferably identical to SEQ ID The amino acid sequence shown in NO:1 has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity.

[0309] The amylase variants according to the invention having amylase activity preferably have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity or sequence similarity, preferably sequence identity, to the amino acid sequence shown in SEQ ID NO:1.

[0310] Preferably, the amylase variant of the present invention having amylase activity comprises:

[0311] (a) an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity to SEQ ID NO:1;

[0312] (b) an amino acid sequence encoded by a polynucleotide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity to SEQ ID NO:2,

[0313] (c) an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions to the complementary sequence of:

[0314] (i) the coding sequence of SEQ ID NO: 1; or

[0315] (ii) the polynucleotide shown in SEQ ID NO: 2;

[0316] (d) an amino acid sequence encoded by a polynucleotide having at least 95% but less than 100% sequence identity to SEQ ID NO: 2, wherein the polynucleotide further differs from SEQ ID NO: 2 only due to the degeneracy of the genetic code, or

[0317] (e) A fragment of (a), (b), (c) or (d) having amylase activity.

[0318] Preferably, the amylase variants according to the invention having amylase activity have at least 91.0%, at least 91.5%, at least 92.0%, at least 92.5%, at least 93.0%, at least 93.5%, 94.0%, at least 94.5%, at least 95.0%, at least 95.5%, at least 96.0%, at least 96.5%, at least 97.0%, at least 97.5%, at least 98.0%, at least 98.5%, at least 99.0%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% but less than 100% sequence identity or sequence similarity, preferably sequence identity, to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4. Particularly preferably, the amylase variants according to the present invention having amylase activity have at least 91.0%, at least 91.5%, at least 92.0%, at least 92.5%, at least 93.0%, at least 93.5%, 94.0%, at least 94.5%, at least 95.0%, at least 95.5%, at least 96.0%, at least 96.5%, at least 97.0%, at least 97.5%, at least 98.0%, at least 98.5%, at least 99.0%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. More preferably, the amylase variants according to the present invention having amylase activity preferably have at least 95.0% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0319] The structure of alpha-amylase comprises three different domains A, B and C, see, e.g., Machius et al., 1995, J. Mol. Biol. [Journal of Molecular Biology] 246: 545-559. The alpha-amylase variants described herein may further comprise one or more non-catalytic CBMs (carbohydrate binding modules, also referred to as carbohydrate binding domains or specifically for amylase starch binding domains). CBMs can improve the association of enzymes with substrates. CBMs are attached to the C domain. Preferably, the amylase of the present invention does not comprise a carbohydrate binding domain. Preferably, the alpha-amylase variant of the present invention consists only of three domains, A, B and C domains.

[0320] As used herein, the "A and B domains" or "AB domains" of alpha-amylase correspond to the amino acids aligned with amino acids 1-399 of SEQ ID NO: 3. As used herein, the "C domain" of alpha-amylase corresponds to the amino acids aligned with amino acids 400-485 of SEQ ID NO: 3.

[0321] Preferably, the amylase variants described above comprising one or more amino acid changes, preferably insertions, deletions, substitutions or combinations thereof, preferably substitutions are hybrid amylases comprising domains thereof, particularly AB domains thereof and C domains thereof, from different parent amylases. Amylase variants can be produced by replacing the C domain or a portion thereof of another amylase with the C domain of an amylase or a portion thereof. Preferably, the A and B domains of the amylase variants described herein have at least 75% identity with the A and B domains of the amylase of SEQ ID NO: 3, e.g., at least 78%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% identity.

[0322] Preferably, the A and B domains of the amylase variants described herein are identical to those of SEQ ID NO:6 has at least 75% identity, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identity, meaning that the amino acid sequence forming the A and B domains is identical to SEQ ID NO:6 has at least 75% identity, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity.

[0323] Preferably, the C domain of the amylase variant described herein comprises %, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identical C domains.

[0324] Preferably, the C domain of the amylase variants described herein is at least 75% identical to SEQ ID NO:8, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identical.

[0325] In one embodiment of the present invention, the amino acid sequences forming the A and B domains are similar to those of SEQ ID NO:6 has at least 75% identity, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identity, and the amino acid sequence forming the C domain is identical to SEQ ID NO:8 has at least 75% identity, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identity.

[0326] In one embodiment of the present invention, the amino acid sequence forming the A and B domains has 100% identity with the amino acid sequence of SEQ ID NO: 6, and the amino acid sequence forming the C domain has 100% identity with the amino acid sequence of SEQ ID NO: NO:8 has at least 75% identity, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identity.

[0327] In one embodiment of the present invention, the amino acid sequences forming the A and B domains are similar to those of SEQ ID NO:6 has at least 75% identity, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identity, and the amino acid sequence forming the C domain is identical to SEQ ID NO:8 has 100% identity.

[0328] In one embodiment, the present invention relates to a method for preparing an amylase variant, the method comprising the step of preparing a hybrid from at least two different amylases, wherein the hybrid comprises A and B domains and a C domain, and wherein the amino acid sequences of the A and B domains are the same as those of SEQ ID NO:6 is at least 75%, preferably at least 78%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identical to the amino acid sequence of SEQ ID %, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100% identical to the amino acid sequence of NO:8, and one or more amino acid changes as described herein are introduced into the hybrid.

[0329] The amylase variants according to the invention having amylase activity preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92.5%, at least 93.5%, at least 94.5%, at least 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 99.6%, at least 100.6%, at least 101.5%, at least 102.5%, at least 103.5%, at least 104.5%, at least 105.5%, at least 106.5%, at least 107.5%, at least 108.5%, at least 109.5%, at least 110.5%, at least 111.5%, at least 112.5%, at least 113.5%, at least 114.5%, at least 115.5%, at least 116.5%, at least 117.5%, at least 118.5%, at least 119.5%, at least 119.5%, at least at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity and comprises A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are the same as those of SEQ ID NO:6 is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identical to the amino acid sequence of SEQ ID NO:6 and the amino acid sequence of the C domain is identical to SEQ ID 8. The amino acid sequence of NO:8 is at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% identical.

[0330] In one embodiment, the amylase variant according to the present invention having amylase activity comprises or consists of: any one of SEQ ID NO: 1, 3, 4 or any one of SEQ ID NO: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably the amino acid sequence shown in SEQ ID NO: 1, with one or more amino acid changes cited herein.

[0331] Preferably, the amylase variant according to the invention having amylase activity comprises or consists of: any one of SEQ ID NO: 1, 3, 4 or any one of SEQ ID NO: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably the amino acid sequence shown in SEQ ID NO: 1, having an amino acid substitution X25H and, preferably any one of amino acid substitutions X195F or X206Y (according to the numbering of SEQ ID NO: 3), and having 1 to 7, preferably 2 to 7 or 3 to 7, such as 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions selected from the group consisting of: X116K, X176K, X181T, X186E, X225A, X320K and X482W (according to the numbering of SEQ ID NO: 3), preferably comprising a deletion at one or more, preferably two or more amino acids corresponding to the positions selected from the group consisting of: 181, 182, 183 and 184 (according to the numbering of SEQ ID NO:3), as described herein.

[0332] In another embodiment, the amylase variant according to the present invention having amylase activity comprises or consists of: any one of SEQ ID NO: 1, 3, 4 or any one of SEQ ID NO: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: The amino acid sequence shown in NO:1 has one or more amino acid changes as described above and further comprises 1 to 50, preferably 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 8, or 2 to 5, preferably 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 8, or 3 to 5, preferably 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10 or 4 to 8 conservative amino acid exchanges, preferably including a deletion at one or more, preferably two or more amino acids corresponding to the positions selected from the group consisting of: 181, 182, 183 and 184 (according to the numbering of SEQ ID NO:3), as described herein.

[0333] Preferably, the amylase variant according to the present invention having amylase activity comprises or consists of: any one of SEQ ID NO: 1, 3, 4 or any one of SEQ ID NO: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably the amino acid sequence shown in SEQ ID NO: 1, having an amino acid substitution X25H and, preferably any one of amino acid substitutions X195F or X206Y (according to the numbering of SEQ ID NO: 3), and having 1 to 7, preferably 2 to 7 or 3 to 7, such as 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions selected from the group consisting of: X116K, X176K, X181T, X186E, X225A, X320K and X482W (according to the numbering of SEQ ID NO: 3). NO:3), and further comprises 1 to 50, preferably 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 8, or 2 to 5, preferably 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 8, or 3 to 5, preferably 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10 or 4 to 8 conservative amino acid exchanges, preferably including deletions at one or more, preferably two or more amino acids corresponding to positions selected from the group consisting of: 181, 182, 183 and 184 (according to the numbering of SEQ ID NO:3), as described herein.

[0334] Preferably, the amylase variant according to the present invention having amylase activity comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, having amino acid substitution X25H and, preferably any of amino acid substitutions X195F or X206Y (according to the numbering of SEQ ID NO: 3), and having 1 to 7, preferably 2 to 7 or 3 to 7, such as 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions selected from the group consisting of: X116K, X176K, X181T, X186E, X225A, X320K and X482W (according to the numbering of SEQ ID NO: 3), and further comprising 1 to 10, preferably 1 to 5 conservative amino acid exchanges.

[0335] Conservative amino acid substitutions may occur over the entire length of the amylase variant sequence. In one embodiment, such mutations do not belong to a functional domain of the amylase variant. In one embodiment, conservative mutations do not belong to the catalytic center of the amylase variant.

[0336] In one embodiment, the amylase variants of the invention exhibit one or more improved properties, preferably compared to a parent amylase, preferably compared to an amylase as shown in SEQ ID NO: 1, 3, 4, or any one of SEQ ID NOs 15-41, preferably SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4, more preferably SEQ ID NO: 1 or 3, most preferably SEQ ID NO: 1.

[0337] Preferably, the improved properties are expressed as an improvement factor (IF) > 1.0. Preferably, for improved thermal stability and improved washing performance, the improvement is expressed as an improvement factor. Preferably, the improvement factor is equal to or greater than 1.1, equal to or greater than 1.2, equal to or greater than 1.3, equal to or greater than 1.4, equal to or greater than 1.5, equal to or greater than 1.6, equal to or greater than 1.7, equal to or greater than 1.8, equal to or greater than 1.9 or equal to or greater than 2.0. Preferably, the IF for washing performance is equal to or greater than 1.1, equal to or greater than 1.2 or equal to or greater than 1.3. Preferably, the IF for thermal stability is equal to or greater than 1.1, equal to or greater than 1.2, equal to or greater than 1.3, equal to or greater than 1.5 or equal to or greater than 2.0.

[0338] Alternatively, the improvement in an amylase property is expressed as a percentage improvement compared to a parent amylase. Preferably, the amylase variants of the invention exhibit at least 0.5%, at least 1%, at least 2%, at least 3%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190% or at least 200% improved properties compared to a parent amylase, preferably compared to an amylase as shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1.

[0339] Alternatively, in particular for stability improvement, the improvement in the amylase property is expressed as residual activity after a stability challenge. Preferably, the storage stability is expressed as residual activity after storage under corresponding storage conditions, preferably in a detergent composition, preferably in a laundry or dishwashing detergent, preferably a laundry detergent. Preferably, the residual activity of the amylase variant is increased compared to the parent amylase. Preferably, the residual activity of the amylase variant is at least 0.5%, at least 1%, at least 2%, at least 3%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190% or at least 200% improved compared to the parent amylase, preferably compared to the amylase as shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1. The residual activity compared to the parent amylase can also be converted into an improvement factor by forming a ratio of the residual activity of the variant compared to the residual activity of the parent amylase.

[0340] Preferably, the improved properties are one or more properties selected from the group consisting of: expression, activity, stability, thermostability, specific activity, substrate specificity, pH-dependent activity, pH stability, oxidative stability, catalytic efficiency, catalytic rate, chemical stability, pH activity, stability under storage conditions, substrate binding, substrate cleavage, substrate stability, surface properties, thermal activity, Ca2+ dependence, performance in detergent, performance in laundry detergent, performance in ADW detergent. Preferably, the improved activity is improved specific activity, substrate specificity, pH-dependent activity, catalytic efficiency, catalytic rate, pH activity, substrate binding, substrate cleavage, thermal activity, Ca2+ dependence, wash performance, wash performance of laundry detergent and / or wash performance of ADW detergent, wash performance at low temperatures (preferably below 40°C, more preferably below 30°C, even more preferably below 25°C). Preferably, the improved stability is improved thermal stability, thermal stability in a detergent composition (preferably in a laundry or dishwashing detergent composition, preferably a laundry detergent composition), pH stability, oxidative stability, chemical stability, stability under storage conditions, substrate stability and / or thermal activity. Preferably, the improved properties are improved expression, improved solubility, improved thermal stability, improved thermal stability in a detergent composition, improved stability under storage in a detergent composition and / or improved washing performance. Preferably, the improved properties are improved thermal stability, preferably improved thermal stability in a detergent composition, improved stability under storage in a detergent composition and / or improved washing performance. Preferably, the improved property is improved thermostability, improved thermostability in detergent compositions, improved stability under storage conditions, preferably improved stability in detergent compositions under storage, preferably improved stability in laundry detergents under storage and / or improved stability in ADW detergents under storage, improved wash performance, preferably improved wash performance of laundry detergents, improved wash performance of ADW detergents, and / or improved wash performance at low temperatures, preferably below 40°C, more preferably below 30°C, even more preferably below 25°C. Preferably, the improved property is improved storage stability and / or improved wash performance. Most preferably, the improved property is improved storage stability, preferably improved stability in detergent compositions, preferably compared to an amylase as shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1 or relative to an amylase as shown in SEQ ID NO: 33.

[0341] Preferably, the present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0342] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3,

[0343] (ii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 176, 181, 186, 195, 206, 225, 320 and 482 according to the numbering of SEQ ID NO: 3,

[0344] (iii) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1, 3, 4 or to any one of SEQ ID NO: 15-41, preferably to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, and

[0345] (iv) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 3), preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3).

[0346] Preferably, the present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0347] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3,

[0348] (ii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 176, 181, 186, 195, 206, 225, 320 and 482 according to the numbering of SEQ ID NO: 3,

[0349] (iii) having at least 80% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1, 3, 4 or to any one of SEQ ID NO: 15-41, preferably to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, and

[0350] (iv) the amylase variant of the present invention comprises a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 3), preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3).

[0351] Preferably, the amylase variant of the present invention comprises

[0352] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3,

[0353] (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K and X482W according to the numbering of SEQ ID NO: 3,

[0354] (iii) with SEQ ID NO: 1, 3, 4 or with any one of SEQ ID NO: 15-41, preferably with SEQ ID NO: 1 or SEQ ID NO: 3, preferably with SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity, and

[0355] (iv) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 3), preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3).

[0356] Preferably, the amylase variant of the present invention comprises

[0357] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3,

[0358] (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K and X482W according to the numbering of SEQ ID NO: 3,

[0359] (iii) with SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity, and

[0360] (iv) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 3), preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3).

[0361] Preferably, the present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0362] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3,

[0363] (ii) an amino acid substitution at either position 195 or 206 according to the numbering of SEQ ID NO: 3,

[0364] (iii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 176, 181, 186, 225, 320 and 482 according to the numbering of SEQ ID NO: 3,

[0365] (iv) has at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1, 3, 4 or to any one of SEQ ID NO: 15-41, preferably to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, and

[0366] (v) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 3), preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3).

[0367] Preferably, the present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0368] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3,

[0369] (ii) any one of the amino acid substitutions X195F or X206Y according to the numbering of SEQ ID NO: 3,

[0370] (iii) one or more, two or more, three or more, four or more, five or more, six or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3,

[0371] (iv) has at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1, 3, 4 or to any one of SEQ ID NO: 15-41, preferably to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, and

[0372] (v) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 3), preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3).

[0373] Preferably, the present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0374] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3,

[0375] (ii) any one of the amino acid substitutions X195F or X206Y according to the numbering of SEQ ID NO: 3,

[0376] (iii) one or more, two or more, three or more, four or more, five or more, six or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3,

[0377] (iv) has at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1, and

[0378] (v) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 3), preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3).

[0379] Preferably, the amylase variant of the present invention comprises

[0380] (i) a combination of amino acid substitutions (according to the numbering of SEQ ID NO: 3) selected from the group consisting of:

[0381] X25H+X116K+X181T+X206Y+X225A+X320K,

[0382] X25H+X116K+X176K+X181T+X206Y+X225A+X320K,

[0383] X25H+X116K+X181T+X186E+X206Y+X225A+X320K,

[0384] X25H+X116K+X181T+X206Y+X225A+X320K+X482W,

[0385] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K,

[0386] X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W,

[0387] X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W,

[0388] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W,

[0389] X25H+X116K+X181T+X195F+X225A+X320K,

[0390] X25H+X116K+X176K+X181T+X195F+X225A+X320K,

[0391] X25H+X116K+X181T+X186E+X195F+X225A+X320K,

[0392] X25H+X116K+X181T+X195F+X225A+X320K+X482W,

[0393] X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K,

[0394] X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W,

[0395] X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W, and

[0396] X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W,

[0397] (ii) with SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity, and

[0398] (iii) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 3), preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3).

[0399] Preferably, the amylase variant of the present invention comprises

[0400] (i) a combination of amino acid substitutions (according to the numbering of SEQ ID NO: 3) selected from the group consisting of:

[0401] X25H+X116K+X181T+X195F+X225A+X320K,

[0402] X25H+X116K+X176K+X181T+X195F+X225A+X320K,

[0403] X25H+X116K+X181T+X186E+X195F+X225A+X320K,

[0404] X25H+X116K+X181T+X195F+X225A+X320K+X482W,

[0405] X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K,

[0406] X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W,

[0407] X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W, and

[0408] X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W,

[0409] (ii) with SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity, and

[0410] (iii) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 3), preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3).

[0411] Preferably, the amylase variant of the present invention comprises

[0412] (i) a combination of amino acid substitutions (according to the numbering of SEQ ID NO: 3) selected from the group consisting of:

[0413] X25H+X116K+X181T+X206Y+X225A+X320K,

[0414] X25H+X116K+X176K+X181T+X206Y+X225A+X320K,

[0415] X25H+X116K+X181T+X186E+X206Y+X225A+X320K,

[0416] X25H+X116K+X181T+X206Y+X225A+X320K+X482W,

[0417] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K,

[0418] X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W,

[0419] X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W, and

[0420] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W,

[0421] (ii) with SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity, and

[0422] (iii) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 3), preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3).

[0423] Preferably, the amylase variant of the present invention comprises

[0424] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3,

[0425] (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K and X482W according to the numbering of SEQ ID NO: 3,

[0426] (iii) with SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity,

[0427] (iv) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (according to the numbering of SEQ ID NO: 3), preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3), and

[0428] (v) wherein the amylase variant exhibits one or more improved properties, preferably relative to said parent amylase, preferably relative to the parent amylase shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, or relative to the amylase shown in SEQ ID NO: 33, preferably the amylase variant has increased stability, thermostability, storage stability, storage stability in detergent compositions, wash performance, wash performance in laundry detergents, and / or wash performance in dishwashing detergents, preferably the improved property is improved storage stability and / or wash performance, preferably improved wash performance on laundry, preferably the improved property is improved storage stability, preferably wherein said improved property is expressed as an improvement factor (IF) > 1.0, and wherein preferably the improvement factor is equal to or greater than 1.1, preferably, equal to or greater than 1.2, more preferably, equal to or greater than 1.3, preferably the amylase variant exhibits improved storage stability in detergent compositions, preferably relative to a reference amylase, preferably relative to said parent amylase, preferably relative to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 33. NO: 1, or relative to the amylase shown in SEQ ID NO: 33, preferably having an improvement factor equal to or greater than 1.1, preferably, equal to or greater than 1.2, more preferably, equal to or greater than 1.3, preferably after storage at 40°C in a detergent composition, preferably for 14 days, 24 days, 56 days or 84 days, preferably for 84 days, preferably as determined in an ES1-C detergent as described herein, preferably containing an additional builder (preferably HEDP), preferably relative to a reference amylase, preferably relative to said parent amylase, preferably relative to SEQ ID NO: 1 or SEQ ID NO: 3, preferably the parent amylase shown in SEQ ID NO: 1, or relative to the amylase shown in SEQ ID NO: 33; and / or preferably, the amylase variant exhibits improved washing performance on laundry, preferably at 40°C, preferably relative to an amylase having the amino acid sequence shown in SEQ ID NO: 33.

[0429] Preferably, the amylase variant of the present invention comprises

[0430] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3,

[0431] (ii) any one of the amino acid substitutions X195F or X206Y according to the numbering of SEQ ID NO: 3,

[0432] (iii) one or more, two or more, three or more, four or more, five or more, six or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3,

[0433] (iv) with SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity,

[0434] (v) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (according to the numbering of SEQ ID NO: 3), preferably a deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3), and

[0435] (vi) wherein the amylase variant exhibits one or more improved properties, preferably relative to said parent amylase, preferably relative to the parent amylase shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, or relative to the amylase shown in SEQ ID NO: 33, preferably the amylase variant has increased stability, thermostability, storage stability, storage stability in detergent compositions, wash performance, wash performance in laundry detergents, and / or wash performance in dishwashing detergents, preferably the improved property is improved storage stability and / or wash performance, preferably improved wash performance on laundry, preferably the improved property is improved storage stability, preferably wherein said improved property is expressed as an improvement factor (IF) > 1.0, and wherein preferably the improvement factor is equal to or greater than 1.1, preferably, equal to or greater than 1.2, more preferably, equal to or greater than 1.3, preferably the amylase variant exhibits improved storage stability in detergent compositions, preferably relative to a reference amylase, preferably relative to said parent amylase, preferably relative to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 33. NO: 1, or relative to the amylase shown in SEQ ID NO: 33, preferably having an improvement factor equal to or greater than 1.1, preferably, equal to or greater than 1.2, more preferably, equal to or greater than 1.3, preferably after storage at 40°C in a detergent composition, preferably for 14 days, 24 days, 56 days or 84 days, preferably for 84 days, preferably as determined in an ES1-C detergent as described herein, preferably containing an additional builder (preferably HEDP), preferably relative to a reference amylase, preferably relative to said parent amylase, preferably relative to SEQ ID NO: 1 or SEQ ID NO: 3, preferably the parent amylase shown in SEQ ID NO: 1, or relative to the amylase shown in SEQ ID NO: 33; and / or preferably, the amylase variant exhibits improved washing performance on laundry, preferably at 40°C, preferably relative to an amylase having the amino acid sequence shown in SEQ ID NO: 33.

[0436] Particularly preferably, the amylase variant of the present invention comprises

[0437] (i) a combination of amino acid substitutions (according to the numbering of SEQ ID NO: 3) selected from the group consisting of:

[0438] X25H+X116K+X181T+X206Y+X225A+X320K,

[0439] X25H+X116K+X176K+X181T+X206Y+X225A+X320K,

[0440] X25H + X116K + X181T + X186E + X206Y + X225A + X320K,

[0441] X25H + X116K + X181T + X206Y + X225A + X320K + X482W,

[0442] X25H + X116K + X176K + X181T + X186E + X206Y + X225A + X320K,

[0443] X25H + X116K + X176K + X181T + X206Y + X225A + X320K + X482W,

[0444] X25H + X116K + X181T + X186E + X206Y + X225A + X320K + X482W,

[0445] X25H + X116K + X176K + X181T + X186E + X206Y + X225A + X320K + X482W,

[0446] X25H + X116K + X181T + X195F + X225A + X320K,

[0447] X25H + X116K + X176K + X181T + X195F + X225A + X320K,

[0448] X25H + X116K + X181T + X186E + X195F + X225A + X320K,

[0449] X25H + X116K + X181T + X195F + X225A + X320K + X482W,

[0450] X25H + X116K + X176K + X181T + X186E + X195F + X225A + X320K,

[0451] X25H + X116K + X176K + X181T + X195F + X225A + X320K + X482W,

[0452] X25H + X116K + X181T + X186E + X195F + X225A + X320K + X482W, and

[0453] X25H + X116K + X176K + X181T + X186E + X195F + X225A + X320K + X482W,

[0454] (ii) has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity to SEQ ID NO: 1, and

[0455] (iii) a deletion of two amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of the amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3).

[0456] Particularly preferred are amylase variants comprising

[0457] (i) Amino acid substitutions (according to the numbering of SEQ ID NO: 3) combination:

[0458] X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K, or X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W

[0459] (ii) has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity to SEQ ID NO: 1, and

[0460] (iii) a deletion of two amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of the amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 3).

[0461] Most preferred are amylase variants comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, having amino acid changes X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+D183*+G184* or X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W (according to the numbering of SEQ ID NO: 3).

[0462] Most preferred is an amylase variant comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, having amino acid changes X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W (according to the numbering of SEQ ID NO: 3).

[0463] Nucleic acid construct

[0464] The present invention also relates to polynucleotides encoding the amylase variants of the present invention.Preferably, the polynucleotide is a codon-optimized polynucleotide for improved expression in a specific host cell, preferably a Bacillus cell.

[0465] Therefore, the present invention also relates to a nucleic acid, preferably an isolated, synthetic and / or recombinant nucleic acid, comprising:

[0466] (a) having a difference of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92.5%, at least 93.5%, at least 94.5%, at least 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 99.5%, at least 100.5%, at least 101.5%, at least 102.5%, at least 103.5%, at least 104.5%, at least 105.5%, at least 106.5%, at least 107.5%, at least 108.5%, at least 109.5%, at least 111.5%, at least 112.5%, at least 113.5%, at least 114.5%, at least 115.5%, at least 116.5%, at least 117.5%, at least 118.5%, at least 119.5%, at least 119.5 %, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% identical to a nucleic acid sequence, wherein the nucleic acid encodes an amylase variant having amylase activity as described herein;

[0467] (b) a nucleic acid sequence encoding a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92.5%, at least 93.5%, at least 94.5%, at least 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 99.6%, at least 100. .5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least to 99.5% but less than 100% identity, wherein the nucleic acid encodes an amylase variant having amylase activity as described herein;

[0468] (c) a polynucleotide that hybridizes under high stringency conditions, preferably under very high stringency conditions, to the complementary sequence of:

[0469] (i) the coding sequence of SEQ ID NO: 1 or the coding sequence of any amylase variant described herein having amylase activity; or

[0470] (ii) the polynucleotide shown in SEQ ID NO: 2;

[0471] (d) a fragment of (a), (b) or (c), wherein the fragment encodes a polypeptide having amylase activity; or

[0472] (e) A nucleic acid sequence that is completely complementary to any one of (a) to (d).

[0473] (f) A polynucleotide that differs from any of the nucleic acid sequences described in (a) to (e) only due to the degeneracy of the genetic code.

[0474] The present invention also relates to a nucleic acid construct (preferably an expression cassette) comprising a polynucleotide as described herein.

[0475] Typically, an expression cassette comprises three elements: a promoter sequence, an open reading frame, and a 3' untranslated region, which in eukaryotes usually contains a polyadenylation site. Additional regulatory elements may include transcriptional and translational enhancers. Intron sequences may also be added to the 5' untranslated region (UTR) or the coding sequence to increase the amount of the mature messenger accumulated in the cytosol. An expression cassette may be a part of a vector or may be integrated into the genome of a host cell and replicated with the genome of its host cell. An expression cassette is usually capable of increasing or decreasing expression.

[0476] The present invention also relates to an expression vector comprising a polynucleotide or nucleic acid construct as described herein.The expression vector may be a low copy number vector or a high copy number vector.

[0477] After being transformed into host cell or host cell organelle, carrier as used herein can provide the section for transcription and translation of foreign polynucleotide.This type of other section can include regulating nucleotide sequence, maintaining and / or duplicating required one or more replication origins, one or more selectable markers, polyadenylation signal, suitable site for insertion of foreign coding sequence, such as multiple cloning site in specific cell type.An example is when needing carrier as free genetic element (such as plasmid or cosmid molecule) to be maintained in bacterial cell.The limiting examples of suitable replication origin include f1-ori and colE1.

[0478] A vector may replicate without integrating into the host cell genome, for example, as a plasmid in a bacterial host cell, or it may integrate part or all of its DNA into the host cell's genome and thereby result in replication and expression of its DNA.

[0479] The polynucleotide encoding the amylase variant can be introduced into the vector by standard recombinant DNA techniques. Once introduced into the vector, the polynucleotide comprising the coding sequence can be suitable for introduction (transformation, transduction, transfection, etc.) into a host cell or a host cell organelle. A cloning vector suitable for expression of the polynucleotide sequence in a host cell or a host cell organelle can be selected.

[0480] Host cells

[0481] The present invention also relates to a host cell comprising a polynucleotide encoding an amylase variant as described herein, a nucleic acid construct as described herein or an expression vector as described herein. In one embodiment of the present invention, the vector is used for transformation of a host cell.

[0482] Polynucleotides encoding amylase variants as described herein can be transiently or stably introduced into host cells and can remain non-integrated, for example as a plasmid. Typically, stable transformation is due to the integration of nucleic acids comprising foreign coding sequences into chromosomes or as episomes (independent nuclear DNA fragments). Typically, transient transformation is due to the non-integration of nucleic acids comprising foreign nucleic acid sequences into chromosomes or as episomes. Alternatively, polynucleotides encoding amylase variants as described herein can be integrated into the host genome.

[0483] Introduction of nucleic acids into host cells can be achieved, for example, but not limited to, by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 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, Journal of Bacteriology 169: 5271-5278). Specific transformation protocols for each type of host cell are known in the art (see, eg, for transformation of E. coli protoplasts, see Hanahan, 1983, J. Mol. Biol. 166:557-580).

[0484] Various host cells can be used to express the nucleic acid constructs described herein. Host cells comprising the genetic constructs described herein can be obtained by one of the methods described herein for introducing polynucleotides into such host cells. The host cells of the present invention do not naturally express the amylase variants. Therefore, the host cell is a recombinant host cell; the nucleic acid constructs described herein are heterologous to the host cell.

[0485] In one embodiment, the host cell is a prokaryotic organism or a eukaryotic organism. In another embodiment, the host cell is a bacterium, an archaea, a fungal cell, a yeast cell, or a eukaryotic cell. In another embodiment, the host cell is a non-human host cell.

[0486] In one embodiment, the host cell is a bacterial cell. The bacterial host cell can be any gram-positive bacteria or gram-negative bacteria. Gram-positive bacteria include but are not limited to Bacillus, Brevibacterium, Corynebacterium, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus and Oceanobacillus. Gram-negative bacteria include, but are not limited to, Escherichia, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Acetobacter, Flavobacterium, Fusobacterium, Gluconobacter. In particular embodiments, the bacterial host cell is an Escherichia coli cell. In one embodiment, the host cell is a bacterial cell. In particular embodiments, the host cell is an Escherichia or Bacillus.

[0487] Preferably, the bacterial host cell is a Bacillus cell.The bacterial host cell may be any Bacillus cell. Bacillus cells useful in the practice of the present invention include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus methylotrophicus, Bacillus cereus, Bacillus paralicheniformis, Bacillus thunbergii ... paralicheniformis), Bacillus subtilis, and Bacillus thuringiensis cells. In one embodiment, the bacterial host cell is a Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell. In a preferred embodiment, the bacterial host cell is a Bacillus licheniformis cell, Bacillus pumilus, or Bacillus subtilis cell. Preferably, the bacterial host cell is a Bacillus licheniformis cell.

[0488] Alternatively, the bacterial host cell can be Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus bulgaricusk, Lactobacillus reuteri, Escherichia coli, Staphylococcus aureus, Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoacidophilum, Corynebacterium callunae, Corynebacterium ammoniagenes, Corynebacterium thermoammoniagenes, thermoaminogenes), Corynebacterium melassecola, Corynebacterium effiziens, Corynebacterium efficiens, Corynebacterium deserti, Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divarecatum, Pseudomonas putida, Pseudomonas syringae, Streptomyces coelicolor, Streptomyces lividans, Streptomyces albus, Streptomyces avermitilis, Gluconobacter oxydans, Gluconobacter moby morbifer), Gluconobacter thailandicus, Acetobacter acetiaceti), Clostridium acetobutylicum, Clostridium saccharobutylicum, Clostridium beijerinckii, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, Streptococcus equi subsp., Zooepidemicus or Basfia succiniciproducens.

[0489] Alternative additional host cells include, but are not limited to, Aspergillus niger, Aspergillus oryzae, Hansenula polymorpha, Thermomyces lanuginosus, fusarium oxysporum, Fusarium heterosporum, Pichia pastoris (also known as Komagataella phaffii), Myceliopthora thermophile, Themothelomyces thermophila, Schizosaccharomyces pombe, Trichoderma (preferably Trichoderma reesei), and Saccharomyces (preferably Saccharomyces cerevisiae), or Rhizomucor.

[0490] In another embodiment, the bacterial host cell may additionally contain modifications (eg, deletions or disruptions) of other genes that may be detrimental to the production, recovery or use of the polypeptide of interest.

[0491] Preparation method

[0492] Another embodiment of the present invention is a method for obtaining an amylase variant of a parent amylase, the method comprising the steps of:

[0493] a) introducing into a parent amylase, preferably into any one of SEQ ID NOs: 1, 3, 4 or any one of SEQ ID NOs: 15-41, preferably into SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1,

[0494] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3,

[0495] (ii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 176, 181, 186, 195, 206, 225, 320 and 482 according to the numbering of SEQ ID NO: 3,

[0496] b) introducing deletions at two or more positions in the parent amylase, preferably selected from positions 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 3;

[0497] thereby providing an amylase variant of the parent amylase,

[0498] wherein the variant has at least 60% but less than 100% sequence identity with the amino acid sequence of SEQ ID NO: 1, 3, 4 or with any one of SEQ ID NO: 15-41, preferably with the polypeptide of SEQ ID NO: 1, and wherein the amylase variant has amylase activity, and preferably wherein the amylase variant has improved properties, preferably relative to the parent.

[0499] A preferred embodiment of the present invention is a method for obtaining an amylase variant of a parent amylase, the method comprising the following steps:

[0500] a) into a parent amylase, preferably into SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1,

[0501] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3,

[0502] (ii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 176, 181, 186, 195, 206, 225, 320 and 482 according to the numbering of SEQ ID NO: 3,

[0503] b) introducing deletions at two or more positions in the parent amylase, preferably selected from positions 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 3;

[0504] thereby providing an amylase variant of the parent amylase,

[0505] wherein the variant has at least 80% but less than 100% sequence identity with the amino acid sequence of the polypeptide of SEQ ID NO: 1, and wherein the amylase variant has amylase activity, and preferably wherein the amylase variant has improved properties, preferably relative to the parent.

[0506] Methods for introducing amino acid changes (e.g., substitutions or deletions) into protein sequences are well known in the art. Variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semisynthetic gene construction, random mutagenesis, shuffling, etc.

[0507] The obtained amylase variants can be produced on an industrial scale and subsequently purified. Industrial production of the enzyme is typically accomplished by culturing a host cell expressing the enzyme (also referred to as fermentation). Suitable host cells are described below. The nucleic acid sequences encoding the amylase variants described herein can be transformed into a host cell, which is then cultured under conditions suitable for the host cell to produce the amylase variant. In a preferred embodiment, the amylase variant is purified from the host cell.

[0508] Therefore, in yet another embodiment, the present invention relates to a method for producing an amylase variant, the method comprising the steps of:

[0509] (a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding an amylase variant as described herein by introducing a nucleic acid construct comprising a polynucleotide encoding an amylase variant as described herein into the host cell;

[0510] (b) culturing the recombinant host cell of step (a) under conditions conducive to expression of the polynucleotide; and

[0511] (c) optionally, recovering the amylase variant encoded by the polynucleotide.

[0512] The host cell culture is usually carried out in a suitable nutrient medium to allow the recombinant cells to grow and express the desired protein. At the end of the fermentation, the fermentation broth is collected and can be further processed, wherein the fermentation broth comprises a liquid portion and a solid portion. The target enzyme can be further purified from the fermentation broth.

[0513] The amylase variants described herein may be secreted (into the liquid portion of the fermentation broth), or may not be secreted from the microbial cells (and therefore contained in the cells of the fermentation broth). According to this, the amylase variants may be recovered from the liquid portion of the fermentation broth or from the cell lysate. Preferably, the amylase variants are secreted from the cells into the fermentation broth, preferably by a secretion signal peptide added to the end of the amino acid sequence of the amylase variant. The recovery of the amylase variants may be achieved by methods known to those skilled in the art. Suitable methods for recovering proteins from the fermentation broth include, but are not limited to, collection, centrifugation, filtration, extraction, and precipitation. If the target product precipitates or crystallizes in the fermentation broth or is at least partially combined with the particulate matter of the fermentation broth, additional processing steps may be required to release the target protein from the biomass or dissolve the target protein crystals and precipitates. WO 0043502 A1, WO 2008110498 A1, and WO 2017097869A1 describe methods for recovering target proteins from fermentation broth, which are precipitated and / or crystallized during fermentation. If the desired protein is contained in the cells of the fermentation broth, it may be necessary to release the target product from the cells. Release from cells can be achieved, for example but not limited to, by cell lysis using techniques well known to those skilled in the art, such as lysozyme treatment, sonication, French press, or a combination thereof.

[0514] Amylase variants can be purified from fermentation broth by methods known in the art. For example, amylase variants can be separated from fermentation broth by conventional procedures, including but not limited to centrifugation, filtration, extraction, spray drying, evaporation or precipitation. The separated polypeptide can then be further purified by various procedures known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing and size exclusion), electrophoresis procedures (e.g., preparative isoelectric focusing (IEF)), differential solubility (e.g., ammonium sulfate precipitation) or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, eds., VCH Press, New York, 1989). The purified polypeptide can then be concentrated by procedures known in the art, including but not limited to ultrafiltration and evaporation, particularly thin film evaporation.

[0515] Amylase preparation

[0516] Purified solutions of the amylase variants described herein can be further processed to form compositions containing amylases.Thus, also claimed herein is a composition comprising an amylase variant described herein and at least one additional component.

[0517] Therefore, the present invention also relates to a method for preparing a composition, which method comprises the following steps: mixing

[0518] a) an amylase variant as described herein; and

[0519] b) one or more components described herein.

[0520] Furthermore, the present invention therefore also relates to a method for improving the stability of an amylase in a composition, the method comprising the steps of: mixing

[0521] a) an amylase variant as described herein; and

[0522] b) one or more components described herein.

[0523] The composition may be a non-complexed formulation (eg, an amylase variant formulation), or a complexed formulation (eg, a detergent composition).

[0524] In one embodiment of the invention, the amylase variant is formulated as an amylase variant formulation, preferably a concentrated amylase variant formulation. The amylase variant formulation can be solid or liquid. The protein formulation can be obtained using techniques known in the art. For example, but not limited to, a solid enzyme formulation can be obtained by extrusion or granulation. Suitable extrusion and granulation techniques are known in the art and are described, for example, in WO 94 / 19444A1 and WO 97 / 43482A1.

[0525] Preferably, the enzyme formulation comprises the enzyme of the present invention in an amount of 2-120 mg active enzyme / g enzyme formulation, preferably 6-80 mg / g, 6-60 mg / g or 10-40 mg / g.

[0526] In one embodiment, the amylase variant formulation, in particular the liquid enzyme formulation, further comprises one or more additional compounds selected from the group consisting of: solvents, salts, pH adjusters, preservatives, enzyme stabilizers and thickeners. Preferably, the amylase variant formulation does not contain a surfactant. The solvent may be water and / or an organic solvent. The aqueous amylase variant formulation of the present invention may contain water in an amount of more than about 50% by weight, more than about 60% by weight, more than about 70% by weight, or more than about 80% by weight, all relative to the total weight of the enzyme formulation. The formulation containing the amylase variant of the present invention may contain an organic solvent in an amount of more than 30% by weight, more than 40%, more than about 50%, more than about 60% by weight, more than about 70% by weight, or more than about 80% by weight, all relative to the total weight of the enzyme formulation. The organic solvent may be a water-miscible solvent. The organic solvent may be one or more selected from the group consisting of glycerol, propylene glycol, polypropylene glycol, and polyethylene glycol.

[0527] In one embodiment, the amylase variant formulation comprises at least one preservative. Preferably, the preservative means a substance added to the liquid composition for the purpose of preservation, meaning that more preferably, the compounds known to have preservative characteristics contained in the liquid composition formed during the production process are excluded from the term preservative. In one embodiment, the preservative is selected from the group consisting of: 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol and formic acid (in acid form or its salt form), and 4,4'-dichloro 2-hydroxydiphenyl ether. Typically, the liquid composition of the present invention comprises at least one preservative in an amount less than 10ppm, for example, relative to the gross weight of the liquid composition, in an amount of 2ppm to 5% by weight. Alternatively, the amylase variant formulation does not contain a preservative, which means that the content of the preservative is less than 1ppm, preferably 0ppm.

[0528] Preferably, the amylase variant formulations comprise an enzyme stabilization system. Preferably, the amylase variant formulations described herein comprise from about 0.001% to about 10%, from about 0.005% to about 8%, or from about 0.01% to about 6% of an enzyme stabilization system by weight of the composition. The enzyme stabilization system can be any stabilization system that is compatible with the amylase.

[0529] Preferably, the enzyme stabilization system comprises at least one compound selected from the group consisting of: a polyol (preferably 1,3-propylene glycol, ethylene glycol, glycerol, 1,2-propylene glycol, or sorbitol), an inorganic salt (preferably CaCl2, MgCl2, or NaCl), a short chain (preferably C1-C3) carboxylic acid or a salt thereof (preferably formic acid, a formate (preferably sodium formate), acetic acid, acetate, or lactate), a borate, boric acid, boronic acid (preferably 4-formylphenylboronic acid (4-FPBA)), a peptide aldehyde (preferably Z-VAL-H or Z-GAY-H), a peptide acetal, and a peptide aldehyde bisulfite adduct. Preferably, the enzyme stabilization system comprises a combination of at least two of the compounds selected from the group consisting of salts, polyols and short chain carboxylic acids, and preferably one or more of the compounds selected from the group consisting of borates, boric acid, boronic acid (preferably, 4-formylphenylboronic acid (4-FPBA)), peptide aldehydes, peptide acetals and peptide aldehyde bisulfite adducts. In a preferred embodiment, in the presence of a protease, the stabilization system comprises a protease inhibitor, preferably selected from borates, boric acid, boronic acid (preferably, 4-FPBA), peptide aldehydes (preferably, peptide aldehydes such as Z-VAL-H or Z-GAY-H), peptide acetals and peptide aldehyde bisulfite adducts, preferably the protease inhibitor is a peptide aldehyde, preferably Z-VAL-H or Z-GAY-H. In one embodiment, the stabilization system does not comprise a protease inhibitor. Preferably, the composition does not contain boron. Preferably, the amylase variant formulation comprises a calcium salt, preferably calcium chloride.

[0530] Preferably, the liquid amylase variant formulation comprises or consists of an amylase variant, a solvent, an enzyme stabilization system and optionally a preservative and optionally a second enzyme different from the amylase variant as described herein. Preferably, the amylase variant formulation contains no surfactant.

[0531] Therefore, the present invention also relates to a method for preparing an amylase variant formulation, preferably a concentrated amylase variant formulation, comprising the steps of: mixing

[0532] a) an amylase variant as described herein; and

[0533] b) one or more components selected from the group consisting of a solvent, an enzyme stabilization system, a preservative, and a second enzyme different from the amylase variant.

[0534] Furthermore, the present invention therefore also relates to a method for improving the stability of an amylase in a formulation, the method comprising the steps of: mixing

[0535] a) an amylase variant as described herein; and

[0536] b) one or more components selected from the group consisting of a solvent, an enzyme stabilization system, a preservative, and a second enzyme different from the amylase variant.

[0537] Second enzyme

[0538] In another embodiment, the composition comprising the amylase variant as described herein further comprises one or more second enzymes different from the amylase variant. Preferably, the second enzyme is selected from the group consisting of proteases, second amylases, lipases, cellulases, mannanases, hemicellulases, phospholipases, esterases, pectinases, lactases, peroxidases, xylanases, cutinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, beta-glucanases, arabinosidases, hyaluronidases, chondroitinase, laccases, nucleases, , DNA enzymes, phosphodiesterases, phytases, carbohydrases, galactanases, xanthanases, xyloglucanases, oxidoreductases, perhydrolases, aminopeptidases, asparaginases, carbohydrases, carboxypeptidases, catalases, chitinases, cyclodextrin glycosyltransferases, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β-glucosidase, invertases, ribonucleases, transglutaminases and dispersins, and combinations of at least two of the foregoing types. More preferably, the second enzyme is selected from the group consisting of proteases, lipases, cellulases, mannanases, xylanases, DNA enzymes, dispersins, pectinases, oxidoreductases and cutinases, and combinations of at least two of the foregoing types. Most preferably, the second enzyme is a protease, preferably a subtilisin.

[0539] The composition of the invention may comprise one type of enzyme or more than one enzyme of different types, e.g. an amylase and a protease, or more than one enzyme of the same type, e.g. two or more different proteases, or a mixture thereof, e.g. an amylase and two different proteases.

[0540] Protease

[0541] Proteases are active proteins that exert "protease activity" or "proteolytic activity." Proteolytic activity relates to the rate at which a protein is degraded by a protease or proteolytic enzyme over a defined time period.

[0542] Preferably, the second enzyme different from the amylase variant is a protease having at least 40% to 100% identity to the full-length polypeptide sequence of any one of SEQ ID NOs: 10 to 14. In one embodiment, the protease comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, 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% sequence identity to the full-length polypeptide sequence shown in SEQ ID NOs: 10, 11, 12, 13 or 14, preferably SEQ ID NO: 10.

[0543] Preferably, the protease used in combination with the amylase variants described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to SEQ ID NO: 10, and further comprises an amino acid substitution at one or more of the following positions: 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95 , 96,97,98,99,100,101,102,103,104,106,118,120,123,128,129,130,131,154,160,167,170,194,199,205,206,217,218,222,224,232,235,236,245,248,252 and 274 (according to BPN' numbering), and has proteolytic activity. In one embodiment, this protease has no mutations at positions Asp32, His64 and Ser221 (according to BPN' numbering). Preferably, the protease used in combination with the amylase variants described herein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, but less than 100% sequence identity with SEQ ID NO: 10, and is further characterized by having an amino acid glutamic acid (E), or aspartic acid (D), or asparagine (N), or glutamine (Q), or alanine (A), or glycine (G) or serine (S), preferably glutamic acid (E), at position 101 (according to BPN' numbering), and having proteolytic activity. Most preferred is a protease having at least 80% but less than 100% sequence identity with SEQ ID NO: 10, and is characterized by having an amino acid glutamic acid (E) at position 101 (according to BPN' numbering), and having proteolytic activity. The protease may comprise an amino acid substitution at position 101, such as R101E alone or in combination with one or more substitutions at positions 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and / or 274 (according to BPN' numbering), and have proteolytic activity.In one embodiment, the protease comprises one or more additional substitutions: (a) threonine at position 3 (3T), (b) isoleucine at position 4 (4I), (c) alanine, threonine or arginine at position 63 (63A, 63T or 63R), (d) aspartic acid or glutamic acid at position 156 (156D or 156E), (e) proline at position 194 (194P), (f) methionine at position 199 (199M), (g) isoleucine at position 205 (205I), (h) aspartic acid, glutamic acid or glycine at position 217 (217D, 217E or 217G), (i) a combination of two or more amino acids according to (a) to (h). Suitable proteases may be at least 80% identical to SEQ ID NO: 10 and characterized in that they comprise one amino acid (according to (a)-(h)) or according to (i) in combination with amino acids 101E, 101D, 101N, 101Q, 101A, 101G or 101S (according to BPN' numbering), and have proteolytic activity. In one embodiment, the protease is at least 80% identical to SEQ ID NO: 10 and characterized in that it comprises mutations (according to BPN' numbering) R101E, or S3T+V4I+V205I, or S3T+V4I+R101E+V205I or S3T+V4I+V199M+V205I+L217D, and has proteolytic activity. In another embodiment, the protease comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 10, and is further characterized in comprising S3T+V4I+S9R+A15T+V68A+D99S+R101S+A103S+I104V+N218D (according to BPN' numbering), and has proteolytic activity. In another embodiment, the protease can have an amino acid sequence at least 80% identical to SEQ ID NO: 10, and is further characterized by comprising R101E, and one or more substitutions selected from the group consisting of S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61 D,R, S87E, G97S, A98D,E,R, S106A,W, N117E, H120V,D,K,N, S125M, P129D, E136Q, S144W, S161T, S163A,G, Y171 L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D and L262N,Q,D (according to BPN' numbering), and has proteolytic activity.

[0544] Lipase

[0545] "Lipase", "lipolytic enzyme", "lipid esterase" all refer to enzymes of EC class 3.1.1 ("carboxylic ester hydrolase"). Lipase means an active protein having lipase activity (or lipolytic activity; triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74; enzymes having cutinase activity may be referred to herein as cutinases), sterol esterase activity (EC 3.1.1.13) and / or wax ester hydrolase activity (EC 3.1.1.50). Lipases include those of bacterial or fungal origin.

[0546] In one aspect of the invention, suitable lipases (component (b)) are selected from the group consisting of a lipase from the genus Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258068, EP 305216, WO 92 / 05249 and WO 2009 / 109500, or a lipase from H. insolens as described in WO 96 / 13580; a lipase derived from Rhizomucor miehei as described in WO 92 / 05249; miehei); lipases from strains of Pseudomonas (some of which are now renamed Burkholderia), for example from Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (EP 218272, WO 94 / 25578, WO 95 / 30744, WO 95 / 35381, WO 96 / 00292), Pseudomonas cepacia (EP 331376), Pseudomonas stutzeri (P.stutzeri) (GB 1372034), Pseudomonas fluorescens, Pseudomonas sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012), Pseudomonas mendocina (WO 95 / 14783), Pseudomonas glumae (WO 95 / 35381, WO96 / 00292); lipases from Streptomyces griseus (WO 2011 / 150157) and Streptomyces spp.pristinaespiralis) (WO 2012 / 137147), a GDSL-type Streptomyces lipase (WO 2010 / 065455); a lipase from Thermobifida fusca as disclosed in WO 2011 / 084412; a lipase from Geobacillus stearothermophilus as disclosed in WO 2011 / 084417; a Bacillus lipase, for example as disclosed in WO 00 / 60063, as disclosed in Dartois et al. (1992), Biochemica et Biophysica Acta [Biochemistry and Biophysics Acta], 1131, 253-360 or WO 2011 / 084599 disclosed in Bacillus subtilis (B.subtilis), Bacillus stearothermophilus (B.stearothermophilus) (JP S64-074992) or Bacillus pumilus (B.pumilus) (WO 91 / 16422); as disclosed in WO 94 / 01541, a lipase from Candida antarctica; a cutinase from Pseudomonas mendocina (US 5389536, WO 88 / 09367); a cutinase from Magnaporthe grisea (WO 2010 / 107560); as disclosed in WO 90 / 09446, WO 00 / 34450 and WO 01 / 92502, a cutinase from Fusarum oleraceum (Fusarum oleraceum) (W00 / 09446, WO 00 / 34450 and WO 01 / 92502); as disclosed in WO 90 / 09446, WO 00 / 34450 and WO 01 / 92502, a cutinase from Pseudomonas mendocina (US 5389536, WO 88 / 09367 ... solani pisi); and a cutinase from Humicola lanuginosa as disclosed in WO 00 / 34450 and WO 01 / 92502.

[0547] Such suitable lipase variants are, for example, those developed by the methods as disclosed in WO 95 / 22615, WO 97 / 04079, WO 97 / 07202, WO 00 / 60063, WO 2007 / 087508, EP 407225 and EP 260105.

[0548] Commercially available lipases include, but are not limited to, those sold under the trade name Lipolase TM 、Lipex TM 、Lipolex TM and Lipoclean TM(Novozymes), Lumafast (originally from Genencor), Preferenz L (DuPont), and Lipomax (Gist-Brocades / now DSM).

[0549] In one embodiment, the lipase is selected from fungal triacylglycerol lipase (EC class 3.1.1.3). The fungal triacylglycerol lipase may be selected from the lipase of Thermomyces lanuginosus. In one embodiment, the Thermomyces lanuginosus lipase is selected from the triacylglycerol lipase of amino acids 1-269 of SEQ ID NO: 2 according to US 5869438 and variants thereof having lipolytic activity.

[0550] The Thermomyces lanuginosus lipase can be selected from a variant having lipolytic activity that is at least 90%, at least 91%, 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% identical to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US 5869438.

[0551] The Thermomyces lanuginosus lipase can be selected from variants with lipolytic activity that contain only conservative mutations that do not involve the functional domain of amino acids 1-269 of SEQ ID NO: 2 of US 5869438. The lipase variants of this embodiment with lipolytic activity can be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US5869438.

[0552] The Thermomyces lanuginosus lipase may be selected from variants having lipolytic activity comprising the following amino acid substitutions when compared to amino acids 1-269 of SEQ ID NO: 2 of US 5869438: T231R and N233R. When compared to amino acids 1-269 of SEQ ID NO: 2 of US 5869438, the lipase variant may further comprise one or more of the following amino acid exchanges: Q4V, V60S, A150G, L227G, P256K.

[0553] The Thermomyces lanuginosus lipase can be selected from variants with lipolytic activity comprising amino acid substitutions T231R, N233R, Q4V, V60S, A150G, L227G, P256K within the polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US 5869438 and being at least 95%, at least 96% or at least 97% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US 5869438.

[0554] The Thermomyces lanuginosa lipase can be selected from variants having lipolytic activity comprising amino acid substitutions T231R and N233R within amino acids 1-269 of SEQ ID NO: 2 of US5869438 and being at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US5869438.

[0555] The Thermomyces lanuginosus lipase may be a variant of amino acids 1-269 of SEQ ID NO: 2 of US 5869438 having lipolytic activity, wherein the variant of amino acids 1-269 of SEQ ID NO: 2 of US 5869438 is characterized in containing amino acid substitutions T231R and N233R.

[0556] The Thermomyces lanuginosus lipase can be selected from a variant having lipolytic activity, preferably comprising at least one, preferably more than one, more preferably all of the following substitutions within the polypeptide sequence of amino acids 1-269 of SEQ ID NO: 1 of WO 2015 / 010009: N11K, A18K, G23K, K24A, V77I, D130A, V154I, V187T, T189Q and being at least 95%, at least 96% or at least 97% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 1 of WO2015 / 010009.

[0557] Amylase

[0558] Amylases other than the amylases described herein include amylases of bacterial or fungal origin (EC 3.2.1.1 and 3.2.1.2, respectively).Preferably, the amylase is selected from the group of alpha-amylases (EC 3.2.1.1).

[0559] The amylase may be from Bacillus licheniformis having SEQ ID NO: 2 as described in WO 95 / 10603 and variants at least 95% identical thereto. Suitable variants are described in WO 95 / 10603 comprising one or more substitutions at the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408 and 444 having amylolytic activity. Variants are described in SEQ ID NO: 4 of WO 94 / 02597, WO 94 / 018314, WO 97 / 043424 and WO 99 / 019467.

[0560] The amylase may further be from Bacillus stearothermophilus having SEQ ID NO:6 as disclosed in WO 02 / 10355, or an amylase optionally having a C-terminal truncation compared to the wild-type sequence. Suitable variants of SEQ ID NO:6 include variants comprising a deletion at position 179 and / or 181 and / or 182 and / or a substitution at position 193.

[0561] The amylase may further be from Bacillus sp. 707 having SEQ ID NO: 6 as disclosed in WO 99 / 19467 and variants at least 95% identical thereto. Preferred variants of SEQ NO: 6 are those having substitutions, deletions or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.

[0562] The amylase may further be from Bacillus halmapalus having SEQ ID NO: 2 or SEQ ID NO: 7 as described in WO 96 / 23872, also described herein as SP-722. Preferred variants are described in WO 97 / 3296, WO 99 / 194671 and WO 2013 / 001078.

[0563] The amylase may further be from Bacillus sp. DSM 12649 having SEQ ID NO: 4 as disclosed in WO 00 / 22103 and variants at least 95% identical thereto.

[0564] The amylase may further be from Bacillus sp. A 7-7 (DSM 12368) having an amino acid sequence at least 95% identical to SEQ ID NO: 2, particularly over the region according to amino acids 32 to 516 of SEQ ID NO: 2, as disclosed in WO 02 / 10356.

[0565] The amylase may further be from Bacillus strain TS-23 having SEQ ID NO: 2 and variants thereof as disclosed in WO 2009 / 061380.

[0566] The amylase may further be from Cytophaga sp. having SEQ ID NO: 1 as disclosed in WO 2013 / 184577 and variants at least 95% identical thereto.

[0567] The amylase may further be from Bacillus megaterium DSM 90 having SEQ ID NO: 1 as disclosed in WO 2010 / 104675 and variants at least 95% identical thereto.

[0568] The amylase may further be from Bacillus sp. comprising amino acids 1 to 485 of SEQ ID NO: 2 as described in WO 00 / 60060 and variants at least 95% identical thereto.

[0569] The amylase may further be from Bacillus amyloliquefaciens or a variant thereof, preferably selected from the amylase according to SEQ ID NO: 3 as described in WO 2016 / 092009.

[0570] The amylase may have SEQ ID NO: 12 as described in WO 2006 / 002643, or an amylase variant thereof comprising the substitutions Y295F and M202LITV within said SEQ ID NO: 12.

[0571] The amylase may have SEQ ID NO:6 as described in WO 2011 / 098531, or amylase variants comprising substitutions at one or more positions selected from the group consisting of 193 [G, A, S, T or M], 195 [F, W, Y, L, I or V], 197 [F, W, Y, L, I or V], 198 [Q or N], 200 [F, W, Y, L, I or V], 203 [F, W, Y, L, I or V], 206 [F, W, Y, N, L, I, V, H, Q, D or E], 210 [F, W, Y, L, I or V], 212 [F, W, Y, L, I or V], 213 [G, A, S, T or M] and 243 [F, W, Y, L, I or V] within said SEQ ID NO:6.

[0572] The amylase may have SEQ ID NO: 1 as described in WO 2013 / 001078, or amylase variants comprising changes at two or more (several) positions corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476 and G477 within said SEQ ID NO: 1.

[0573] The amylase may have SEQ ID NO: 2 as described in WO 2013 / 001087, or amylase variants comprising a deletion of positions 181+182, or 182+183, or 183+184 within said SEQ ID NO: 2, optionally comprising one or two or more modifications at any position corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476 and G477 within said SEQ ID NO: 2.

[0574] The amylase may be a hybrid alpha-amylase derived from the above amylases, eg, as described in WO 2006 / 066594.

[0575] According to WO 2014 / 183920, the hybrid amylase may have A and B domains and a C domain, wherein the A and B domains are at least 90% identical to SEQ ID NO: 2 of WO 2014 / 183920, and the C domain is at least 90% identical to SEQ ID NO: 6 of WO 2014 / 183920, wherein the hybrid amylase has amylolytic activity; preferably, the hybrid α-amylase is at least 95% identical to SEQ ID NO: 23 of WO 2014 / 183920 and has amylolytic activity.

[0576] According to WO 2014 / 183921, the hybrid amylase may have A and B domains and a C domain, the A and B domains having at least 75% identity with SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 26, SEQ ID NO: 32 and SEQ ID NO: 39 as disclosed in WO 2014 / 183921, and the C domain having at least 90% identity with SEQ ID NO: 6 of WO 2014 / 183921, wherein the hybrid amylase has amylolytic activity; preferably, the hybrid alpha-amylase is at least 95% identical to SEQ ID NO: 30 as disclosed in WO 2014 / 183921 and has amylolytic activity;

[0577] According to WO 2021 / 032881, the hybrid amylase may comprise A and B domains derived from an alpha amylase derived from Bacillus species A7-7 (DSM 12368), and a C domain derived from an alpha amylase derived from Bacillus cereus; preferably, the A and B domains are at least 75% identical to the amino acid sequence of SEQ ID NO: 42, and the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44 - both sequences are as disclosed in WO 2021 / 032881; more preferably, the hybrid amylase is at least 80% identical to SEQ ID NO: 54 as disclosed in WO 2021 / 032881.

[0578] In one embodiment, the at least one amylase is selected from commercially available amylases including but not limited to those sold under the trade name Duramyl TM Termamyl TM 、Fungamyl TM 、Stainzyme TM 、Stainzyme Plus TM、Natalase TM , Liquozyme X and BAN TM 、Amplify TM 、Amplify Prime TM (from Novozymes), and Rapidase TM 、Purastar TM 、Powerase TM 、Effectenz TM (M100 from DuPont), Preferenz TM (S1000, S110 and F1000; from DuPont), PrimaGreen TM (ALL; DuPont), Optisize TM Those sold by (DuPont).

[0579] Mannanase

[0580] As described herein, "mannanase" is an enzyme selected from the group of mannan degrading enzymes. The mannan degrading enzyme may be selected from β-mannosidase (EC 3.2.1.25), endo-1,4-β-mannosidase (EC 3.2.1.78) and 1,4-β-mannobiose glycosidase (EC 3.2.1.100). Preferably, the mannan degrading enzyme is selected from the group of endo-1,4-β-mannosidase (EC3.2.1.78), which may be referred to herein as endo-β-1,4-D-mannanase, β-mannanase or mannanase.

[0581] The mannanase may be selected from alkaline mannanases of family 5 or 26 (ie GH5 or GH26). The term "alkaline mannanase" is intended to encompass a mannanase having an enzyme activity of at least 40% of its maximum activity at a given pH of 7 to 12, preferably 7.5 to 10.5.

[0582] The mannanase may be selected from the group consisting of mannanases derived from Bacillus organisms, such as those described in JP-0304706 [β-mannanase from Bacillus species], JP-63056289 [alkaline, thermostable β-mannanase], JP-63036774 [Bacillus microorganism FERM P-8856 producing β-mannanase and β-mannosidase at alkaline pH], JP-08051975 [alkaline β-mannanase from alkalophilic Bacillus species AM-001], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 91 / 18974 [mannanase active at extreme pH and temperature], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 97 / 11164 [mannanase active at extreme pH and temperature], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 97 / 11164 [mannanase active at extreme pH and temperature], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 97 / 11164 [mannanase active at extreme pH and temperature], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 97 / 11164 [mannanase active at extreme pH and temperature], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 2014 / 100018 [endo-(3-mannanase 1 (Bleman 1; see US 5,476,775) cloned from Bacillus circulans or Bacillus lentus strain CMG1240]. Suitable mannanases are described in WO 99 / 064619.

[0583] The mannanase may be selected from mannanases derived from Trichoderma organisms, such as disclosed in WO 93 / 24622.

[0584] The mannanase may be selected from commercially available mannanases, such as (Novozymes) or (M100)(DuPont).

[0585] Cellulase

[0586] "Cellulase" is an enzyme that can hydrolyze cellulose. Cellulase can be selected from cellobiohydrolase (1,4-PD-glucan cellobiohydrolase, EC 3.2.1.91), endo-ss-1,4-glucanase (EC 3.2.1.4) and ss-glucosidase (EC 3.2.1.21). Endoglucanase of EC class 3.2.1.4 can be named endoglucanase, endo-1,4-ss-D-glucan 4-glucanohydrolase, endo-1,4-β-glucanase, carboxymethyl cellulase and β-1,4-glucanase.

[0587] Endoglucanases can be classified by amino acid sequence similarity under Family 5 which contains more than 20 endoglucanases of EC 3.2.1.4 (Henrissat, B., accessed at UniProt 10 / 26 / 2011). Reference is also made to T.-M. Enveri, "Microbial Cellulases," in WM Fogarty, Microbial Enzymes and Biotechnology, Applied Science Publishers, pp. 183-224 (1983); Methods in Enzymology, (1988) Vol. 160, pp. 200-391 (edited by Wood, WA and Kellogg, ST); Béguin, P., "Molecular Biology of Cellulose Degradation," Annu. Rev. Microbiol. (1990), Vol. 44, pp. 219-248; Begun, P. and Aubert, JP., "The biological degradation of cellulose," FEMS Microbiology Reviews [FEMS Microbiology] 13 (1994) pp. 25-58; Henrissat, B., "Cellulases and their interaction with cellulose", Cellulose [Cellulose] (1994), Vol. 1, pp. 169-196.

[0588] Preferably, the at least one cellulase is selected from the glycosyl hydrolase family 7 (GH7, pfam00840), preferably from the endoglucanases (EC 3.2.1.4).

[0589] Preferably, alkaline cellulases are used, wherein "alkaline cellulases" is intended to cover cellulases having enzymatic activity at a given pH in the range of 7 to 12, preferably 7.5 to 10.5.

[0590] In one embodiment, the cellulase is selected from cellulase comprising a cellulose binding domain. In another embodiment, the cellulase comprises a catalytic domain but does not comprise a cellulose binding domain.

[0591] In one embodiment, at least one endoglucanase of EC class 3.2.1.4 is derived from

[0592] Bacillus, for example Bacillus species CBS 670.93 and CBS 669.93

[0593] Melanocarpus, for example Melanocarpus albomyces as disclosed in WO 97 / 14804

[0594] Clostridium, such as Clostridium thermocellum

[0595] Humicola, for example Humicola insolens (DSM 1800) as disclosed in EP 0495257, EP 0531315, EP 0531372, US 4435307, US 5648263, US 5776757, WO 89 / 09259, WO 91 / 17244, WO 94 / 07998 (sequence shown in Figure 1 for the 43 kd human variant thereof), WO 95 / 24471, WO 96 / 11262 and WO 98 / 12307.

[0596] Fusarium, for example Fusarium oxysporum, for example strain J79 (DSM 2672) as disclosed in EP 0 495 257, EP 0 531 315, EP 0 531 372, US 5 648 263, US 5 776 757, WO 89 / 09259, WO 91 / 17244, WO 95 / 24471 and WO 96 / 11262

[0597] Thielavia, for example Thielavia terrestris or Myceliophthora thermophila strain CBS11765 as disclosed in EP 0531315, US 5648263, US 5776757, WO 89 / 09259, WO 91 / 17244, WO 95 / 24471, WO 96 / 11262, WO 96 / 29397 (SEQ ID NO: 9 and variants thereof) and WO 98 / 12307.

[0598] - Trichoderma, for example Trichoderma reesei, Trichoderma longibrachiatum or Trichoderma harzianum as disclosed in EP 1305432, EP 1240525, WO 92 / 06165, WO 94 / 21801, WO 94 / 26880, WO 95 / 02043, WO 95 / 24471 and WO 02 / 099091.

[0599] Aspergillus, for example Aspergillus aculeatus as disclosed in WO 93 / 17244

[0600] • Erwinia, for example Erwinia chrysanthermi as described by MH Boyer et al., European Journal of Biochemistry, Vol. 162, pp. 311-316 (1987).

[0601] Acremonium, e.g., Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pinkertoniae, Acremonium roseogriseum, Acremonium incoloratum, and Acremonium furatum as disclosed in WO 96 / 11262 and WO 96 / 29397 (SEQ ID NO: 5 and variants thereof).

[0602] - Cellvibrio genus, for example Cellvibrio mixtus DSM 11683, Cellvibrio mixtus DSM 11684, Cellvibrio mixtus DSM 11685, Cellvibrio mixtus ACM 2601, Cellvibrio mixtus DSM 1523 and Cellvibrio gilvus DSM 11686, as disclosed in WO 98 / 08940.

[0603] - Cephalosporium, for example Cephalosporium sp. RYM-202 as disclosed in WO 96 / 11262.

[0604] Suitable cellulases also include those of variants of the above cellulases having cellulolytic activity. In one embodiment, the cellulase variants include variants having at least 40% to 100% identity when compared to the full-length polypeptide sequence of the parent enzyme as disclosed above. In one embodiment, the cellulase variants having cellulolytic activity are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar and / or identical to the full-length polypeptide sequence of the parent enzyme as disclosed above.

[0605] The cellulase may be a Humicola insolens DSM 1800 cellulase complex having endoglucanase, cellobiohydrolase and beta-glucosidase activities.

[0606] The cellulase may be a Humicola insolens DSM 1800 endoglucanase (EC 3.2.1.4), preferably having a polypeptide sequence according to positions 21-435 of SEQ ID NO: 2 as disclosed in WO 2018 / 224544, or a variant at least 95% identical thereto.

[0607] The cellulase may be a Humicola insolens endoglucanase with 43 kD (EC 3.2.1.4), preferably according to the polypeptide sequence disclosed in Figure 1a of WO 94 / 07998 ("43 kD hum") or a variant thereof (preferably at least 90% identical thereto), preferably those disclosed in WO 94 / 07998.

[0608] The cellulase may be a Bacillus species cellulase (EC 3.2.1.4) selected from a polypeptide or a catalytically active fragment thereof that is at least 80% similar and / or identical to the amino acid sequence of position 1 to position 773 of SEQ ID NO: 2 of WO 2004 / 053039. In one embodiment, the cellulase is a mature polypeptide that is at least 95% identical to SEQ ID NO: 1 of WO 2018 / 224544.

[0609] The cellulase may be a Thielavia terrestris cellulase (EC 3.2.1.4) having a polypeptide or a catalytically active fragment thereof having an amino acid sequence at least 80% similar and / or identical to that of position 1 to position 299 of SEQ ID NO: 4 of WO 2004 / 053039. In one embodiment, the cellulase is a mature polypeptide at least 95% identical to SEQ ID NO: 4 of WO 2018 / 224544.

[0610] The cellulase may be a mature Sordaria fimicola cellulase, preferably having a polypeptide sequence according to SEQ ID NO: 5 of WO 2018 / 224544 or a variant thereof that is at least 95% identical.

[0611] At least one cellulase may be selected from and (Novozymes), Clazinase TM and Puradax HA TM (Genencor Int. Inc.) and KAC-500(B) TM (Kao Corporation).

[0612] Detergent composition

[0613] In one embodiment, the present invention relates to the use of the amylase variants in detergent compositions. Accordingly, the present invention also relates to detergent compositions comprising the amylase variants described herein and one or more detergent components.

[0614] Therefore, the present invention also relates to a method for preparing a detergent composition, the method comprising the steps of: mixing

[0615] a) an amylase variant as described herein; and

[0616] b) one or more detergent ingredients as described herein.

[0617] Furthermore, the present invention therefore also relates to a method for preparing a detergent composition having improved amylase stability and / or providing a detergent composition having improved washing performance, the method comprising the steps of: mixing

[0618] c) an amylase variant as described herein; and

[0619] d) one or more detergent ingredients as described herein.

[0620] The one or more detergent components may be selected from the group consisting of additional enzymes other than the amylase variant, enzyme stabilization systems, surfactants, defoamers, builders, polymers, bleaching systems (bleaches), rheology modifiers, hydrotropes, softeners, desiccants, brighteners, buffers, preservatives, anti-corrosion additives, dyes and fragrances.

[0621] Preferably, at least one component of the detergent is selected from the group consisting of a surfactant, a builder, a polymer, a preservative and a second enzyme different from the amylase variant.

[0622] Preferably, one or more detergent components, preferably surfactants and / or builders, are biodegradable and / or bio-based.

[0623] Detergent components may have more than one function in the final application of the detergent composition, so any detergent component mentioned in the context of a specific function herein may also have another function in the final application of the detergent composition. The function of a specific detergent component in the final application of the detergent composition generally depends on its amount in the detergent composition, i.e., the effective amount of the detergent component. The type and / or amount of the detergent component in the detergent composition depends on the desired application, such as washing white textiles, colored textiles and wool. The selected one or more components also depend on the physical form of the detergent composition (liquid, solid, gel, provided in a small bag or as a sheet, etc.). For example, the one or more components selected for washing a preparation further depend on regional practices, which themselves are related to the following aspects: such as the washing temperature used, the mechanical principle of the washing machine (vertical axis compared to the horizontal axis machine), the water consumption of each washing cycle, etc., and geographical features such as the average hardness of water.

[0624] In one embodiment, the detergent composition is a formulation of more than two detergent components, wherein at least one component is effective in removing stains, at least one component is effective in providing optimal cleaning conditions, and at least one component is effective in maintaining the physical properties of the detergent.

[0625] The detergent composition may be a liquid or solid detergent composition or a combination of a liquid and solid detergent composition. The liquid detergent composition is preferably a gel detergent composition. The solid detergent composition may be a soap bar or a powder detergent composition, preferably a powder detergent composition, wherein the powder detergent composition may be compressed into tablets.

[0626] The detergent composition may be a unit dose or a multi-unit dose composition. The detergent composition may be in the form of a pouch (including a multi-compartment pouch). The detergent composition may be a laundry or dishwashing detergent composition suitable for home care and / or industrial and institutional (I&I) cleaning. Both laundry and dishwashing compositions may be in the form of hand wash or automatic washing compositions. Preferably, the dishwashing composition is an automatic dishwashing (ADW).

[0627] The detergent pouch can be any form, shape and material suitable for accommodating the composition, for example, before contacting with water, the composition is not allowed to be released from the pouch. The pouch is made of a water-soluble film, which surrounds an internal volume. The internal volume can be divided into the compartments of the pouch. Preferred films are polymeric materials, preferably polymers forming films or sheets, such as polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC). The pouch can include solid laundry detergent compositions or partial components and / or liquid detergent compositions or partial components separated by a water-soluble film. The compartment of the liquid component can be different from the compartment comprising a solid in composition (see, for example, US2009 / 0011970).

[0628] Preferably, the amylase variant according to the invention can be added to the detergent composition in an amount corresponding to 0.002 mg to 6 mg active enzyme variant / g detergent composition, preferably 0.005 mg / g-5 mg / g, 0.005 mg / g-3 mg / g, 0.01 mg / g-2 mg / g or 0.05 mg / g-2 mg / g.

[0629] In one embodiment, the pH of the detergent composition is in the range of 5-12, preferably in the range of 6-11, more preferably in the range selected from 6-10, 7-9 and 7.5-8.5. In one embodiment, the formulation is a detergent composition, preferably a liquid detergent composition.

[0630] In one embodiment, the detergent composition according to the present invention comprises one or more surfactants.Depending on their ionic charge, surfactants are referred to as nonionic surfactants, anionic surfactants, cationic surfactants or amphoteric surfactants.

[0631] The detergent composition of the present invention may include one or more surfactants, which may be anionic and / or cationic and / or nonionic and / or semipolar and / or zwitterionic, or a mixture thereof. In a preferred embodiment, the detergent composition of the present invention includes at least one surfactant. In a particular embodiment, the detergent composition of the present invention includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. One or more surfactants are typically present at a level of about 0.1wt.-% to 60wt.-% (e.g., 1wt.-% to 40wt.-%, 3wt.-% to 20wt.-% or 3wt.-% to 10wt.-%). One or more surfactants are selected based on the desired cleaning application, and include one or more any conventional surfactants known in the art. Any surfactant known in the art for detergents can be used. Non-limiting examples of surfactants are disclosed in McCutcheon's 2016 Detergents and Emulsifiers, and McCutcheon's 2016 Functional Materials, both North American and International editions, MC Publishing Co, 2016 edition. Other useful examples are disclosed in earlier editions of the same publications known to those skilled in the art.

[0632] When included therein, the detergent typically comprises from about 1 wt.-% to 40 wt.-% (e.g., 5 wt.-% to 30 wt.-%, 5 wt.-% to 15 wt.-%, or 20 wt.-% to 25 wt.-%) of anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular linear alkylbenzene sulfonate (LAS), isomers of LAS, branched alkylbenzene sulfonate (BABS), phenylalkane sulfonate, alpha-olefin sulfonate (AOS), olefin sulfonate (olefinsulfonate), alkene sulfonate (alkene sulfonate), alkane-2,3-diyl bis(sulfates), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkane sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfo fatty acid methyl esters (α-SFMe or SES) (including methyl ester sulfonate (MES)), alkyl or alkenyl succinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinic acid or soaps, and combinations thereof.

[0633] When included therein, the detergent typically contains about 0 to 10 wt.-% of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyl dimethylethanolamine quaternary ammonium salts (ADMEAQ), cetyl trimethyl ammonium bromide (CTAB), dimethyl distearyl ammonium chloride (DSDMAC) and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

[0634] When included therein, the detergent typically comprises about 0.2 wt.-% to 40 wt.-% of nonionic surfactant, for example 0.5 wt.-% to 30 wt.-%, in particular 1 wt.-% to 20 wt.-%, 3 wt.-% to 10 wt.-%, 3 wt.-% to 5 wt.-% or 8 wt.-% to 12 wt.-%. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (e.g., ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polyglycosides (APG), alkoxylated amines, fatty acid monoethanolamide (FAM), fatty acid diethanolamide (FADA), ethoxylated fatty acid monoethanolamide (EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamide, GA, or fatty acid glucamide, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

[0635] When included therein, the detergent typically contains about 0 to 10 wt.-% of a semi-polar surfactant. Non-limiting examples of semi-polar surfactants include amine oxides (AOs), such as alkyl dimethyl amine oxides, N-(coconut alkyl)-N,N-dimethyl amine oxides, and N-(tallow alkyl)-N,N-bis-(2-hydroxyethyl) amine oxides, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.

[0636] When included therein, the detergent typically comprises about 0 to 10 wt.-% of a zwitterionic surfactant.Non-limiting examples of zwitterionic surfactants include betaines, alkyl dimethyl betaines, sulfobetaines, and combinations thereof.

[0637] The detergent composition according to the invention may contain one or more compounds selected from complexing agents (chelating agents), chelants, sequestrating agents, precipitants and ion exchange compounds, which can form water-soluble complexes with calcium and magnesium. Such compounds may be referred to herein as "builders" or "building agents", but it is not intended to limit such compounds to this function in the final application of the detergent composition.

[0638] In one embodiment, the detergent composition of the present invention comprises at least one builder selected from non-phosphate-based builders, such as sodium gluconate, one or more sodium citrates, one or more sodium silicates, one or more sodium carbonates, one or more sodium phosphonates, one or more sodium aminocarboxylates, one or more sodium polycarboxylates, one or more sodium polysulfonates and one or more sodium polyphosphonates. In one embodiment, the detergent composition of the present invention comprises a strong chelating builder. Preferably, the detergent composition of the present invention is phosphate-free, meaning substantially free of phosphate-based builders. In this context, "substantially phosphate-free" is understood to mean that the sum of the contents of phosphate and polyphosphate is in the range of 10 ppm to 1% by weight, as determined by a weight method and with reference to the corresponding detergent composition of the present invention. In another preferred embodiment, the detergent composition comprises a phosphonate, wherein the phosphonate is preferably DTPMP and / or HEDP.

[0639] In one embodiment, the detergent composition of the present invention comprises at least one "citrate" selected from monoalkali metal salts and dialkali metal salts of citric acid, in particular monosodium and preferably trisodium citric acid, ammonium or substituted ammonium salts of citric acid and citric acid itself. Citrate can be used as an anhydrous compound or as a hydrate, for example as sodium citrate dihydrate. The content of citrate can be in the range of 0% to about 20% by weight, in the range of about 0.5% to about 10% by weight, or in the range of 1%-5% by weight, all relative to the total weight of the detergent composition. In one embodiment, the detergent composition of the present invention comprises a total amount of citrate in the range of about 1%-3% relative to the total weight of the detergent composition.

[0640] The detergent compositions of the present invention may contain one or more silicates. "One or more silicates" in the context of the present invention include in particular sodium disilicate and sodium metasilicate, aluminosilicates such as sodium aluminosilicate such as zeolite A (i.e. Na 12 (AlO2) 12 (SiO2) 12 *27H2O) and sheet silicates, in particular those having the formula α-Na2Si2O5, β-Na2Si2O5 and δ-Na2Si2O5.

[0641] The detergent composition of the present invention may contain one or more carbonates. The term "one or more carbonates" includes alkali metal carbonates and alkali metal bicarbonates, preferably sodium salts. Particularly suitable is sodium carbonate (Na2CO3).

[0642] The detergent compositions of the present invention may contain one or more phosphonates. "Phosphonates" include, but are not limited to, 2-phosphinobutane-1,2,4-tricarboxylic acid (PBTC); ethylenediaminetetrakis(methylenephosphonic acid) (EDTMPA); 1-hydroxyethane-1,1-diphosphonic acid (HEDP), CH2C(OH)[PO(OH)2]2; aminotris(methylenephosphonic acid) (ATMP), N[CH2PO(OH)2]3; aminotris(methylenephosphonic acid) sodium salt (ATMP), N[CH2PO(ONa)2]3; 2-hydroxyethyliminobis(methylenephosphonic acid), HOCH2CH2N[CH2PO(OH)2]2; diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), (HO)2POCH2N[CH2CH2N[CH2PO(OH)2]2]2; diethylenetriaminepenta(methylenephosphonate) sodium salt, C9H (28-x) N3Na x O 15 P5 (x = 7); Hexamethylenediamine (tetramethylenephosphonic acid) potassium salt, C 10 H (28-x) N2K x O 12 P4 (x = 6); and bis(hexamethylene)triamine (pentamethylenephosphonic acid), (HO2)POCH2N[(CH2)2N[CH2PO(OH)2]2]2. Salts thereof may also be suitable.

[0643] The detergent composition of the present invention may contain one or more aminocarboxylates. Non-limiting examples of suitable "aminocarboxylates" include, but are not limited to, diethanolglycine (DEG), dimethylglycine (DMG), nitrilotriacetic acid (NTA), N-hydroxyethylaminodiacetic acid, ethylenediaminetetraacetic acid (EDTA), N-(2-hydroxyethyl)iminodiacetic acid (HEIDA), hydroxyethylenediaminetriacetic acid, N-hydroxyethyl-ethylenediaminetriacetic acid (HEDTA), hydroxyethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid (DTPA) and methylglycinediacetic acid (MGDA), glutamic acid-diacetic acid (GLDA), iminodisuccinic acid (IDS), hydroxyiminodisuccinic acid, ethylenediaminedisuccinic acid (EDDS), aspartic acid-diacetic acid and alkali metal or ammonium salts thereof. Also suitable are aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamate (SMGL), N-(2-sulfoethyl)glutamate (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), para-aminobenzenesulfonic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and sulfomethyl-N,N-diacetic acid (SMDA) and their alkali metal or ammonium salts. Preferred are MGDA or EDDS. The term "ammonium salt" as used in this context refers to a salt having at least one cation carrying a nitrogen atom that is permanently or temporarily quaternized. Examples of cations carrying at least one nitrogen atom that is permanently quaternized include tetramethylammonium, tetraethylammonium, dimethyldiethylammonium and nC 10 -C 20 Examples of cations carrying at least one nitrogen atom that is temporarily quaternized include protonated amines and ammonia, such as monomethylammonium, dimethylammonium, trimethylammonium, monoethylammonium, diethylammonium, triethylammonium, nC 10 -C 20 - alkyldimethylammonium 2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium, tris(2-hydroxyethyl)ammonium, N-methyl 2-hydroxyethylammonium, N,N-dimethyl-2-hydroxyethylammonium, and especially NH4 + .

[0644] In one embodiment, the detergent composition of the present invention comprises more than one builder.Preferably, the detergent composition of the present invention contains less than 0.2% by weight of nitrilotriacetic acid (NTA), or 0.01% to 0.1% by weight of NTA relative to the total weight of the detergent composition.

[0645] In one embodiment, the detergent composition of the present invention comprises at least one aminocarboxylate selected from methylglycine diacetate (MGDA), glutamic acid diacetate (GLDA) and corresponding salts thereof, for example, an alkali metal (such as sodium) salt thereof, in an amount in the range of 0.1% to 25.0% by weight, in the range of 1.0% to 18.0% by weight, in the range of 3.0% to 15.0% by weight, in the range of 3.0% to 10.0% by weight, or in the range of 5.0% to 8.0% by weight, relative to the total weight of the detergent composition.

[0646] The detergent composition of the present invention may include one or more hydrotropes. One or more hydrotropes may be selected from organic solvents, such as ethanol, isopropanol, ethylene glycol, 1,2-propylene glycol, and other organic solvents known in the art that are miscible with water under normal conditions, but are not limited thereto. In one embodiment, the detergent composition of the present invention includes 1,2-propylene glycol, and its total amount is within the range of 5%-10% by weight, preferably about 6% by weight, all relative to the gross weight of the detergent composition. Other non-limiting examples of hydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium isopropylbenzene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate and combinations thereof.

[0647] In one embodiment, the detergent composition comprises at least one preservative. Preferably, the preservative means a substance added to the liquid composition for the purpose of preservation, meaning that more preferably, the compounds known to have preservative characteristics contained in the liquid composition formed during the production process are excluded from the term preservative. In one embodiment, the preservative is selected from the group consisting of: 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol and formic acid (in acid form or its salt form), and 4,4'-dichloro 2-hydroxydiphenyl ether. Typically, the liquid composition of the present invention comprises at least one preservative in an amount less than 10ppm, for example, relative to the gross weight of the liquid composition, in an amount of 2ppm to 5% by weight. Alternatively, the detergent composition does not contain a preservative, which means that the content of the preservative is less than 1ppm, preferably 0ppm.

[0648] In one embodiment, the detergent composition comprising an amylase variant as described herein further comprises one or more second enzymes different from the amylase variant. Preferably, the second enzyme is selected from the group consisting of proteases, second amylases, lipases, cellulases, mannanases, hemicellulases, phospholipases, esterases, pectinases, lactases, peroxidases, xylanases, cutinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidases, chondroitinase, laccases, nucleases. , DNA enzymes, phosphodiesterases, phytases, carbohydrases, galactanases, xanthanases, xyloglucanases, oxidoreductases, perhydrolases, aminopeptidases, asparaginases, carbohydrases, carboxypeptidases, catalases, chitinases, cyclodextrin glycosyltransferases, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β-glucosidase, invertases, ribonucleases, transglutaminases and dispersins, and combinations of at least two of the foregoing types. More preferably, the second enzyme is selected from the group consisting of proteases, lipases, cellulases, mannanases, xylanases, DNA enzymes, dispersins, pectinases, oxidoreductases and cutinases, and combinations of at least two of the foregoing types. Most preferably, the second enzyme is a protease, preferably a subtilisin.

[0649] Particularly preferred additional enzymes are disclosed elsewhere herein, and that description is also incorporated by reference into this part of the specification.

[0650] The composition of the invention may comprise one type of enzyme or more than one enzyme of different types, e.g. an amylase and a protease, or more than one enzyme of the same type, e.g. two or more different proteases, or a mixture thereof, e.g. an amylase and two different proteases.

[0651] The detergent composition may comprise a water-soluble source of calcium and / or magnesium ions. In one embodiment, the detergent composition comprises an enzyme stabilization system as described herein. Preferably, in particular in the case of a liquid detergent composition, the detergent composition may comprise at least one protease inhibitor as described herein, preferably selected from boronic acid derivatives (preferably 4-FPBA), and peptide aldehydes (preferably Z-VAL-H or Z-GAY-H). Preferably, the detergent composition does not contain boron.

[0652] In one embodiment, the present invention relates to a method for providing a detergent composition, preferably a liquid detergent composition, more preferably a liquid laundry detergent composition, the method comprising mixing in one or more steps:

[0653] (a) at least one amylase variant according to the present invention, preferably wherein the amylase is provided in an amylase variant formulation as described herein; and

[0654] (b) at least one detergent component, preferably selected from surfactants, builders, polymers, preservatives and a second enzyme different from the amylase variant, present in an amount effective for cleaning performance and / or maintaining the physical properties of the detergent.

[0655] In one embodiment, the present invention relates to a detergent composition comprising

[0656] a) an amylase variant as described herein;

[0657] b) one or more surfactants, preferably in a concentration of 0.2% to 65%, preferably 0.2% to 40%,

[0658] c) one or more builders, preferably in a concentration of 0.01% to 25%, and

[0659] d) optionally one or more additional compounds selected from the group consisting of: additional enzymes different from the amylase in a), defoamers, polymers, bleaching systems (bleaches), rheology modifiers, hydrotropes, softeners, desiccants, brighteners, buffers, preservatives, anti-corrosion additives, dyes and fragrances;

[0660] Preferably, the detergent composition is a liquid, powder, sachet or capsule detergent composition.

[0661] Preferably, the detergent composition of the present invention, preferably the powder detergent composition of the present invention, comprises, in addition to the amylase variant as described herein, one or more compounds selected from the group consisting of alcohol ethoxylate 7EO, coconut fatty acid C12-18, C12-C14 fatty alcohol ether sulfate (1-3EO, preferably 2EO), linear alkylbenzene sulfonic acid, sodium acetate, sodium citrate, sodium silicate, sodium carbonate, sodium phosphate, sodium bicarbonate, zeolite 4A, HEDP, MGDA, sodium sulfate, sodium chloride, optical brighteners and polymers, and optionally bleach activators and percarbonates.

[0662] Preferably, the detergent composition, preferably a powder detergent composition, of the present invention comprises, in addition to the amylase variant described herein,

[0663] b) one or more surfactants selected from the group consisting of: alcohol ethoxylate 7EO, coconut fatty acid C12-18, C12-C14 fatty alcohol ether sulfate (1-3EO, preferably 2EO), linear alkylbenzene sulfonic acid, preferably in a concentration of 0.2%-65%,

[0664] c) one or more builders selected from the group consisting of HEDP, MGDA, GLDA and DTPMP, preferably in a concentration of 0.01% to 25%, and

[0665] d) one or more compounds selected from the group consisting of sodium acetate, sodium citrate, sodium silicate, sodium carbonate, sodium phosphate, sodium bicarbonate, zeolite 4A, sodium sulfate, sodium chloride, optical brighteners and polymers, and optionally bleach activators and percarbonates.

[0666] Preferably, the detergent composition of the present invention, preferably the liquid detergent composition of the present invention, comprises, in addition to the amylase variant as described herein, one or more compounds selected from the group consisting of alcohol ethoxylate 7EO, coconut fatty acid C12-18, C12-C14 fatty alcohol ether sulfate (1-3EO, preferably 2EO), linear alkylbenzene sulfonic acid, sulfonic acid, 1,2-propylene glycol, triethanolamine, monoethanolamine, NaOH, glycerol, ethanol, sodium citrate and polymers.

[0667] Preferably, the detergent compositions, preferably liquid detergent compositions, of the present invention comprise, in addition to the amylase variants described herein,

[0668] b) one or more surfactants selected from the group consisting of: alcohol ethoxylate 7EO, coconut fatty acid C12-18, C12-C14 fatty alcohol ether sulfate (1-3EO, preferably 2EO), linear alkylbenzene sulfonic acid, preferably in a concentration of 0.2%-65%,

[0669] c) one or more builders selected from the group consisting of HEDP, MGDA, GLDA and DTPMP, preferably in a concentration of 0.01% to 25%, and

[0670] d) one or more compounds selected from the group consisting of sulfonic acid, 1,2 propylene glycol, triethanolamine, monoethanolamine, NaOH, glycerol, ethanol, sodium citrate, and polymers.

[0671] In one embodiment, the amylase variants described herein are included in a detergent composition comprising one or more, preferably all, compounds selected from the group consisting of (all percentages are w / w):

[0672] a formulation comprising an amylase variant described herein, 0.05% to 1.0%;

[0673] Anionic detersive surfactants (e.g., alkyl benzene sulfonates, alkyl ethoxylated sulfates, and mixtures thereof), 8% to 15%;

[0674] Nonionic detersive surfactant (such as alkyl ethoxylated alcohol), 0.5% to 4%;

[0675] Cationic detersive surfactant (such as quaternary ammonium compound), 0%-4%;

[0676] Other detersive surfactants (e.g., zwitterionic detersive surfactants, amphoteric surfactants, and mixtures thereof), 0% to 4%;

[0677] Carboxylic acid polymers (e.g., copolymers of maleic acid and acrylic acid), 1% to 4%;

[0678] Polyethylene glycol polymers (e.g., polyethylene glycol polymers containing polyvinyl acetate side chains), 0.5% to 4%;

[0679] Polyester soil release polymers (such as Repel-o-tex and / or Texcare polymers), 0.1% to 2%;

[0680] Cellulosic polymers (e.g., carboxymethylcellulose, methylcellulose, and combinations thereof), 0.5% to 2%;

[0681] Other polymers (e.g., amine polymers, dye transfer inhibitor polymers, hexamethylenediamine derivative polymers, and mixtures thereof), 0% to 4%;

[0682] Zeolite builders and phosphate builders (e.g. zeolite 4A and / or sodium tripolyphosphate), 0% to 4wt%;

[0683] Other builders (such as sodium citrate and / or citric acid), 0% to 3%;

[0684] Carbonates (e.g. sodium carbonate and / or sodium bicarbonate), 15% to 30%;

[0685] Silicates (such as sodium silicate), 0% to 10%;

[0686] Fillers (e.g. sodium sulfate and / or biofillers), 10% to 40%;

[0687] Available oxygen source (such as sodium percarbonate), 10% to 20%;

[0688] Bleach activators (e.g. tetraacetylethylenediamine (TAED) and / or nonanoyloxybenzenesulfonate (NOBS), 2% to 8%;

[0689] bleach catalysts (e.g. quaternary oxaziridinium-based bleach catalysts and / or transition metal bleach catalysts), 0% to 0.1%;

[0690] Other bleaching agents (e.g., reducing bleach and / or preformed peracids), 0% to 10%;

[0691] Chelating agents (e.g., ethylenediamine-N'N'-disuccinic acid (EDDS) and / or hydroxyethanediphosphonic acid (HEDP), 0.2% to 1%;

[0692] Photobleaches (e.g., sulfonated zinc phthalocyanine and / or sulfonated aluminum phthalocyanine), 0% to 0.1%;

[0693] Colorants (e.g., Direct Violet 99, Acid Red 52, Acid Blue 80, Direct Violet 9, Solvent Violet 13, and any combination thereof), 0% to 1%;

[0694] Brightener (e.g. Brightener 15 and / or Brightener 49), 0.1% to 0.4%;

[0695] Fabric softeners (e.g., montmorillonite clay and / or polydimethylsiloxane (PDMS)), 0% to 4%;

[0696] Flocculants (such as polyethylene oxide), 0% to 1%;

[0697] Foam inhibitors (e.g. silicones and / or fatty acids), 0% to 0.1%;

[0698] Perfume (e.g., perfume microcapsules, spray perfumes, starch encapsulated perfume notes, perfume-loaded zeolites, and any combination thereof), 0.1% to 1%; and

[0699] Aesthetic agents (e.g., colored soap rings and / or colored spots / strips), 0% to 1%; and

[0700] Optionally, a protease (e.g., Savinase, Coronase, Ovozyme, Kannase, Liquanase, Polarzyme, Purafect, Purafast, Properase, Excellase, FN3, FN4, Effectenz P, PreferenzP, Progress Uno, Progress Excel, Blaze, Excellenz P), about 0.05 wt % to about 0.2 wt %;

[0701] Optionally, an additional amylase different from the amylase variants described herein (e.g., Termamyl(R), Termamyl Ultra(R), Natalase(R), Optisize HT Plus(R), Purastar, Powerase(R), Stainzyme(R), Preferenz S, Effectenz S, Amplify, Amplify Prime, Achieve alpha, Excellenz S, and any combination thereof), about 0.05 wt % to about 0.2 wt %;

[0702] Optionally, cellulase (e.g., Carezyme, Celluclean, Puradax, Biotouch, Whitezyme, Revitalenz, and combinations thereof), 0.05% to 0.2%;

[0703] Optionally, lipase (e.g., Lipex, Lipolex, Lipoclean, Preferenz L, and any combination thereof), 0.05% to 0.2%;

[0704] Optionally, other enzymes (e.g., xyloglucanases, cutinases, pectin lyases (e.g., Xpect), mannanases (e.g., Mannanway, Mannastar, Marvellenz, Effectenz M, Preferenz M, Preferenz F, and combinations thereof) bleaching enzymes and combinations thereof), 0.05% to 0.2%;

[0705] In another embodiment, the amylase variants described herein are included in a detergent composition comprising one or more, preferably all, compounds selected from the group consisting of (all percentages are w / w):

[0706] a formulation comprising an amylase variant described herein, 0.05% to 1.0%;

[0707] A carboxyl group-containing polymer (comprising about 60% to about 70% by mass of an acrylic acid-based monomer (A); and about 30% to about 40% by mass of a sulfonic acid group-containing monomer (B); and wherein the average molecular weight is about 23,000 to about 50,000, preferably in the range of about 25,000 to about 38,000 (as described in WO 2014032269), about 0.5 wt% to about 1.5 wt%;

[0708] Anionic detersive surfactant (e.g., alkyl benzene sulfonates, alkyl ethoxylated sulfates, and mixtures thereof), about 8 wt % to about 15 wt %;

[0709] Nonionic detersive surfactant (e.g., alkyl ethoxylated alcohol) about 0.5 wt% to 4 wt%;

[0710] Cationic detersive surfactant (e.g., quaternary ammonium compound), about 0 wt % to about 4 wt %;

[0711] Other detersive surfactants (e.g., zwitterionic detersive surfactants, amphoteric surfactants, and mixtures thereof), about 0 wt % to 4 wt %;

[0712] Carboxylate polymers (e.g., copolymers of maleic acid and acrylic acid), from about 1 wt % to about 4 wt %;

[0713] Polyethylene glycol polymer (e.g., polyethylene glycol polymer containing polyvinyl acetate side chains), about 0 wt % to about 4 wt %;

[0714] Polyester soil release polymer (e.g., Repel-O-Tex(R) and / or Texcare(R) polymer), about 0.1 wt % to about 2 wt %;

[0715] Cellulosic polymers (e.g., carboxymethyl cellulose, methyl cellulose, and combinations thereof), about 0.5 wt % to about 2 wt %;

[0716] Other polymers (e.g., amine polymers, dye transfer inhibitor polymers, hexamethylenediamine derivative polymers, and mixtures thereof), from about 0 wt % to about 4 wt %;

[0717] Zeolite builders and phosphate builders (e.g., zeolite 4A and / or sodium tripolyphosphate), from about 0 wt % to about 4 wt %;

[0718] Other additives (such as sodium citrate and / or citric acid), about 0 wt % to about 3 wt %;

[0719] Carbonate (such as sodium carbonate and / or sodium bicarbonate), about 15 wt % to about 30 wt %;

[0720] Silicate (e.g., sodium silicate), about 0 wt % to about 10 wt %;

[0721] Filler (such as sodium sulfate and / or biological filler), about 10 wt % to about 40 wt %;

[0722] Available oxygen source (such as sodium percarbonate), about 10 wt % to about 20 wt %;

[0723] Bleach activators (e.g., tetraacetylethylenediamine (TAED) and / or nonanoyloxybenzenesulfonate (NOBS), about 2 wt % to about 8 wt %;

[0724] bleach catalyst (e.g., quaternary oxaziridinium-based bleach catalyst and / or transition metal bleach catalyst), from about 0 wt % to about 0.1 wt %;

[0725] Other bleaching agents (e.g., reducing bleach and / or preformed peracids), from about 0 wt % to about 10 wt %;

[0726] Chelating agents (e.g., ethylenediamine-N'N'-disuccinic acid (EDDS) and / or hydroxyethanediphosphonic acid (HEDP), about 0.2 wt % to about 1 wt %;

[0727] Photobleach (e.g., sulfonated zinc phthalocyanine and / or sulfonated aluminum phthalocyanine), from about 0 wt % to about 0.1 wt %;

[0728] Colorants (e.g., Direct Violet 99, Acid Red 52, Acid Blue 80, Direct Violet 9, Solvent Violet 13, and any combination thereof), about 0 wt % to about 0.5 wt %;

[0729] Brightener (e.g., Brightener 15 and / or Brightener 49), about 0.1 wt % to about 0.4 wt %;

[0730] Fabric softener (e.g. montmorillonite clay and / or polydimethylsiloxane (PDMS)), 0 wt% to 15 wt%;

[0731] Flocculant (such as polyethylene oxide), 0wt% to 1wt%;

[0732] Foam inhibitors (e.g. silicones and / or fatty acids), 0 wt% to 0.1 wt%;

[0733] Perfumes (e.g., perfume microcapsules, spray perfumes, starch encapsulated perfume notes, perfume-loaded zeolites, and any combination thereof), 0.1 wt% to 1 wt%; and

[0734] Aesthetic agents (e.g., colored soap rings and / or colored spots / strips), 0wt% to 1wt%;

[0735] Optionally, a protease (e.g., Savinase, Coronase, Ovozyme, Kannase, Liquanase, Polarzyme, Purafect, Purafast, Properase, Excellase, FN3, FN4, Effectenz P, PreferenzP, Progress Uno, Progress Excel, Blaze, Excellenz P), about 0.05 wt % to about 0.2 wt %,

[0736] Optionally, an additional amylase different from the amylase variants described herein (e.g., Termamyl(R), Termamyl Ultra(R), Natalase(R), Optisize HT Plus(R), Purastar, Powerase(R), Stainzyme(R), Preferenz S, Effectenz S, Amplify, Amplify Prime, Achieve alpha, Excellenz S), about 0.05 wt % to about 0.2 wt %,

[0737] Optionally, cellulase (e.g., Carezyme(R), Celluzyme(R), Puradax, Celluclean(R), Biotouch, Whitezyme, Revitalenz, and combinations thereof, typically having an enzyme activity of about 10 to 50 mg active enzyme / g), about 0.05 wt % to 0.5 wt %

[0738] Optionally, lipase (e.g., Lipex(R), Lipolex(R), Lipoclean(R), Preferenz L, and any combination thereof, typically having an enzyme activity of about 10 mg to about 50 mg active enzyme / g), about 0.2 wt % to about 1 wt %

[0739] Optionally, other enzymes (e.g., xyloglucanases (e.g., Whitezyme(R)), cutinases, pectin lyases (e.g., Xpect), mannanases (e.g., Mannanway, Mannastar, Marvellenz, Effectenz M, Preferenz M, Preferenz F, and combinations thereof), bleaching enzymes, typically having an enzyme activity of about 10 mg to about 50 mg active enzyme / g), 0 wt % to 2 wt %,

[0740] Further preferred detergent compositions include the components listed below (all percentages are w / w):

[0741] - water, alcohol ethoxysulfate, alcohol ethoxylate, amino oxide, citric acid, C12-18 topped palm kernel fatty acids, ethanol, 1,2 propylene glycol, sodium formate, calcium chloride, sodium hydroxide, silicone emulsion, trans-sulfated EHDQ, amylase variants as described herein;

[0742] - sodium linear alkylbenzene sulfonate 8.8%. Ethoxylated fatty alcohol C12-18 (7EO) 4.7%. Sodium soap 3.2%. Defoamer DC2-4248S 3.9%. Sodium aluminosilicate zeolite 4A 28.3%. Sodium carbonate 11.6%. Sodium salt of a copolymer of acrylic acid and maleic acid (Sokalan CP5) 2.4%. Sodium silicate 3.0%. Carboxymethylcellulose 1.2%. Dequest 2066 2.8%. Optical brightener 0.2%. Sodium sulfate 6.5%. 0.4% of a formulation comprising an amylase variant as described herein;

[0743] -12% LAS, 11% AEO Biosoft N25-7 (NI), 7% AEOS (SLES), 6% MPG (monopropylene glycol), 3% ethanol, 3% TEA, 2.75% cocoa soap, 2.75% soy soap, 2% glycerol, 2% sodium hydroxide, 2% sodium citrate, 1% sodium formate, 0.2% DTM PA and 0.2% PCA, an amylase variant as described herein;

[0744] -5%-15% anionic surfactants; <5% nonionic surfactants, phosphonates, soaps; enzymes, optical brighteners, benzisothiazolinone, methylisothiazolinone, fragrances, α-isomethylionone, citronellol, geraniol, linalool, amylase variants described herein;

[0745] - Water, Sodium Dodecylbenzene Sulfonate, C14-C15 Pareth-7, Sodium Citrate, Propylene Glycol, Sodium Palm Kernel Oleate, Sodium Laureth Sulfate, MEA Dodecylbenzene Sulfonate, Quaternized Sulfated Ethoxylated Hexamethylene Diamine, Sodium Cumene Sulfonate, Fragrance, PEG / Vinyl Acetate Copolymer, Sodium Formate, Hydrogenated Castor Oil, Sodium Diethylenetriamine Pentamethylene Phosphonate, PEG / PPG-10 / 2 Propylheptyl Ether, Butylphenyl Methylpropional, Polyvinylpyridine-N-oxide, Sorbitol, Glycerin, Ethanolamine, Sodium Hydroxide, α-Isomethyl Ionone, Calcium Chloride, Geraniol, Linalool, Citronellol, Tripropylene Glycol, Benzisothiazolinone, Dimethicone, Sodium Acetate, Cellulase, Colorant, Glyceryl Stearate, Hydroxyethylcellulose, Silica, Amylase Variants as described herein;

[0746] - Water, Sodium Laureth Sulfate, Propylene Glycol, C14-C15 Pareth-7, Sodium Citrate, Sodium Palm Kernel Oleate, Ethanol, Sodium Formate, Quaternized Sulfated Ethoxylated Hexamethylenediamine, Sodium Hydroxide, Fragrance, Polyvinylpyridine-N-oxide, Sorbitol, Calcium Chloride, Glycerin, Sodium Acetate, Colorant, Cellulase, Amylase Variants as Described herein;

[0747] - Water, Sodium Laureth Sulfate, Propylene Glycol, C14-C15 Pareth-7, Sodium Citrate, Sodium Palm Kernel Oleate, Ethanol, Sodium Formate, Quaternized Sulfated Ethoxylated Hexamethylene Diamine, Sodium Hydroxide, Fragrance, Sorbitol, Calcium Chloride, Glycerin, Sodium Acetate, Colorant, Cellulase, Amylase Variants as Described herein;

[0748] - Water, Sodium Laureth Sulfate, Propylene Glycol, C14-C15 Pareth-7, Sodium Citrate, Sodium Palm Kernel Oleate, Ethanol, Sodium Formate, Quaternized Sulfated Ethoxylated Hexamethylene Diamine, Sodium Hydroxide, Sorbitol, Calcium Chloride, Glycerin, Sodium Acetate, Cellulase, Silica, Amylase Variants as described herein;

[0749] - Water, Sodium Dodecylbenzene Sulfonate, C14-C15 Pareth-7, Sodium Citrate, Propylene Glycol, Sodium Palm Kernel Oleate, Sodium Laureth Sulfate, MEA Dodecylbenzene Sulfonate, Quaternized Sulfated Ethoxylated Hexamethylene Diamine, Sodium Cumene Sulfonate, Fragrance, PEG / Vinyl Acetate Copolymer, Sodium Formate, C12-C14 Pareth-7, Hydrogenated Castor Oil, Sodium Diethylenetriamine Pentamethylene Phosphonate, PEG / PPG-10 / 2 Propylheptyl Ether, Butylphenyl Methylpropional, Fluorescent Brightener, Sorbitol, Glycerin, Ethanolamine, Sodium Hydroxide, α-Isomethyl Ionone, Calcium Chloride, Geraniol, Linalool, Citronellol, Tripropylene Glycol, Sodium Chloride, Benzisothiazolinone, Dimethicone, Sodium Acetate, Cellulase, Colorant, Glyceryl Stearate, Hydroxyethylcellulose, Silica, Amylase Variants as Described herein;

[0750] -15%-30% anionic surfactants, nonionic surfactants, 5%-15% soap, <5% polycarboxylates, fragrances, phosphates, optical brighteners, amylase variants as described herein;

[0751] - 15-30% anionic surfactant, 5-15% nonionic surfactant, soap, benzisothiazolinone, methylisothiazolinone, perfume, amylase variant as described herein;

[0752] - 11% LAS, 2% AS / AEOS, 2% soap, 3% AEO, 15.15% sodium carbonate, 3% sodium silicate, 18.75% zeolite, 0.15% chelating agent, 2% sodium citrate, 1.65% AA / MA copolymer, 2.5% CMC and 0.5% SRP, an amylase variant as described herein;

[0753] - 16.5% LAS, 15% zeolite, 12% sodium disilicate, 20% sodium carbonate, 1% sokalan, 35.5% sodium sulfate, an amylase variant as described herein;

[0754] -15%-30% anionic surfactants, <5% nonionic surfactants, phosphonates, polycarboxylates, zeolites; enzymes, fragrances, hexylcinnamaldehyde, amylase variants as described herein;

[0755] -15%-30% of the following: anionic surfactants, oxygen-based bleach and zeolite, less than 5% of the following: nonionic surfactants, phosphonates, polycarboxylates, soaps, other ingredients: fragrance, hexyl cinnamaldehyde, benzyl salicylate, linalool, optical brighteners, enzymes and citronellol, amylase variants as described herein;

[0756] - Water, Alcohol Ethoxysulfate, Diethylene Glycol, Alcohol Ethoxylate, Ethanolamine, Linear Alkylbenzene Sulfonate, Sodium Fatty Acid, Polyethyleneimine Ethoxylate, Citric Acid, Sodium Cumene Sulfonate, Propylene Glycol, DTPA, Disodium Diaminostilbene Disulfonate, Dipropyl Ethyltetramine, Sodium Hydroxide, Sodium Formate, Calcium Formate, Polydimethylsiloxane, Liquitint TM , hydrogenated castor oil, flavor, amylase variant as described herein;

[0757] -Linear alkylbenzene sulfonate, propylene glycol, citric acid, sodium hydroxide, ethanolamine, ethanol, alcohol sulfate, polyethyleneimine ethoxylate, sodium fatty acid, diquaternium ethoxysulfate, diethylene glycol, laureth-9, alkyl dimethylamine oxide, fragrance, disodium diaminostilbene disulfonate, DTPA, sodium formate, calcium formate, polyethylene glycol 4000, mannanase, Liquitint TM Blue, polydimethylsiloxane, an amylase variant as described herein;

[0758] - water, sodium alcohol ethoxysulfate, propylene glycol, ethanol, linear alkylbenzene sulfonate sodium salt, polyethyleneimine ethoxylate, diethylene glycol, trans-sulfated and ethoxylated hexamethylenediamine, alcohol ethoxylate, linear alkylbenzene sulfonate MEA salt, sodium formate, sodium alkyl sulfate, DTPA, amine oxide, calcium formate, disodium diaminostilbene, disulfonates, polydimethylsiloxane, benzisothiazolinone, amylase variants as described herein;

[0759] -Water, Alcohol Ethoxysulfate, Linear Alkylbenzene Sulfonate, Diethylene Glycol, Propylene Glycol, Ethanolamine, Citric Acid, Alcohol Sulfate, Sodium Hydroxide, Polyethyleneimine, Ethoxylate, Sodium Fatty Acid, Ethanol, Laureth-9, Diquaternium Ethoxysulfate, Lauramine Oxide, Sodium Cumene, Sulfonate, Fragrance, DTPA, Disodium Diaminostilbene Disulfonate, Sodium Formate, Disodium Distyryl Biphenyl Disulfonate, Calcium Formate, Polyethylene Glycol 4000, Mannanase, Pectinase, Liquitint TM Blue, polydimethylsiloxane, an amylase variant as described herein;

[0760] - Water, alcohol ethoxysulfate, propylene glycol, sodium fatty acid, lauryl trimethyl ammonium chloride, ethanol, sodium hydroxide, sodium cumene sulfonate, citric acid, ethanolamine, diethylene glycol, silicone polyether, fragrance, polyethyleneimine ethoxylate, laureth-9, DTPA, polyacrylamide quaternary ammonium chloride, disodium diaminostilbene disulfonate, sodium formate, Liquitint TM Orange, dipropylethyltetramine, polydimethylsiloxane, cellulase, an amylase variant as described herein;

[0761] - Water, Sodium Alcohol Ethoxysulfate, Sodium Alkyl Sulfate, MEA Citrate, Linear Alkylbenzene Sulfonate MEA Salt, Propylene Glycol, Diethylene Glycol, Polyethyleneimine Ethoxylate, Ethanol, Sodium Fatty Acid, Ethanolamine, Lauramine Oxide, Laureth-9, DTPA, Sodium Cumene Sulfonate, Sodium Formate, Calcium Formate, Linear Alkylbenzene Sulfonate Sodium Salt, Alcohol Sulfate, Sodium Hydroxide, Diquaternium Ethoxysulfate, Fragrance, Mannanase, Pectinase, Disodium Diaminostilbene Disulfonate, Benzisothiazolinone, Liquitint TM Blue, polydimethylsiloxane, dipropylethyltetramine, an amylase variant as described herein;

[0762] -Water, Sodium Alcohol Ethoxysulfate, MEA Citrate, Sodium Alkyl Sulfate, Alcohol Ethoxylate, Linear Alkylbenzene Sulfonate MEA Salt, Sodium Fatty Acid, Polyethyleneimine Ethoxylate, Diethylene Glycol, Propylene Glycol, Diquaternium Ethoxysulfate, Polyethyleneimine, Ethoxylate Propoxylate, Ethanol, Sodium Cumene Sulfonate, Fragrance, DTPA, Disodium Diaminostilbene Disulfonate, Mannanase, Cellulase, Sodium Formate, Calcium Formate, Laurylamine Oxide, Liquitint TM Blue, Dimethicone / polydimethyl silicone, amylase variants as described herein;

[0763] - Water, alcohol ethoxy sulfate, linear alkylbenzene sulfonate, alcohol ethoxylate, citric acid, ethanolamine, sodium fatty acid, diethylene glycol, propylene glycol, sodium hydroxide, polyethyleneimine ethoxylate, silicone polyether, ethanol, sodium cumene sulfonate, diquaternium ethoxy sulfate, laureth-9, fragrance, DTPA, disodium diaminostilbene disulfonate, disodium distyryl biphenyl disulfonate, sodium formate, calcium formate, mannanase, Liquitint TM Orange, polydimethylsiloxane, polyacrylamide quaternary ammonium chloride, cellulase, dipropylethyltetramine, an amylase variant as described herein;

[0764] - water, alcohol ethoxysulfate, diethylene glycol, monoethanolamine citrate, sodium formate, propylene glycol, linear alkylbenzene sulfonate, ethanolamine, ethanol, polyethyleneimine ethoxylate, benzisothiazoline, calcium formate, citric acid, sodium diethylenetriamine pentaacetate, polydimethylsiloxane, diquaternary ammonium ethoxysulfate, disodium diaminostilbene disulfonate, laureth-9, mannanase, sodium cumene sulfonate, sodium fatty acid, amylase variants as described herein;

[0765] -Water, alcohol ethoxysulfate, MEA citrate, alcohol sulfate, alcohol ethoxylate, linear alkylbenzene sulfonate MEA salt, fatty acid sodium, polyethyleneimine ethoxylate, diethylene glycol, propylene glycol, diquaternium ethoxysulfate, polyethyleneimine, ethoxylate propoxylate, ethanol, sodium cumene sulfonate, fragrance, DTPA, disodium diaminostilbene disulfonate, mannanase, cellulase, sodium formate, calcium formate, laurylamine oxide, liquitint TM Blue, polydimethylsiloxane, an amylase variant as described herein;

[0766] - water, sodium alcohol ethoxysulfate, MEA citrate, linear alkylbenzene sulfonate: sodium salt, alcohol ethoxylate, linear alkylbenzene sulfonate: MEA salt, sodium fatty acid, polyethyleneimine ethoxylate, diethylene glycol, propylene glycol, diquaternary ammonium ethoxysulfate, polyethyleneimine ethoxylate propoxylate, ethanol, sodium cumene sulfonate, citric acid, DTPA, disodium diaminostilbene disulfonate, sodium formate, calcium formate, polydimethylsiloxane, amylase variants as described herein;

[0767] - Water, alcohol ethoxylate sulfate, linear alkylbenzene sulfonate sodium / Mea salt, propylene glycol, diethylene glycol, sodium formate, ethanol, sodium fatty acid, fragrance, laurylamine oxide, DTPA, polyethyleneamine ethoxylate, calcium formate, diaminobenzene disulfonate disodium, polydimethylsiloxane, tetramine, Liquitint TM Blue, an amylase variant as described herein;

[0768] - linear alkylbenzene sulfonate, C12-16 Pareth-9, propylene glycol, alcohol ethoxysulfate, water, polyethyleneimine ethoxylate, glycerin, fatty acid salts, PEG-136 polyvinyl acetate, ethylenediamine disuccinate, monoethanolamine citrate, sodium bisulfite, sodium diethylenetriamine pentaacetate, disodium distyrylbiphenyl disulfonate, calcium formate, mannanase, xyloglucanase, sodium formate, hydrogenated castor oil, dye, subtilisin, benzisothiazoline, fragrance, amylase variants as described herein;

[0769] - deionized water, dipropylene glycol butyl ether, sodium alkyl sulfate, hydrogen peroxide, ethanol, magnesium sulfate, alkyl dimethyl amine oxide, citric acid, sodium hydroxide, trimethoxybenzoic acid, flavor, amylase variant as described herein;

[0770] - Water, alkyl ethoxylate, linear alkylbenzene sulfonate, hydrogen peroxide, diquaternary ammonium ethoxy sulfate, ethanolamine, disodium distyrylbiphenyl disulfonate, tetrabutylethylenebisphenol, F&DC Yellow 3, fragrance, amylase variants as described herein;

[0771] - sodium percarbonate, sodium sulfate, sodium carbonate, sodium aluminosilicate, nonanoyloxybenzene sulfonate, sodium polyacrylate, water, sodium alkylbenzene sulfonate, DTPA, polyethylene glycol, sodium palmitate, modified starch, FD&C Blue 1, flavor, amylase variants as described herein;

[0772] - Water, alkyl ethoxylate, MEA borate, linear alkylbenzene sulfonate, propylene glycol, diquaternary ammonium ethoxy sulfate, calcium chloride enzyme (Calcium Chlorideenzyme), ethanolamine, benzisothiazolinone, sodium citrate, sodium hydroxide, flavor, amylase variants as described herein;

[0773] - Water, alkylamine oxide, dipropylene glycol phenyl ether, hydrogen peroxide, citric acid, ethylenediamine disuccinate sodium salt, sodium alkyl sulfate, flavor, amylase variant as described herein;

[0774] - sodium bicarbonate, sodium carbonate, sodium percarbonate, alcohol ethoxylate, sodium chloride, maleic acid / acrylic acid copolymer, nonanoyloxybenzene sulfonate, sodium sulfate, coloring agent, sodium diethylenetriamine pentaacetate, hydrated aluminum silicate (zeolite), polyethylene glycol, sodium alkylbenzene sulfonate, sodium palmitate, starch, water, flavor, amylase variants as described herein;

[0775] - Polyvinyl alcohol bag film, in which the liquid part and the powder part are packaged: liquid ingredients: dipropylene glycol, diquaternary ammonium ethoxysulfate, water, glycerol, LiquitintTM Orange, amylase variants described herein;

[0776] - Powder ingredients: sodium percarbonate, nonanoyloxybenzene sulfonate, sodium carbonate, sodium sulfate, sodium aluminosilicate, sodium polyacrylate, sodium alkylbenzene sulfonate, maleic acid / acrylic acid copolymer, water, polyethylene glycol, sodium palmitate, modified starch, glycerol, DTPA, flavor, amylase variant as described herein;

[0777] - Water, Sodium Alcohol Ethoxysulfate, Sodium Linear Alkylbenzene Sulfonate / MEA Salt, MEA Citrate, Propylene Glycol, Polyethyleneimine Ethoxylate, Ethanol, Diethylene Glycol, Polyethyleneimine Propoxyethoxylate, Sodium Fatty Acid, Sodium Cumene Sulfonate, DTPA, Fragrance, Disodium Diaminostilbene Disulfonate, Calcium Formate, Sodium Formate, Dextranase, Polydimethylsiloxane, Liquitint TM Blue, mannanase, amylase variant as described herein;

[0778] -Sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, bentonite, water, sodium percarbonate, sodium polyacrylate, silicate, alkyl sulfate, nonanoyloxybenzene sulfonate, DTPA, polyethylene glycol 4000, silicone, ethoxylate, fragrance, polyethylene oxide, palmitic acid, disodium diaminostilbene disulfonate, liquitint TM Red, FD&C Blue 1, cellulase, amylase variants as described herein;

[0779] -Water, Sodium Alcohol Ethoxysulfate, MEA Citrate, Linear Alkylbenzene Sulfonate: Sodium / MEA Salt, Propylene Glycol, Polyethyleneimine Ethoxylate, Ethanol, Diethylene Glycol, Polyethyleneimine, Propoxyethoxylate, Diquaternium Ethoxysulfate, Alcohol Sulfate, Polydimethylsiloxane, Fragrance, Sodium Fatty Acid, DTPA, Sodium Bisulfite, Disodium Diaminostilbene Disulfonate, Dextranase, Castor Oil, Calcium Formate, MEA, Styrene Acrylic Acid Copolymer, Sodium Formate, Liquitint TM Blue, an amylase variant as described herein;

[0780] - Water, Sodium Alcohol Ethoxysulfate, MEA Citrate, Linear Alkylbenzene Sulfonate: Sodium / MEA Salt, Propylene Glycol, Ethanol, Diethylene Glycol, Polyethyleneimine Propoxyethoxylate, Polyethyleneimine Ethoxylate, Alcohol Sulfate, Polydimethylsiloxane, Fragrance, Sodium Fatty Acid, DTPA, Sodium Bisulfite, Disodium Diaminostilbene Disulfonate, Castor Oil, Calcium Formate, MEA, Styrene Acrylic Acid Copolymer, Propyl Ammonium Propionamide, Dextranase, Sodium Formate, Liquitint TM Blue, an amylase variant as described herein;

[0781] - Water, Sodium Alcohol Ethoxysulfate, MEA Citrate, Linear Alkylbenzene Sulfonate: Sodium / MEA Salt, Propylene Glycol, Polyethyleneimine Ethoxylate, Ethanol, Diethylene Glycol, Polyethyleneimine, Propoxyethoxylate, Diquaternium Ethoxysulfate, Alcohol Sulfate, Polydimethylsiloxane, Fragrance, Sodium Fatty Acid, DTPA, Sodium Bisulfite, Disodium Diaminostilbene Disulfonate, Dextranase, Castor Oil, Calcium Formate, MEA, Styrene Acrylic Acid Copolymer, Propyl Ammonium Propionamide, Sodium Formate, Liquitint TM Blue, an amylase variant as described herein;

[0782] - sodium carbonate, sodium aluminosilicate, alkyl sulfates, sodium sulfate, linear alkylbenzene sulfonates, water, sodium polyacrylate, silicates, ethoxylates, sodium percarbonate, polyethylene glycol 4000, disodium diaminostilbene disulfonate, silicones, cellulases, amylase variants as described herein;

[0783] - sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, alkyl sulfate, sodium percarbonate, water, sodium polyacrylate, silicate, nonanoyloxybenzene sulfonate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, disodium diaminostilbene disulfonate, palmitic acid, silicone, cellulase, amylase variants as described herein;

[0784] - sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, water, nonanoyloxybenzene sulfonate, alkyl sulfate, sodium polyacrylate, silicate, sodium percarbonate, ethoxylate, polyethylene glycol 4000, flavor, DTPA, palmitic acid, disodium diaminostilbene disulfonate, silicone, cellulase, amylase variants as described herein;

[0785] - sodium carbonate, sodium aluminosilicate, sodium sulfate, sodium percarbonate, alkyl sulfates, linear alkylbenzene sulfonates, water, nonanoyloxybenzene sulfonate, sodium polyacrylate, silicates, ethoxylates, polyethylene glycol 4000, DTPA, fragrance, palmitic acid, disodium diaminostilbene disulfonate, FD&C Blue 1, silicone, cellulase, alkyl ether sulfates, amylase variants as described herein;

[0786] - sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, sodium percarbonate, nonanoyloxybenzene sulfonate, alkyl sulfate, water, silicate, sodium polyacrylate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, disodium diaminostilbene disulfonate, silicone, FD&C Blue 1, cellulase, alkyl ether sulfate, amylase variants as described herein;

[0787] - sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, sodium percarbonate, alkyl sulfate, water, sodium polyacrylate, silicate, nonanoyloxybenzene sulfonate, ethoxylate, polyethylene glycol 4000, DTPA, flavor, cellulase, disodium diaminostilbene disulfonate, silicone, FD&C Blue 1, amylase variants as described herein;

[0788] -Water, Sodium Alcohol Ethoxysulfate, MEA Citrate, Sodium Linear Alkylbenzene Sulfonate, Linear Alkylbenzene Sulfonate: MEA Salt, Alcohol Ethoxylate, Sodium Fatty Acid, Propylene Glycol, Diethylene Glycol, Polyethyleneimine Ethoxylate Propoxylate, Diquaternium Ethoxysulfate, Ethanol, Sodium Cumene Sulfonate, Fragrance, DTPA, Sodium Bisulfate, Disodium Diaminostilbene Disulfonate, Mannanase, Cellulase, Sodium Formate, Calcium Formate, Laurylamine Oxide, Liquitint TM Blue, mannanase, cellulase, dimethicone / polydimethyl silicone, amylase variants as described herein;

[0789] -Water, Sodium Alcohol Ethoxysulfate, Linear Alkylbenzene Sulfonate: Sodium / MEA Salt, MEA Citrate, Propylene Glycol, Polyethyleneimine Ethoxylate, Fragrance, Ethanol, Diethylene Glycol, Polyethyleneimine Propoxyethoxylate, Alcohol Sulfate, Sodium Fatty Acid, DTPA, Disodium Diaminostilbene Disulfonate, MEA, Mannanase, Glucose Enzyme, Sodium Formate, Polydimethylsiloxane, Liquitint TM Blue, tetramine, an amylase variant as described herein;

[0790] -Water, Sodium Alcohol Ethoxysulfate, MEA Citrate, Sodium Linear Alkylbenzene Sulfonate, Linear Alkylbenzene Sulfonate: MEA Salt, Alcohol Ethoxylate, Sodium Fatty Acid, Propylene Glycol, Diethylene Glycol, Polyethyleneimine Ethoxylate Propoxylate, Diquaternium Ethoxysulfate, Ethanol, Sodium Cumene Sulfonate, Fragrance, DTPA, Sodium Bisulfate, Disodium Diaminostilbene Disulfonate, Mannanase, Cellulase, Sodium Formate, Calcium Formate, Laurylamine Oxide, Liquitint TM Blue, mannanase, cellulase, dimethicone / polydimethyl silicone, amylase variants as described herein;

[0791] - sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, sodium percarbonate, nonanoyloxybenzene sulfonate, alkyl sulfate, water, silicate, sodium polyacrylate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, disodium diaminostilbene disulfonate, silicone, FD&C Blue 1, cellulase, alkyl ether sulfate, amylase variant as described herein; or

[0792] - water, dodecylbenzenesulfonic acid, laureth-11, peg-75 lanolin, propylene glycol, denatured alcohol (alcoholdenat.), potassium soyate, potassium hydroxide, disodium cocoamphodiacetate, ethylenediaminetriacetate cocoalkylacetamide, fragrance, zinc ricinoleate, sodium chloride, benzisothiazolinone, methylisothiazolinone, ci 16255, benzyl alcohol, amylase variants as described herein.

[0793] Preferably, the above composition comprising a protease further comprises 4-FPBA and / or a peptide aldehyde protease inhibitor, most preferably Z-GAY or Z-VAL.

[0794] The amylase variants described herein can be comprised in one of the following detergent compositions.

[0795]

[0796]

[0797]

[0798]

[0799]

[0800] How to use

[0801] The present invention also relates to the use of the amylase variants as described herein in cleaning processes (e.g. laundry or hard surface cleaning), preferably for home care or I&I cleaning, including manual and automatic dishwashing and medical device cleaning. Thus, the present invention also relates to the use of the amylase variants as described herein for providing a detergent composition with improved enzyme stability (preferably amylase stability) and / or for providing a detergent composition with improved washing performance (preferably against stains sensitive to amylases).

[0802] The present invention therefore also relates to a method for cleaning, preferably laundry or hard surface cleaning, comprising the step of contacting an object, preferably a textile or a hard surface, with a composition comprising an amylase variant as described herein, preferably wherein the composition comprises at least one additional detergent component, preferably a surfactant and / or a builder.

[0803] In addition, the present invention also relates to a method for improving the stability of amylase in a detergent composition and / or for improving the washing performance of a detergent composition (preferably for stains sensitive to amylase), which method comprises the step of formulating an amylase variant as described herein in a detergent composition.

[0804] Preferred Embodiments

[0805] Specifically, preferred herein are:

[0806] 1. An amylase variant of a parent amylase, wherein the amylase variant comprises

[0807] (i) amino acid substitutions at two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all of the amino acid positions corresponding to the amino acid positions selected from the group consisting of 25, 116, 176, 181, 186, 195, 206, 225, 320, and 482, according to the numbering of SEQ ID NO: 3, and

[0808] (ii) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1, 3, 4 or to any one of SEQ ID NO: 15-41, preferably to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1.

[0809] 2. The amylase variant of embodiment 1, wherein the amylase variant comprises

[0810] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3,

[0811] (ii) an amino acid substitution at one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 176, 181, 186, 195, 206, 225, 320, and 482, according to the numbering of SEQ ID NO: 3,

[0812] (iii) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1, 3, 4 or to any one of SEQ ID NO: 15-41, preferably to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1.

[0813] 3. The amylase variant of any of the preceding embodiments, wherein the amylase variant comprises an amino acid substitution at one or more positions selected from the group consisting of: 116, 181, 225, and 320.

[0814] 4. The amylase variant of any of the preceding embodiments, wherein the amylase variant comprises an amino acid substitution at one or more positions selected from the group consisting of 195 or 206, preferably the amylase variant comprises an amino acid substitution at either position 195 or position 206.

[0815] 5. The amylase variant of any of the preceding embodiments, wherein the amylase variant, the amylase variant of any of the preceding claims, wherein the amylase variant comprises an amino acid substitution at position 482.

[0816] 6. The amylase variant of any one of the preceding embodiments, wherein the amino acid substitution at position 25 is X25H or an amino acid substitution similar to X25H.

[0817] 7. The amylase variant of any of the preceding embodiments, wherein the amino acid substitution at position 116 is X116K, wherein the amino acid substitution at position 176 is X176K, wherein the amino acid substitution at position 181 is X181T, wherein the amino acid substitution at position 186 is X186E, wherein the amino acid substitution at position 195 is X195F, wherein the amino acid substitution at position 206 is X206Y, wherein the amino acid substitution at position 225 is X225A, wherein the amino acid substitution at position 320 is X320K, and wherein the amino acid substitution at position 482 is X482W, or amino acid substitutions similar to these substitutions.

[0818] 8. The amylase variant of any preceding embodiment, wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1.

[0819] 9. In one embodiment, the amylase variant of the present invention comprises

[0820] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3,

[0821] (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3, and

[0822] (iii) has at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1, 3, 4 or to any one of SEQ ID NO: 15-41, preferably to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1.

[0823] 10. In one embodiment, the amylase variant of the present invention comprises

[0824] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 3,

[0825] (ii) any one of the amino acid substitutions X195F or X206Y according to the numbering of SEQ ID NO: 3,

[0826] (iii) one or more, two or more, three or more, four or more, five or more, six or more, or all amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 3, and

[0827] (iv) has at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1, 3, 4 or to any one of SEQ ID NO: 15-41, preferably to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1.

[0828] 11. The amylase variant of any of the preceding embodiments, wherein the amylase variant has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least to 99.5% but less than 100% sequence identity to SEQ ID NO: 1.

[0829] 12. The amylase variant of any one of the preceding embodiments, wherein the amylase variant comprises

[0830] a) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and a substitution at one or more positions selected from 176 and 186; preferably, one or more substitutions selected from X176K and X186E, or

[0831] b) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and a substitution at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K, or

[0832] c) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and a substitution at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and a substitution at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K.

[0833] 13. The amylase variant of any of the preceding embodiments, wherein the variant comprises an amino acid substitution X195F and a substitution at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and a substitution at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K.

[0834] 14. The amylase variant of any of the preceding embodiments, wherein the variant comprises an amino acid substitution X206Y and a substitution at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and a substitution at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K.

[0835] 15. The amylase variant of any one of the preceding embodiments, wherein the variant comprises a combination of amino acid substitutions selected from the group consisting of:

[0836] X25H+X176K+X186E,

[0837] X25H+X176K+X186E+X195F,

[0838] X25H+X116K+X181T+X195F+X225A+X320K,

[0839] X25H+X116K+X176K+X181T+X195F+X225A+X320K,

[0840] X25H+X116K+X181T+X186E+X195F+X225A+X320K,

[0841] X25H+X116K+X181T+X195F+X225A+X320K+X482W,

[0842] X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K,

[0843] X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W,

[0844] X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W, and

[0845] X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W.

[0846] 16. The amylase variant of any one of the preceding embodiments, wherein the variant comprises a combination of amino acid substitutions selected from the group consisting of:

[0847] X25H+X176K+X186E,

[0848] X25H+X176K+X186E+X206Y,

[0849] X25H+X116K+X181T+X206Y+X225A+X320K,

[0850] X25H+X116K+X176K+X181T+X206Y+X225A+X320K,

[0851] X25H+X116K+X181T+X186E+X206Y+X225A+X320K,

[0852] X25H+X116K+X181T+X206Y+X225A+X320K+X482W,

[0853] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K,

[0854] X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W,

[0855] X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W, and

[0856] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W.

[0857] 17. The amylase variant of any of the preceding embodiments, comprising the amino acid substitutions X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K, or X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W (according to the numbering of SEQ ID NO: 3).

[0858] 18. The amylase variant of any of the preceding embodiments, further comprising a deletion of two amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion corresponding to the amino acids at positions 181 and 182, 182 and 183, or 183 and 184, preferably D183* and G184*, wherein the numbering is according to the amino acid sequence shown in SEQ ID NO: 3.

[0859] 19. An amylase variant as described in any of the preceding embodiments, wherein the amylase variant has at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5% but less than 100% sequence identity to any one of SEQ ID NO: 1, 3 or 4, preferably SEQ ID NO: 1.

[0860] 20. The amylase variant of any of the preceding embodiments, wherein the variant comprises A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75% but less than 100% identical to the amino acid sequence of SEQ ID NO:6, and the amino acid sequence of the C domain is at least 75% but less than 100% identical to the amino acid sequence of SEQ ID NO:8.

[0861] 21. The amylase variant of any of the preceding embodiments, wherein the amylase variant comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 1, 3, 4, or any one of SEQ ID NOs: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, having amino acid substitution X25H and having any of amino acid substitutions X195F or X206Y and having 1 to 7, preferably 2 to 7 or 3 to 7, such as 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K and X482W, and preferably having 1 to 10, preferably 1 to 5 additional conservative amino acid substitutions, and preferably a deletion of two amino acids corresponding to positions selected from the group consisting of: 181, 182, 183 and 184.

[0862] 22. The amylase variant of any preceding embodiment, wherein the amylase variant further comprises 1 to 50, preferably 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 8, or 2 to 5, preferably 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 8, or 3 to 5, preferably 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10, or 4 to 8 conservative amino acid exchanges, preferably wherein the conservative mutations do not belong to the catalytic center of the amylase variant.

[0863] 23. The amylase variant of any of the preceding embodiments, wherein the amylase variant comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, has NO.3 numbered changes X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K, or X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W, preferably X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W and optionally having 1 to 10, preferably 1 to 5 additional conservative amino acid substitutions.

[0864] 24. The amylase variant of any of the preceding embodiments, wherein the substitution is selected from N25H, W116K, R176K, R181T, G186E, N195F, I206Y, T225A, R320K, and Y482W.

[0865] 25. The amylase variant of any of the preceding embodiments, wherein the amylase variant exhibits one or more improved properties, preferably relative to the parent amylase, preferably relative to SEQ ID NO: 1 or SEQ ID NO: 3, preferably the parent amylase shown in SEQ ID NO: 1, or relative to the amylase shown in SEQ ID NO: 33, preferably the amylase variant has increased stability, thermostability, storage stability, storage stability in detergent compositions, wash performance, wash performance in laundry detergents, and / or wash performance in dishwashing detergents, preferably the improved properties are improved storage stability and / or improved wash performance, preferably improved wash performance on laundry, most preferably the improved properties are improved storage stability, preferably wherein the improved properties are expressed as an improvement factor (IF) > 1.0, and wherein preferably the improvement factor is equal to or greater than 1.1, preferably, equal to or greater than 1.2, more preferably, equal to or greater than 1.3, preferably the amylase variant exhibits improved storage stability in detergent compositions, preferably relative to a reference amylase, preferably relative to the parent amylase, preferably relative to SEQ ID NO: 33. NO:1 or SEQ ID NO:3, preferably the parent amylase shown in SEQ ID NO:1, or relative to the amylase shown in SEQ ID NO:33, preferably having an improvement factor equal to or greater than 1.1, preferably equal to or greater than 1.2, more preferably equal to or greater than 1.3, preferably after storage at 40°C in a detergent composition, preferably for 14 days, 24 days, 56 days or 84 days, preferably for 84 days, preferably as determined in an ES1-C detergent as described herein, preferably containing additional builder (preferably HEDP), preferably relative to a reference amylase, preferably relative to said parent amylase, preferably relative to SEQ ID NO:1 or SEQ ID NO:3, preferably the parent amylase shown in SEQ ID NO:1, or relative to the amylase shown in SEQ ID NO:33; and / or preferably the amylase variant exhibits improved wash performance on laundry, preferably at 40°C, preferably relative to an amylase having the amino acid sequence shown in SEQ ID NO:33.

[0866] 26. A polynucleotide encoding the amylase variant of any preceding embodiment.

[0867] 27. A formulation comprising the amylase variant of any one of embodiments 1 to 25 and at least one additional component, preferably a solvent.

[0868] 28. A composition as described in embodiment 27, wherein the composition comprises one or more second enzymes different from the amylase variant mentioned in any of the preceding claims, preferably one or more second enzymes selected from the group consisting of: protease, second amylase, lipase, cellulase, hemicellulase, mannanase, xylanase, DNase, dispersin, pectinase, oxidoreductase and cutinase, preferably selected from protease, mannanase and lipase, most preferably protease.

[0869] 29. The formulation of embodiment 27 or 28, comprising an enzyme stabilization system, preferably comprising a calcium salt, and optionally a protease inhibitor in the presence of a protease.

[0870] 30. A detergent composition comprising the amylase variant of any one of embodiments 1 to 25, preferably a laundry detergent composition or a hard surface cleaning detergent composition, most preferably a laundry detergent composition.

[0871] 31. The detergent composition of embodiment 30, wherein the composition comprises one or more surfactants and / or one or more builders, preferably one or more strong chelating builders.

[0872] 32. A detergent composition as described in any of embodiments 30 or 31, wherein the detergent composition comprises components selected from the group consisting of: enzyme stabilization systems, surfactants, defoamers, builders, polymers, bleaching systems (bleaches), rheology modifiers, hydrotropes, softeners, desiccants, brighteners, buffers, foam inhibitors, preservatives, anti-corrosion additives, dyes and fragrances.

[0873] 33. The detergent composition of any one of embodiments 30 to 32, comprising a builder, wherein the builder is selected from MDGA, GLDA, DTPMP, HEDP and EDDS, preferably MDGA or EDDS.

[0874] 34. The detergent composition of embodiment 33, wherein the builder is a non-phosphate based builder.

[0875] 35. The detergent composition of any one of embodiments 30 to 34, comprising a surfactant, wherein the surfactant is selected from the group consisting of nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, and combinations thereof.

[0876] 36. The detergent composition of any one of embodiments 30 to 35, wherein the detergent composition is free of anionic surfactants.

[0877] 37. The detergent composition of any one of embodiments 30 to 36, wherein the surfactant and / or the builder is biodegradable and / or bio-based.

[0878] 38. The detergent composition of any one of embodiments 30 to 37, wherein the detergent composition is a liquid or a solid.

[0879] 39. The detergent composition of any one of embodiments 30 to 38, wherein the detergent composition is in the form of a sachet.

[0880] 40. The detergent composition of any one of embodiments 30 to 39, wherein the detergent composition is a liquid laundry detergent composition.

[0881] 41. The detergent composition of any one of embodiments 30 to 40, wherein the detergent composition is free of boron.

[0882] 42. The detergent composition of any one of embodiments 30 to 41, wherein the detergent composition does not contain a preservative.

[0883] 43. A method for cleaning, the method comprising the steps of contacting a textile or a hard surface, preferably a textile, with an amylase variant as described in any of embodiments 1-25 or a formulation as described in any of embodiments 27-29 or a detergent composition as described in any of embodiments 30-42, preferably at low temperature, preferably at 30°C or 40°C, preferably at 30°C.

[0884] 44. Use of an amylase variant as described in any of embodiments 1-25 or a formulation as described in any of embodiments 27-29 or a detergent composition as described in any of embodiments 30-42 to improve amylase stability in a detergent composition and / or to improve the wash performance of a detergent composition, preferably on stains sensitive to amylases, preferably relative to the amylase shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, or relative to the amylase shown in SEQ ID NO: 33, preferably at 30°C or 40°C, preferably at 40°C, preferably as determined in an ES1-C detergent as described herein, preferably containing an additional builder (preferably HEDP).

[0885] Examples

[0886] General method:

[0887] Amylase expression

[0888] By the standard scheme based on restriction endonuclease digestion-connection, the gene encoding amylase is cloned into a Gram-positive expression vector, which contains a promoter sequence, a sequence encoding a secretion signal peptide, and a ribosome binding site. After the reaction, the plasmid assembly mixture is transformed into Bacillus subtilis PY79 by the natural competence transformation method established. Successful transformation is selected by plating on LB agar plates supplemented with 20 μg / ml kanamycin sulfate and incubating overnight at 37 degrees Celsius. After overnight selection, a single bacterium colony is obtained, and then in a rich culture medium (e.g., LB liquid culture medium) with 20 μg / ml kanamycin sulfate, shaken overnight at 250rpm at 37 degrees Celsius. The next morning, cells are precipitated by centrifugation, and the Macherey-Nagel NucleoSpin test kit is used to separate plasmid DNA by alkaline lysis. The DNA of separation can then be transformed into electrocompetent Bacillus licheniformis cells.

[0889] Bacillus licheniformis cells are made electrocompetent by standard Gram-positive electrocompetent cell preparation techniques. For example, cells are prepared by culturing Bacillus licheniformis strains in a rich medium containing a concentrated osmotic agent (e.g., LB liquid medium containing 0.5M D-sorbitol) and harvesting cells in the early exponential growth phase. Cells are harvested by cooling on ice and precipitating by centrifugation. After harvesting, cells are washed by 3 suspension-precipitation (by centrifugation) cycles with a wash buffer rich in an osmotic agent (e.g., 10% glycerol with 0.5M D-sorbitol and 0.5M D-mannitol) to remove salts. Finally, cells are concentrated by being resuspended in a wash buffer at 1% to 10% of the original culture volume.

[0890] Once prepared, plasmid DNA is added to Bacillus licheniformis electrocompetent cells in a 0.2 cm Bio-Rad electroporation cuvette. According to the manufacturer's instructions, cells are electroporated using a Bio-Rad Gene Pulser Xcell. Cells are immediately rescued by the addition of 1 ml of a rich medium containing a concentrated osmotic agent after the electroshock. After two hours of recovery at 37 degrees Celsius, successful transformants are then selected by plating on LB agar plates supplemented with 20 μg / ml kanamycin sulfate and incubating overnight at 37 degrees Celsius.

[0891] Amylase expression and protein quantification

[0892] The single colony of the expression strain is picked into 60 μL rich culture medium (e.g., LB liquid medium) supplemented with 20 μg / mL kanamycin sulfate in 96-well plates (GE Life Sciences part 28403943). The culture is grown at 37 degrees Celsius for 16 hours, and then 15 μL culture is used to inoculate 600 μL of defined glucose-mineral medium and 20 μg / mL kanamycin sulfate in 96-well plates. The culture is grown at 37 degrees Celsius for 48 hours, and then the supernatant is harvested by using centrifugation to precipitate cells and remove residual culture fluid. A larger amount is produced by merging the supernatant of the same clone. The expression level (concentration is in mg / mL) of alpha-amylase activity is identified by LabChip (LC) analysis to allow equal addition in laundry detergent.

[0893] Amylase activity measurement

[0894] Alpha-amylase activity can be determined by the method using ethylidene-4-nitrophenyl-α-D-maltoseptaglycoside (EPS) as substrate. D-maltoseptaglycoside is a closed oligosaccharide that can be cut by endoamylase. After cutting, the α-glucosidase digests the substrate contained in the test kit, releases yellow free PNP molecules, and can therefore be measured at 405nm by visible spectrophotometry. The test kit containing EPS substrate and α-glucosidase is manufactured by Roche Costum Biotech (Cat. No. 10880078103) or Thermo Scientific (TR25421). The slope of the time-dependent absorption curve is directly proportional to the specific activity (activity / mg enzyme) of the α-amylase discussed under given conditions. The residual activity is calculated by dividing the activity after the storage time by the activity of the sample at time zero. The residual activity is compared with the residual activity of the reference given in the following example.

[0895] Example 1: Storage stability in a detergent containing builder at 40°C for up to 84 days.

[0896] Table 2: Testing of amylase variants of the invention compared to a reference amylase (SEQ ID NO: 33) using standard detergent.

[0897]

[0898]

[0899] (*) Amylase variant of SEQ ID NO: 1, whose mutations are given with the numbering of SEQ ID NO: 3.

[0900] The amylase was formulated in a liquid standard detergent (ES1-C) and tested for residual activity after storage at 40°C for 84 days. The final dose was 0.1 g / L amylase. To evaluate the stability of the amylase in the presence of strong chelators, HEDP and / or CaCl2 were supplemented as indicated in the table. No additional CaCl2 was supplemented by the enzyme. 2+ .

[0901] Table 3: ES1-C formulation:

[0902] ES1-C wt% Maranil DBS / LC Linear alkylbenzene sulfonate, anionic surfactant 5.5 Edenor Coconut Fatty Acid <![CDATA[C 12 -C 18 Coconut fatty acid]]> 2.4 Lutensol AO7 Alkyl polyglycol ether, nonionic surfactant 5.5 Texapon N70 Sodium Laureth Sulfate 2EO, anionic surfactant 5.5 1,2-Propanediol 6.0 <![CDATA[C2H5OH]]> 2.0 NaOH 2.2 Citrate 3.0 pH 8-8.5 <![CDATA[H2O]]> Top up to 100%

[0903] After the specified time point, samples were taken and residual activity was measured using EPS as substrate, as described above. The residual activity was calculated by dividing the activity after the storage time by the activity of the sample at time point zero. The residual activity was compared to the residual activity of a reference amylase (SEQ ID NO: 33).

[0904] Table 4:

[0905]

[0906] As can be concluded from the table above, the amylase variants of the present invention are more stable than the reference amylase.

[0907] Example 2: Improved washing performance of amylase

[0908] Determination of washing performance using a Launder-o-meter:

[0909] At 40°C, several soiled swatches were washed with a liquid laundry formulation along with a cotton ballast fabric and 20 steel balls. After washing, the fabric was rinsed, spun and air-dried. Washing performance was determined by measuring the CIELab values ​​of the soiled fabric before and after washing using MACH5 multi-region color measurement. For data evaluation, the ΔE between unwashed and washed stains was calculated, and the ΔΔE between enzyme detergent and non-enzyme detergent was calculated. The data represent the average of two separate experiments.

[0910] Table 6: Washing conditions

[0911]

[0912] 1) CFT-CS28, CFT-CS129 Manufacturer: Center for Testmaterials BV, NL-3130AC Vlaardingen

[0913] 2) EMPA 161, manufacturer: Swissatest Testmaterialien AG, Seagull Street 12, 9015 St.Gallen, Switzerland

[0914] Table 7: Initial performance in the Washfastness Tester. The data show the aggregate performance for three stains at 40°C.

[0915]

[0916] (*) Mutations in SEQ ID NO: 1 are given with the numbering of SEQ ID NO: 3

[0917] From the above table it can be concluded that the amylase variants of the present invention provide better cleaning results compared to the reference amylase.

[0918] Determination of washing performance in full-size washing machines:

[0919] At 40 ℃, wash multi-stain monitors in the presence of cotton ballast fabric in full-size washing machines with liquid laundry formulations containing different amylases.Each stain is washed in quadruplicate (2 machines, two / stain / machine).After washing, the stain monitor is air-dried.Determination of washing performance is carried out by measuring the CIELab value of dirty fabric before and after washing using MACH5 multi-region color measurement.For data evaluation, mean value is determined, the ΔY between unwashed and washed stains is calculated, and the ΔΔY between enzyme detergent and non-enzyme detergent is calculated.

[0920] Table 8: Washing conditions

[0921]

[0922]

[0923] 1) Obtained from Center for Testmaterials BV, NL-3130AC Vlaardingen

[0924] 2) Obtained from Swissatest Testmaterialien AG, Seagull Street 12, 9015 St.Gallen, Switzerland

[0925] Table 9: Full scale wash performance of ES1C formulation at 40°C. The data show the aggregate performance on all 11 stains, expressed as ΔΔY:

[0926]

[0927] (*) Mutations in SEQ ID NO: 1 are given with the numbering of SEQ ID NO: 3

[0928] From the above table it can be concluded that the amylase variants of the present invention provide better cleaning results compared to the reference amylase.

Claims

1. An amylase variant of a parent amylase, wherein the amylase variant (i) comprises an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 3, (ii) comprises an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 176, 181, 186, 195, 206, 225, 320 and 482 according to the numbering of SEQ ID NO: 3, and (iii) having at least 60% but less than 100% sequence identity with SEQ ID NO: 1, 3, 4 or with any one of SEQ ID NO: 15-41, preferably with SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO:

1.

2. The amylase variant of claim 1, wherein the amylase variant comprises an amino acid substitution at one or more positions selected from the group consisting of: 116, 181, 225 and 320.

3. The amylase variant of claim 1 or claim 2, wherein the amylase variant comprises an amino acid substitution at one or more positions selected from the group consisting of 195 or 206, preferably the amylase variant comprises an amino acid substitution at either position 195 or position 206.

4. The amylase variant of any one of the preceding claims, wherein the amylase variant comprises an amino acid substitution at position 482.

5. The amylase variant of any one of the preceding claims, wherein the amino acid substitution at position 25 is X25H.

6. The amylase variant of any of the preceding claims, wherein the amino acid substitution at position 116 is X116K, wherein the amino acid substitution at position 176 is X176K, wherein the amino acid substitution at position 181 is X181T, wherein the amino acid substitution at position 186 is X186E, wherein the amino acid substitution at position 195 is X195F, wherein the amino acid substitution at position 206 is X206Y, wherein the amino acid substitution at position 225 is X225A, wherein the amino acid substitution at position 320 is X320K, and wherein the amino acid substitution at position 482 is X482W.

7. The amylase variant of any one of the preceding claims, wherein the amylase variant comprises a) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and a substitution at one or more positions selected from 176 and 186; preferably, one or more substitutions selected from X176K and X186E, or b) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and a substitution at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K, or c) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and a substitution at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and a substitution at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K.

8. The amylase variant of any one of the preceding claims, wherein the variant comprises a combination of amino acid substitutions selected from the group consisting of: X25H+X176K+X186E, X25H+X176K+X186E+X195F, X25H+X176K+X186E+X206Y, X25H+X116K+X181T+X195F+X225A+X320K, X25H+X116K+X176K+X181T+X195F+X225A+X320K, X25H+X116K+X181T+X186E+X195F+X225A+X320K, X25H+X116K+X181T+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K, X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W, X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W, X25H+X116K+X181T+X206Y+X225A+X320K, X25H+X116K+X176K+X181T+X206Y+X225A+X320K, X25H+X116K+X181T+X186E+X206Y+X225A+X320K, X25H+X116K+X181T+X206Y+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K, X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W, X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W, and X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W.

9. The amylase variant of any of the preceding claims, further comprising a deletion at one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion corresponding to two or more amino acids at positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion corresponding to amino acids at positions 181 and 182, 182 and 183, or 183 and 184, wherein the numbering is according to the amino acid sequence shown in SEQ ID NO:

3.

10. An amylase variant as claimed in any of the preceding claims, wherein the amylase variant has at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5% but less than 100% sequence identity to any one of SEQ ID NOs: 1, 3, 4 or to any one of SEQ ID NOs: 15-41, preferably to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO:

1.

11. A polynucleotide encoding the amylase variant as claimed in any one of the preceding claims.

12. A composition comprising the amylase variant of any one of claims 1 to 10, and at least one additional component.

13. A composition as claimed in claim 12, wherein the composition comprises one or more second enzymes different from the amylase variant mentioned in any of the preceding claims, preferably one or more second enzymes selected from the group consisting of: proteases, second amylases, lipases, cellulases, hemicellulases, mannanases, xylanases, DNases, dispersins, pectinases, oxidoreductases and cutinases, preferably selected from proteases, mannanases and lipases, most preferably proteases.

14. A composition as claimed in claim 12 or claim 13, wherein the composition is a detergent composition, preferably a laundry detergent composition or a hard surface cleaning detergent composition.

15. A composition according to any one of claims 12 to 14, wherein the composition comprises one or more surfactants and / or one or more builders, preferably one or more strong chelating builders.

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