Amylase variants

Abstract

The present invention provides novel amylase enzymes, more specifically genetically engineered amylase enzymes, compositions containing the enzymes, and methods of making and using the enzymes or compositions containing the enzymes.

Description

[Technical Field]

[0001] The present invention provides novel amylase enzymes, more specifically genetically engineered amylase enzymes, compositions containing the enzymes, and methods of making and using the enzymes or compositions containing the enzymes. [Background technology]

[0002] Enzymes are increasingly being used in a variety of applications as a sustainable alternative to petrochemicals. Enzymes are biodegradable and can be catalytically active even at low temperatures, resulting in reduced energy consumption. In particular, in the detergent industry, enzymes are being incorporated into cleaning formulations to improve cleaning efficiency and reduce energy consumption in the washing process. Summary of the Invention [Problem to be solved by the invention]

[0003] Amylases are enzymes that can hydrolyze starch. Therefore, amylases are utilized in the removal of starch stains and are added to detergent compositions for this purpose. In detergent applications, amylases must be stable at high temperatures and / or under the denaturing conditions of detergents and wash liquors. Therefore, there is a need for new amylase enzymes that meet these requirements. [Means for solving the problem]

[0004] The present invention relates to amylase variants of a parent amylase, said variants comprising: (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 3; (ii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to amino acid positions selected from the group consisting of 25, 176, 186, 251, 405, and 482 according to the numbering of SEQ ID NO: 3; and (iii) any of SEQ ID NOs: 1, 3, 4, or 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, preferably at least 91% but less than 100% sequence identity to SEQ ID NO: 1 (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) Includes:

[0005] In one embodiment, the amylase variant of the invention comprises: (i) the amino acid substitution X4G according to the numbering of SEQ ID NO: 3; (ii) one or more, two or more, three or more, four or more, five or more, or all of the amino acid substitutions selected from the group consisting of X25H, X176K, X186E, X251E, X405M, and X482W according to the numbering of SEQ ID NO: 3; and (iii) any of SEQ ID NOs: 1, 3, 4, or SEQ ID NOs: 15 to 41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1; 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%, or at least 89% similar to SEQ ID NO: 1 , 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. Includes:

[0006] In another embodiment, the present invention relates to polynucleotides encoding amylase variants.

[0007] Furthermore, the present invention relates to a composition comprising the amylase variant, preferably a detergent composition.

[0008] The present invention further relates to methods of making the amylase variants and methods of using the amylase variants, particularly for cleaning objects. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention may be understood more readily by reference to the following more detailed description of embodiments of the invention and examples included herein.

[0010] While the present invention has been described with reference to specific embodiments, this description is not intended to be construed in a limiting sense.

[0011] definition Unless otherwise noted, terms used herein are to be understood according to conventional usage by those of ordinary skill in the art.

[0012] Before describing exemplary embodiments of the present invention in detail, definitions important for understanding the present invention are provided. Unless otherwise stated or apparent from the nature of the definition, the definitions apply to all compounds, methods and uses described herein.

[0013] As used in this specification and the appended claims, the singular forms "a" and "an" include the respective plural forms as well, unless the context clearly dictates otherwise.

[0014] In the context of the present invention, the terms "about" and "approximately" refer to a margin of accuracy that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically denotes a deviation of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5% from the indicated numerical value.

[0015] Furthermore, the terms "first," "second," "third," or "(a)," "(b)," "(c)," "(d)," etc. in the specification and claims are used to distinguish between similar components and do not necessarily describe a sequential or chronological order. Terms so used are interchangeable under appropriate circumstances, and it is understood that the embodiments of the invention described herein can be performed in orders other than those described or illustrated herein. When the terms "first," "second," "third," or "(a)," "(b)," "(c)," "(d)," "i," "ii," etc. refer to steps of a method, use, or assay, unless otherwise specified in this application, as described herein above or below, there is no time gap or inconsistency between the steps, i.e., the steps may be performed simultaneously, or there may be a time gap of seconds, minutes, hours, days, weeks, months, or even years between such steps.

[0016] Various articles are referenced throughout this application. The entire disclosures of these articles and the references cited within them 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.

[0017] The term "comprising" should be understood to be open-ended. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising." Hereinafter, when a group is defined as comprising at least a certain number of members, this means that it also encompasses groups consisting only of these members.

[0018] A "variant" enzyme differs from a "parent" enzyme by specific amino acid modifications, preferably amino acid substitutions at one or more amino acid positions.

[0019] In describing the variants of the present invention, abbreviations for single amino acids are used according to accepted IUPAC single-letter or three-letter amino acid abbreviations.

[0020] "Amino acid modification," as used herein, refers to an amino acid substitution, deletion, or insertion.

[0021] A "substitution" is described by the original amino acid followed by the number of the position in the amino acid sequence, followed by the amino acid that replaces the original amino acid. For example, a substitution of histidine with alanine at position 120 would be designated "His120Ala" or "H120A." A substitution may also be described by simply naming the resulting amino acid in the variant, without specifying the parent amino acid at this position, e.g., "X120A," or "120A," or "Xaa120Ala," or "120Ala."

[0022] A "deletion" is described by adding the original amino acid followed by the number of the position in the amino acid sequence, followed by an *. Thus, a deletion of glycine at position 150 would be designated "Gly150*" or "G150*." Alternatively, a deletion is indicated by, for example, "deletion of D183 and G184."

[0023] An "insertion" is described by the original amino acid followed by the number of the position in the amino acid sequence, followed by the original amino acid and the added amino acid. For example, an insertion of a lysine at position 180 next to a glycine would be designated as "Gly180GlyLys" or "G180GK." If two or more amino acid residues are inserted, e.g., Lys and Ala after Gly180, this can be indicated as "Gly180GlyLysAla" or "G195GKA."

[0024] When a substitution and an insertion occur at the same position, this can be designated "S99SD+S99A" or, for short, "S99AD". Variants containing multiple modifications are separated by "+", for example, "Arg170Tyr+Gly195Glu", "R170Y+G195E", or "X170Y+X195E" represent substitutions of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively, multiple modifications can be separated by spaces or commas, for example, "R170Y G195E" or "R170Y,G195E", respectively. When different distinct modifications can be introduced at a position, the different modifications are separated by commas, for example, "Arg170Tyr,Glu" and "R170T,E" represent substitutions of arginine at position 170 with tyrosine or glutamic acid, respectively. Alternative substitutions at specific positions may also be indicated as "X120A,G,H", "120A,G,H", "X120A / G / H" or "120A / G / H". Alternatively, different modifications or optional substitutions may be indicated in square brackets, for example "Arg170[Tyr,Gly]" or "Arg170{Tyr,Gly}" or, in summary, "R170[Y,G]" or "R170{Y,G}".

[0025] The term "native" (or naturally occurring 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 (i.e., without any human intervention).

[0026] The term "heterologous" (or exogenous or foreign or recombinant or non-natural or non-natural) polypeptide is defined herein as a polypeptide that is not native to a host cell, a polypeptide native to a host cell in which structural modifications, e.g., deletions, substitutions, and / or insertions, have been created by recombinant DNA techniques to modify the native polypeptide, or a polypeptide native to a host cell in which expression is quantitatively altered or expression is directed from a different genomic location than in the native host cell as a result of manipulation of the host cell's DNA by recombinant DNA techniques, e.g., by a stronger promoter. Similarly, the term "heterologous" (or exogenous or foreign or recombinant or non-natural or non-natural) polynucleotide refers to a polynucleotide that is not native to a host cell, a polypeptide native to a host cell in which structural modifications, e.g., deletions, substitutions, and / or insertions, have been created by recombinant DNA techniques to modify the native nucleotide, or a polynucleotide native to a host cell in which expression is quantitatively altered as a result of manipulation of the polynucleotide's regulatory elements by recombinant DNA techniques, e.g., by a stronger promoter, or a polynucleotide that is native to a host cell but is not integrated into its natural genetic environment as a result of 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 the fact that the two or more polynucleotide sequences or the two or more amino acid sequences do not naturally occur in that particular combination with each other.

[0027] For 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 man using genetic techniques. With respect to a polynucleotide, "recombinant" means (a) the sequence of a polynucleotide or a portion thereof, or (b) one or more gene control sequences operably linked to the polynucleotide, including, but not limited to, a promoter therefor; or (c) Both a) and b) This includes all constructs produced by using genetic / recombinant DNA techniques in which either the gene is not placed in its wild-type genetic environment or has been modified by man.

[0028] A "synthetic" compound is obtained by in vitro chemical and / or enzymatic synthesis.

[0029] Variant polynucleotide and variant polypeptide sequences can be defined by their sequence identity when compared to another sequence. Sequence identity is usually provided as "% sequence identity" or "% identity". To calculate sequence identity, the first step is to generate a sequence alignment. According to the present invention, a pairwise global alignment is generated, which means that two sequences are aligned over their entire length, usually by using a mathematical method called an alignment algorithm.

[0030] According to the present invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, pp. 443-453). Preferably, the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) is used for the present invention, using the program's default parameters (polynucleotides: gap open = 10.0, gap extension = 0.5, and matrix = EDNAFULL; polypeptides: gap open = 10.0, gap extension = 0.5, and matrix = EBLOSUM62). After aligning the two sequences, in a second step, an identity value is determined from the resulting alignment. For this purpose, the % identity is calculated by dividing the number of identical residues by the length of the alignment region representing each of the sequences of the present invention over its entire length, and multiplying by 100: % identity = (identical residues / length of the alignment region representing each of the sequences of the present invention over its entire length) * 100.

[0031] To calculate the percent identity of two nucleic acid sequences, the same applies as for calculating the percent identity of two amino acid sequences, with some criteria. For nucleic acid sequences encoding proteins, pairwise alignment shall be performed over the entire length of the coding region of the sequence of the present invention, from the start codon to the stop codon, excluding introns. Introns present in the other sequence to which the sequence of the present invention is compared shall also be removed for pairwise alignment. After aligning the two sequences, in the second step, the identity value is determined from the resulting alignment. The percent identity is calculated by %-identity = (identical residues / length of the aligned region representing the sequence of the present invention, from the start codon to the stop codon, excluding introns, over its entire length) * 100.

[0032] Furthermore, a preferred alignment program for nucleic acid sequences that implements the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, pp. 443-453) is "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) with the program's default parameters (gap open=10.0, gap extension=0.5, and matrix=EDNAFULL).

[0033] Various polypeptides can be defined by their sequence similarity when compared to another sequence. Sequence similarity is usually provided as "% sequence similarity" or "%-similarity". % sequence similarity considers that a defined set of amino acids share similar properties, e.g., due to their size, hydrophobicity, charge, or other characteristics. Herein, the replacement of one amino acid with a similar amino acid may be referred to as a "conservative mutation". Similar amino acids according to the present invention are defined as follows and shall also be applied for the determination of %-similarity according to the present invention, according to the BLOSUM62 matrix, as used, for example, by the program "NEEDLE", which is one of the most used amino acid similarity matrices for database searching and sequence alignment: Amino acid A is similar to amino acid S, Amino acid D is similar to amino acids E and N, The amino acid E is similar to the amino acids D, K, and Q. The amino acid F is similar to the amino acids W and Y. The amino acid H is similar to the amino acids N and Y. Amino acid I is similar to amino acids L, M, and V, The amino acid K is similar to the amino acids E, Q, and R. The amino acid L is similar to the amino acids I, M, and V, The amino acid M is similar to the amino acids I, L, and V, The amino acid N is similar to the amino acids D, H, and S, The amino acid Q is similar to the amino acids E, K, and R, The amino acid R is similar to the amino acids K and Q. The amino acid S is similar to the amino acids A, N, and T, The amino acid T is similar to the amino acid S, Amino acid V is similar to amino acids I, L, and M, Amino acid W is similar to amino acids F and Y, The amino acid Y is similar to the amino acids F, H, and W.

[0034] For calculating sequence similarity, in the first step, a sequence alignment is generated as described above.After aligning two sequences, in the second step, a similarity value is determined from the generated alignment.For this purpose, %-similarity is calculated by dividing the number of identical residues plus similar residues by the length of the alignment region that represents the sequence of the present invention over its entire length, and multiplying by 100: %-similarity=[(identical residues+similar residues) / length of the alignment region that represents the sequence of the present invention over its entire length]*100.

[0035] For nucleic acids, similar sequences can also be determined by hybridization using the respective stringency conditions. The term "high stringency conditions" refers to prehybridization and hybridization for 12-24 hours according to standard Southern blotting procedures, for probes at least 100 nucleotides in length, in 5x SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 50% formamide at 42°C. The carrier material is finally washed three times for 15 minutes each using 2x SSC, 0.2% SDS at 65°C. The term "very stringency conditions" refers to prehybridization and hybridization for 12-24 hours according to standard Southern blotting procedures, for probes at least 100 nucleotides in length, in 5x SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 50% formamide at 42°C. The carrier material is finally washed three times with 2x SSC, 0.2% SDS at 70°C for 15 minutes each.

[0036] As used herein, a "fragment" or "subsequence" refers to a portion of a polynucleotide or amino acid sequence. The term "functional fragment" refers to any nucleic acid or amino acid sequence that contains only a small portion of a full-length amino acid sequence, respectively, but still has the same or similar activity and / or function. Preferably, a 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. A functional fragment contains consecutive nucleotides or amino acids compared to the original nucleic acid or original amino acid sequence, respectively.

[0037] A "genetic construct" or "expression cassette," as used herein, is a nucleic acid molecule comprised of at least one sequence of interest to be expressed operably linked to one or more control sequences (at least one promoter) as described herein.

[0038] The term "vector," as used herein, includes any type of construct suitable for carrying an exogenous polynucleotide sequence for transfer to another cell or for stable or transient expression within a given cell. The term "vector," as used herein, encompasses any type of cloning vehicle, such as, but not limited to, plasmids, phagemids, viral vectors (e.g., phages), bacteriophages, baculoviruses, cosmids, fosmids, artificial chromosomes, and any other vector specific to a particular host of interest. The exogenous polynucleotide sequence typically includes a coding sequence, which may be referred to herein as a "gene of interest." The gene of interest may include introns and exons, depending on the type of origin or destination host cell.

[0039] The term "introduction of a polynucleotide" or "transformation of a polynucleotide," as referred to herein, encompasses the transfer of an exogenous polynucleotide into a host cell, regardless of the method used for the transfer. That is, the term "transformation of a polynucleotide," as used herein, is independent of vectors, shuttle systems, or host cells; it not only relates to the polynucleotide transfer method of transformation as 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, NY), but also encompasses any additional type of polynucleotide transfer method, such as, but not limited to, transduction or transfection.

[0040] A polynucleotide encoding a polypeptide may be "expressed." The term "expression" or "gene expression" refers to the transcription of a gene or gene construct into structural RNA (e.g., rRNA, tRNA) or mRNA, with or without subsequent translation of the latter into a protein. This process includes transcription of DNA and processing of the resulting mRNA product.

[0041] 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 a different compartment or phase, which compartment or phase does not necessarily have to be separated by a physical barrier. Examples of such different compartments are two compartments, i.e., a filtrate and a retentate, separated by a filtration membrane or cloth, and examples of such different phases are a pellet and a supernatant or a cake and a filtrate, respectively. The solution obtained after purifying the protein of interest from the fermentation broth is referred to herein as a "purified enzyme solution."

[0042] A "protein formulation" (or "enzyme preparation"), e.g., a "protein variant formulation," refers to any non-complex formulation containing a small number of ingredients, which serve the purpose of stabilizing the proteins contained in the protein formulation and / or the protein formulation itself. Preferably, a non-complex protein formulation contains proteins at higher concentrations than complex formulations, e.g., detergent compositions. Thus, preferably, a non-complex protein formulation is a concentrated protein variant formulation. Preferably, a non-complex protein formulation contains 2-120 mg / g of active enzyme, while a complex formulation, such as a detergent composition, contains 0.002-6 mg / g of active enzyme.

[0043] "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" refers to the catalytic effect exerted by an enzyme, expressed as units per milligram of enzyme (specific activity) or molecules of substrate converted per minute per molecule of enzyme (molecular activity). Enzyme activity can be specified by the actual function of the enzyme, such as proteases that exert proteolytic activity by catalyzing the hydrolytic cleavage of peptide bonds, lipases that exert lipolytic activity by hydrolytic cleavage of ester bonds, and amylase activity, which is involved in the hydrolysis of glycosidic bonds in polysaccharides.

[0044] 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.

[0045] To determine and quantify changes in the catalytic activity of an enzyme stored or used under specific conditions over time, the "initial enzyme activity" is measured under defined conditions at time zero (100%) and at a later specific time point (x%). By comparing the measured values, the extent of potential decrease in enzyme activity can be determined. The extent of decrease in enzyme activity determines the stability or instability of the enzyme.

[0046] An "enzyme inhibitor," as used herein, is a compound that slows or stops enzyme activity. Enzyme inhibitors often also stabilize the enzyme in its three-dimensional structure. Thus, enzyme inhibitors usually also act as "enzyme stabilizers."

[0047] "pH stability" refers to the ability of an enzyme to exhibit enzymatic activity after exposure to a particular pH value.

[0048] The terms "thermostable", "thermostable" or "thermo-dependent activity" preferably refer to the ability of an enzyme to exhibit catalytic activity or cleaning performance in a detergent composition (preferably in Model ES1-C detergent) after exposure to elevated temperatures, preferably 40°C for 28 days, preferably 56 days, or 92°C for at least 10 minutes.

[0049] The term "detergent stability" or "stability under storage in a detergent composition" refers to the ability of an enzyme to exhibit catalytic activity or cleaning performance in a detergent composition, preferably in a detergent composition (preferably in Model ES1-C detergent) at a temperature of 40°C or 50°C after storage for 28 days, preferably 56 days.

[0050] The "improvement factor" (IF) is preferably the degree of improvement of an enzyme variant in a particular property over its respective parent enzyme. The improvement of an enzyme variant relative to its respective parent enzyme is preferably characterized by an improvement factor (IF) of >1.0. Alternatively, the improvement factor can be expressed as a percentage, e.g., an IF of 1.1 is equivalent to 110%.

[0051] 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 enzyme's performance. The term "washing performance" is used herein equally for laundry and hard surface cleaning. Washing performance is compared under appropriate wash conditions. The term "appropriate wash conditions" is used herein to refer to the conditions, particularly the wash temperature, time, wash dynamics, foam concentration, detergent type and water hardness, that are actually used in homes in the detergent market segment. The term "improved wash performance" is used to indicate that a better end result is obtained in stain removal under appropriate wash conditions, or that less enzyme by weight is required to obtain the same end result relative to the corresponding control conditions.

[0052] As used herein, the term "specific performance" refers to the cleaning and removal of a particular stain or soil per unit of active enzyme. In some embodiments, specific performance is determined using stains or soils such as egg, egg yolk, milk, grass, minced meat blood, chocolate sauce, baby food, sebum, etc.

[0053] "Detergent composition," "detergent formulation," or "detergent" refers to a composition designed for cleaning soiled materials. Detergent compositions according to the present invention include detergent compositions for various 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 a detergent composition. A typical detergent component is a surfactant. "Surfactant" (used synonymously herein with "surface active agent") refers to an organic chemical that, when added to a liquid, alters the interfacial properties of that liquid. Depending on their ionic charge, surfactants are called nonionic, anionic, cationic, or amphoteric. The term "effective amount of detergent component" includes the amount of a particular component that provides effective stain removal and / or effective cleaning conditions (e.g., pH, temperature, water hardness), the amount of a particular component that effectively provides optical benefits (e.g., optical brightness, dye transfer prevention, color care), and the amount of a particular component that effectively facilitates processing (maintaining physical characteristics during processing, storage, and use; e.g., rheology modifiers, hydrotropes, drying agents).

[0054] The term "laundry" or "washing" refers to the process of treating textiles and / or fabrics with a solution containing the detergent composition of the present invention, both in domestic and industrial laundry. The laundry process can be carried out by using a technical device such as a domestic washing machine or an industrial washing machine. Alternatively, the laundry process can be done manually.

[0055] The term "textile" means any textile material, including yarns (yarns made from natural or synthetic fibers used for knitting or weaving), intermediate products of yarns, fibers, nonwoven materials, natural materials, synthetic materials, and fabrics made from these materials, such as garments, cloth, and other articles. The terms "fabric" (a fabric made by weaving, knitting, or felting fibers) or "garment" (any article of clothing made from a textile), as used herein, are also intended to include the broader term textile.

[0056] The term "fiber" includes natural fibers, synthetic fibers, and mixtures thereof. Examples of natural fibers are of plant (such as flax, jute, and cotton) or animal origin and contain proteins such as collagen, keratin, and fibroin (e.g., silk, sheep's wool, angora, mohair, cashmere). Examples of fibers of synthetic origin are polyurethane fibers such as Spandex® or Lycra®, polyester fibers, polyolefins such as Elastofine, or polyamide fibers such as nylon. A fiber can be a single fiber or part of a textile, such as knitwear, woven fabric, or nonwoven fabric.

[0057] The term "hard surface cleaning" refers to both household and industrial hard surface cleaning, and refers to the process of treating a hard surface 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, medical equipment, cutlery and metal surfaces, including tableware.A specific form of hard surface cleaning is dishwashing, which can be manual dishwashing (MDW) or automatic dishwashing (ADW).

[0058] The term "dishwashing" refers to all forms of washing dishes, for example, by manual or automatic dishwashing. Washing dishware includes, but is not limited to, cleaning all forms of ceramics, such as plates, cups, glasses, bowls, etc., all forms of cutlery, such as spoons, knives, forks, and serving utensils, as well as ceramics, plastics such as melamine, metals, china, glass, and acrylic.

[0059] Cleaning performance is evaluated under relevant cleaning conditions. In this specification, the term "relevant washing conditions" refers to the conditions actually used in a washing machine, automatic dishwasher or manual washing process, in particular the washing temperature, time, washing equipment, foam concentration, detergent type and water hardness.

[0060] The term "medical device cleaning" refers to the cleaning process when reusable medical devices are reprocessed. Medical device cleaning methods can be divided into two categories: manual and mechanical / automatic cleaning methods. Manual cleaning is used when a mechanical unit is not available or when the medical device to be cleaned is too fragile or too difficult to clean with a mechanical unit. Mechanical / automatic cleaning methods remove soils and microorganisms through an automatic cleaning and rinsing process, including ultrasonic cleaning and washing.

[0061] In the detergent field, the term "stain" is typically used in reference to laundry, e.g., cleaning for textiles, fabrics, or fibers, while the term "soil" is typically used in reference to hard surface cleaning, e.g., cleaning dishes and cutlery. However, in this specification, the terms "stain" and "soil" will be used interchangeably.

[0062] "Metal ion-scavenging builders," as used herein, differ from precipitating builders in that when the builder is initially used in an amount sufficient to combine with all of the calcium ions in an aqueous solution at 7° dH hardness (German hardness) at neutral pH, a modest amount of precipitate is formed. "Strong builders" are those with a logarithmic stability constant (Log K) of the cation / chelator complex greater than 4, particularly greater than 5, greater than 6, or greater than 7. Ca ) and is classified as a highly efficient chelating agent that can strongly bind divalent cations such as Ca2+. The stability constant is determined at an ionic strength of 0.1M and a temperature of 25°C. A "strong metal ion-trapping builder" combines both of the above properties.

[0063] Detailed Description The present invention provides novel amylase enzymes, and more particularly, amylase variants, methods of making the amylase variants, compositions comprising the amylase variants, and methods of using the amylase variants or compositions comprising the amylase variants.

[0064] Amylase variants The present invention relates to amylase variants of a parent amylase, said variants comprising: (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 3; (ii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to amino acid positions selected from the group consisting of 25, 176, 186, 251, 405, and 482 according to the numbering of SEQ ID NO: 3; and (iii) any of SEQ ID NOs: 1, 3, 4, or 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, preferably at least 91% but less than 100% sequence identity to SEQ ID NO: 1 (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) Includes:

[0065] The alpha-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, synthetic, and / or recombinant amylase variants. Preferably, the amylase variants of the present invention are purified and recombinant amylase variants.

[0066] The amylases and amylase variants according to the present invention have "amylolytic activity" or "amylase activity." "Amylolytic activity" or "amylase activity" describes the ability to hydrolyze glucosidic bonds in polysaccharides. Amylase activity can be determined by assays for measuring amylase activity known to those skilled in the art. 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 utilizing Phadebas tablets as a substrate (Phadebas Amylase Test, provided by Magle Life Science). Starch is hydrolyzed by amylase to produce 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 of pure amylase protein) of the amylase of interest under a given set of conditions.

[0067] Alternatively, amylase activity can be determined by a method utilizing ethylidene-4-nitrophenyl-alpha-D-maltoheptaoside (EPS). D-maltoheptaoside is a block oligosaccharide that can be cleaved by endo-amylase. After cleavage, the alpha-glucosidase included in the kit digests the substrate, liberating free PNP molecules with a yellow color, which can be measured by visible spectrophotometry at 405 nm. A kit containing the EPS substrate and alpha-glucosidase is manufactured, for example, by Roche Costum Biotech (cat. No. 10880078103). The slope of the time-dependent absorption curve is directly proportional to the specific activity (activity per mg of enzyme) of the amylase of interest under a given set of conditions.

[0068] 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.

[0069] Preferably, the parent amylase for the amylase variant of the present invention is an amylase having at least 60% sequence identity with any of SEQ ID NOs: 1, 3, 4 or any of SEQ ID NOs: 15 to 41, and more preferably, the parent amylase for the amylase variant of the present invention is an amylase according to any of SEQ ID NOs: 1, 3, 4 or any of SEQ ID NOs: 15 to 41. Most preferably, the parent amylase for the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1.

[0070] The present invention relates to amylase variants comprising amino acid modifications, preferably insertions, deletions, substitutions or a combination thereof, most preferably substitutions, at two or more positions corresponding to positions selected from the group consisting of 4, 25, 176, 186, 251, 405 and 482 according to the numbering of the amino acid sequence set forth in SEQ ID NO: 3.

[0071] In an alternative manner, amino acid positions 4, 25, 176, 186, 251, 405 and 482 (according to the numbering of SEQ ID NO:3) can be written with respect to the numbering of SEQ ID NO:1, i.e., according to the numbering of SEQ ID NO:1. In this alternative manner of writing amino acid positions of the invention, amino acid positions 4, 25, 176, 186, 251, 405 and 482 according to the numbering of SEQ ID NO:3 correspond to amino acid positions 4, 25, 176, 184, 249, 403 and 480 according to the numbering of SEQ ID NO:1.

[0072] Preferably, the parent amylase for the amylase variant of the present invention is an amylase having at least 60% sequence identity with any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NOs: 15 to 41, and more preferably, the parent amylase for 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 SEQ ID NOs: 15 to 41. Most preferably, the parent amylase for the amylase variant is an amylase according to SEQ ID NO: 1.

[0073] Preferably, the present invention relates to amylase variants comprising two or more amino acid substitutions selected from the group consisting of X4Q, X25H, X176K, X186E, X251E, X405M and X482W according to the numbering of SEQ ID NO: 3.

[0074] Preferably, the present invention relates to an amylase variant comprising, compared to the parent sequence, two or more amino acid substitutions selected from the group consisting of X4Q, X25H, X176K, X186E, X251E, X405M, X482W and amino acid substitutions at these positions for amino acids similar to the designated amino acid substitutions at these positions according to the numbering of SEQ ID NO: 3, i.e. amino acid substitutions at these positions for amino acids similar to designated target amino acids, wherein said variants have amylase activity; preferably, the parent amylase for the amylase variant of the present invention is the 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 for the amylase variant is the amylase according to SEQ ID NO: 1; Amino acid A is similar to amino acid S, Amino acid D is similar to amino acids E and N, The amino acid E is similar to the amino acids D, K, and Q. The amino acid F is similar to the amino acids W and Y. The amino acid H is similar to the amino acids N and Y. Amino acid I is similar to amino acids L, M, and V, The amino acid K is similar to the amino acids E, Q, and R. The amino acid L is similar to the amino acids I, M, and V, The amino acid M is similar to the amino acids I, L, and V, The amino acid N is similar to the amino acids D, H, and S, The amino acid Q is similar to the amino acids E, K, and R, The amino acid R is similar to the amino acids K and Q. The amino acid S is similar to the amino acids A, N, and T, The amino acid T is similar to the amino acid S, Amino acid V is similar to amino acids I, L, and M, Amino acid W is similar to amino acids F and Y, The amino acid Y is similar to the amino acids F, H, and W.

[0075] Preferably, the present invention relates to amylase variants comprising two or more amino acid substitutions selected from the group consisting of X4Q, X25H, X176K, X186E, X251E, X405M and X482W according to the numbering of SEQ ID NO: 3 compared to the parent sequence, wherein said variants have amylase activity; preferably, the parent amylase for the amylase variants of the present invention is the 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 for the amylase variants is the amylase according to SEQ ID NO: 1.

[0076] Preferably, in this embodiment, the amino acid residues in the parent amylase at the positions recited above (i.e., X) correspond to the amino acid residues shown in SEQ ID NO: 1 at each position according to the numbering of SEQ ID NO: 3.

[0077] Thus, preferably, the present invention relates to amylase variants comprising amino acid substitutions at two or more positions corresponding to positions selected from the group consisting of G4, N25, R176, G186, T251, L405 and Y482 according to the numbering of SEQ ID NO: 3.

[0078] 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 G4Q, N25H, R176K, G186E, T251E, L405M and Y482W according to the numbering of SEQ ID NO: 3.

[0079] The present invention preferably relates to amylase variants comprising an amino acid substitution at one or more positions corresponding to position 4 and positions selected from the group consisting of 25, 176, 186, 251, 405 and 482 according to the numbering of SEQ ID NO:3.

[0080] The present invention relates to amylase variants, preferably comprising amino acid substitutions at two or more, three or more, four or more, five or more or all positions corresponding to position 4 and positions selected from the group consisting of 25, 176, 186, 251, 405 and 482 according to the numbering of SEQ ID NO: 3.

[0081] Preferably, the present invention relates to amylase variants comprising the amino acid substitutions X4Q according to the numbering of SEQ ID NO: 3 and one or more amino acid substitutions selected from the group consisting of X25H, X176K, X186E, X251E, X405M and X482W.

[0082] Preferably, the present invention relates to amylase variants comprising, compared to the parent sequence, the amino acid substitutions X4Q according to the numbering of SEQ ID NO: 3 and one or more amino acid substitutions selected from the group consisting of X25H, X176K, X186E, X251E, X405M and X482W, wherein said variants have amylase activity; preferably, the parent amylase for the amylase variants of the present invention is the 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 for the amylase variants is the amylase according to SEQ ID NO: 1.

[0083] Preferably, in this embodiment, the amino acid residues in the parent amylase at the positions recited above (i.e., X) correspond to the amino acid residues shown in SEQ ID NO: 1 at each position according to the numbering of SEQ ID NO: 3.

[0084] Preferably, therefore, the present invention relates to amylase variants comprising amino acid substitutions at one or more positions corresponding to amino acid position G4 according to the numbering of SEQ ID NO: 3 and positions selected from the group consisting of N25, R176, G186, T251, L405 and Y482.

[0085] In a preferred embodiment, the present invention relates to an amylase variant comprising an amino acid substitution at one or more positions corresponding to the amino acid substitution G4Q according to the numbering of SEQ ID NO: 3 and a position selected from the group consisting of N25H, R176K, G186E, T251E, L405M and Y482W.

[0086] The present invention preferably relates to amylase variants comprising amino acid substitutions at one or more positions corresponding to positions 4 and 25 and positions selected from the group consisting of 176, 186, 251, 405 and 482 according to the numbering of SEQ ID NO:3.

[0087] Preferably, the present invention relates to amylase variants comprising the amino acid substitutions X4Q and X25H according to the numbering of SEQ ID NO: 3 and one or more amino acid substitutions selected from the group consisting of X176K, X186E, X251E, X405M and X482W.

[0088] Preferably, therefore, the present invention relates to amylase variants comprising amino acid substitutions at one or more positions corresponding to amino acid positions G4 and N25 according to the numbering of SEQ ID NO: 3 and positions selected from the group consisting of R176, G186, T251, L405 and Y482.

[0089] In a preferred embodiment, the present invention relates to an amylase variant comprising an amino acid substitution at one or more positions corresponding to the amino acid substitutions G4Q and N25H according to the numbering of SEQ ID NO: 3 and positions selected from the group consisting of R176K, G186E, T251E, L405M and Y482W.

[0090] In one embodiment, the amylase variant further comprises an amino acid substitution at position 439, preferably X439K, more preferably W439K (according to the numbering of SEQ ID NO: 3).

[0091] Preferably, the amylase variant comprises an amino acid substitution at one or more of the following amino acid positions (according to the numbering of SEQ ID NO: 3) compared to the parent amylase: Preferably, the parent amylase for the amylase variant is an amylase according to any of SEQ ID NOs: 1, 3, 4 or any of SEQ ID NOs: 15-41, and most preferably, the parent amylase for the amylase variant is an amylase according to SEQ ID NO: 1, 3 or 4, preferably SEQ ID NO: 1. Preferably, the amino acid residue of the parent amylase at the recited position (i.e., X) corresponds to the amino acid residue shown in SEQ ID NO: 1 at the respective position (according to the numbering of SEQ ID NO: 3).

[0092] The amylase variant comprises an amino acid substitution at position 4 (according to the numbering of SEQ ID NO: 3), preferably the substitution X4Q.

[0093] Preferably, 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 amino acid substitution at position 25 is X25Y or X25D.

[0094] 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.

[0095] 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.

[0096] Preferably, the amylase variant comprises an amino acid substitution at position 251 (according to the numbering of SEQ ID NO: 3), preferably the substitution X251E.

[0097] Preferably, the amylase variant comprises an amino acid substitution at position 405 (according to the numbering of SEQ ID NO: 3), preferably the substitution X405M.

[0098] 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.

[0099] The amylase variant of the present invention is (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 3, and (ii) amino acid substitutions at one or more, two or more, three or more, four or more, five or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 25, 176, 186, 251, 405, and 482 according to the numbering of SEQ ID NO: 3. Includes:

[0100] In a preferred embodiment, the amylase variant of the present invention comprises: (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 3, and (ii) amino acid substitutions at one or more, two or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 25, 176, and 186 according to the numbering of SEQ ID NO:3 Includes:

[0101] In a preferred embodiment, the amylase variant of the present invention comprises: (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 3, and (ii) amino acid substitutions at one or more, two or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 176 and 186 according to the numbering of SEQ ID NO:3 Includes:

[0102] In a preferred embodiment, the amylase variant of the present invention comprises: (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 3, and (ii) an amino acid substitution at one or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 251 and 405 according to the numbering of SEQ ID NO: 3. Includes:

[0103] In a preferred embodiment, the amylase variant of the present invention comprises: (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 3; (ii) amino acid substitutions at one or more, two or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 25, 176, and 186 according to the numbering of SEQ ID NO: 3; and (iii) an amino acid substitution at one or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 251 and 405 according to the numbering of SEQ ID NO: 3. Includes:

[0104] In a particularly preferred embodiment, the amylase variant of the present invention comprises: (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 3; (ii) amino acid substitutions at one or more, two or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 25, 176, and 186 according to the numbering of SEQ ID NO:3; (iii) an amino acid substitution at one or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 251 and 405 according to the numbering of SEQ ID NO: 3; and (iv) an amino acid substitution at an amino acid position corresponding to amino acid position 482 according to the numbering of SEQ ID NO: 3 Includes:

[0105] Preferably, the amylase variants of the invention comprise amino acid substitutions at one of the following combinations of amino acid positions (according to the numbering of SEQ ID NO: 3):

[0106] [Table 1]

[0107] Particularly preferred are combinations of amino acid substitutions at amino acid positions that include at least amino acid position 25 from the table above.

[0108] Even more preferred are combinations of amino acid substitutions at amino acid positions including at least one, preferably two, and more preferably all of the amino acid positions selected from the group consisting of 176 and 186 from the table above.

[0109] Even more preferred are combinations of amino acid substitutions at amino acid positions including at least one, preferably two, and more preferably all of the amino acid positions selected from the group consisting of 25, 176 and 186 from the table above.

[0110] The amylase variant of the present invention is (i) an amino acid substitution at position X4G according to the numbering of SEQ ID NO: 3, and (ii) amino acid substitutions at one or more, two or more, three or more, four or more, five or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of X25H, X176K, X186E, X251E, X405M, and X482W according to the numbering of SEQ ID NO: 3. Includes:

[0111] In a preferred embodiment, the amylase variant of the present invention comprises: (i) an amino acid substitution at position X4G according to the numbering of SEQ ID NO: 3; (ii) amino acid substitutions at one or more, two or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of X25H, X176K, and X186E according to the numbering of SEQ ID NO: 3; and (iii) an amino acid substitution at one or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of X251E and X405M according to the numbering of SEQ ID NO: 3. Includes:

[0112] In a particularly preferred embodiment, the amylase variant of the present invention comprises: (i) an amino acid substitution at position X4G according to the numbering of SEQ ID NO: 3; (ii) amino acid substitutions at one or more, two or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of X25H, X176K, and X186E according to the numbering of SEQ ID NO: 3; (iii) an amino acid substitution at one or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of X251E and X405M according to the numbering of SEQ ID NO: 3; and (iv) an amino acid substitution at an amino acid position corresponding to amino acid position X482W according to the numbering of SEQ ID NO: 3 Includes:

[0113] Preferably, the amylase variants of the present invention comprise one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 3):

[0114] [Table 2]

[0115] From the table above, combinations of amino acid substitutions containing at least the amino acid substitution X25H are particularly preferred.

[0116] [Table 3]

[0117] Particularly preferred are combinations of amino acid substitutions containing at least one, preferably two, and more preferably all of the amino acid substitutions selected from the group consisting of X25H, X176K and X186E from the table above.

[0118] From the table above, a combination of amino acid substitutions containing at least one, preferably two, and more preferably all, of the amino acid substitutions selected from the group consisting of X176K and X186E is particularly preferred.

[0119] Preferably, the amylase variants of the present invention comprise one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 3):

[0120] [Table 4]

[0121] In one embodiment, the amylase variant further comprises an amino acid substitution at position 439, preferably X439K, more preferably W439K (according to the numbering of SEQ ID NO: 3), preferably the combination of amino acid substitutions (according to the numbering of SEQ ID NO: 3) X4Q+X25H+X176K+X186E+X251E+X405M+X439K+X482W.

[0122] Most preferably, the amylase variant of the invention comprises the following combination of amino acid substitutions X4Q+X25H+X176K+X186E+X251E+X405M+X482W (according to the numbering of SEQ ID NO: 3).

[0123] Preferably, the amylase variant further comprises a deletion of 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 both 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*, the numbering being according to the amino acid sequence set forth in SEQ ID NO: 3.

[0124] Preferably, the amylase variant comprises a deletion in two 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 the deletions D183* and G184*, the numbering being according to SEQ ID NO: 3.

[0125] 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 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%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, at least 510%, at least 520%, at least 530%, at least 540%, at least 550%, at least 560%, at least 570%, at least 580%, at least 590%, at least 610%, at least 610%, at least 620%, at least 630%, at 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.

[0126] Preferably, amylase variants according to the present invention having one or more of the amino acid substitutions described herein preferably have an amino acid sequence that is 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 identical to the amino acid sequence set forth in SEQ ID NO: 1, 3, 4 or any of SEQ ID NOs: 15 to 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. at least 97.5%, 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.

[0127] Preferably, amylase variants according to the present invention having one or more of the amino acid substitutions 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 set forth in any of SEQ ID NOs: 1, 3, 4 or 15 to 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.

[0128] In one embodiment, amylase variants according to the invention having one or more of the amino acid substitutions described herein preferably have an amino acid sequence which is 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% identical to the amino acid sequence set forth in SEQ ID NO: 1. , 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.

[0129] In another embodiment, the amylase variant according to the invention having one or more of the amino acid substitutions described herein preferably has an amino acid sequence which is 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 99.5%, at least 99.5%, at least 95%, at least 96%, at least 97.5%, at least 98%, at least 99.5 ... %, 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.

[0130] In another embodiment, the amylase variant according to the invention having one or more of the amino acid substitutions described herein preferably has an amino acid sequence which is 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 99.5%, at least 99.5%, at least 95%, at least 96%, at least 97.5%, at least 98%, at least 99.5 ... %, 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.

[0131] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 15.

[0132] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 16.

[0133] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 17.

[0134] In another embodiment, an amylase variant according to the present invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 18.

[0135] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 19.

[0136] In another embodiment, an amylase variant according to the present invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 20.

[0137] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 21.

[0138] In another embodiment, an amylase variant according to the present invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 22.

[0139] In another embodiment, an amylase variant according to the present invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 23.

[0140] In another embodiment, an amylase variant according to the present invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 24.

[0141] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 25.

[0142] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 26.

[0143] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 27.

[0144] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 28.

[0145] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 29.

[0146] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 30.

[0147] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 31.

[0148] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 32.

[0149] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 33.

[0150] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 34.

[0151] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 35.

[0152] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 36.

[0153] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 37.

[0154] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 38.

[0155] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 39.

[0156] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 40.

[0157] In another embodiment, an amylase variant according to the invention having one or more of the amino acid substitutions 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 to the amino acid sequence set forth in SEQ ID NO: 41.

[0158] Preferably, amylase variants according to the present invention having one or more of the amino acid substitutions described herein preferably have an amino acid sequence that is 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% identical to the amino acid sequence set forth in SEQ ID NO: 4. , 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.

[0159] Preferably, amylase variants according to the present invention having one or more of the amino acid substitutions described herein preferably have an amino acid sequence that is 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% identical to the amino acid sequence set forth in SEQ ID NO: 3. , 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.

[0160] More preferably, amylase variants according to the present invention having one or more of the 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 set forth in SEQ ID NO:1.

[0161] The amylase variants according to the present 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 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99.5%, 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 1109%, at least 1111%, 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 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 129.5%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 1 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% sequence identity or similarity, but less than 100%, preferably sequence identity.

[0162] Preferably, the amylase variant of the present invention having amylase activity comprises: (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 99.5% sequence identity to SEQ ID NO: 1, but less than 100%; (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% sequence identity to SEQ ID NO:2, but less than 100%. (c) under high stringency conditions, (i) the coding sequence of SEQ ID NO: 1, or (ii) a polynucleotide represented by SEQ ID NO: 2 an amino acid sequence encoded by a polynucleotide that hybridizes to the complement of (d) an amino acid sequence encoded by a polynucleotide having at least 95% but less than 100% sequence identity to SEQ ID NO:2 (the polynucleotide further differs from SEQ ID NO:2 solely due to the degeneracy of the genetic code); or (e) a fragment of (a), (b), (c), or (d) having amylase activity Includes:

[0163] Preferably, an amylase variant according to the present invention having amylase activity has 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% sequence identity or similarity, preferably sequence identity, to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4, but less than 100%. Particularly preferably, 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 with the amino acid sequence set forth in SEQ ID NO: 1. More preferably, amylase variants according to the invention having amylase activity preferably have at least 95.0%, but less than 100% sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0164] The structure of an alpha-amylase comprises three separate domains, A, B, and C; see, e.g., Machius et al., 1995, J. Mol. Biol. 246:545-559. The alpha-amylase variants described herein may further comprise one or more non-catalytic CBMs (carbohydrate-binding modules, also called carbohydrate-binding domains or, particularly for amylases, starch-binding domains). The CBMs can improve the association of the enzyme with the substrate. The CBMs are attached to the C-domain. Preferably, the amylases of the invention do not comprise a carbohydrate-binding domain. Preferably, the alpha-amylase variants of the invention consist of only three domains, the A, B, and C domains.

[0165] As used herein, the "A and B domains" or "AB domain" of an 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 an alpha-amylase corresponds to the amino acids aligned with amino acids 400-485 of SEQ ID NO: 3.

[0166] Preferably, the amylase variants comprising one or more of the above-described amino acid modifications, preferably insertions, deletions, substitutions or a combination thereof, preferably substitutions, are hybrid amylases comprising domains thereof from different parent amylases, in particular the A and B domains thereof. Amylase variants can be generated by substituting the C domain or a portion thereof of one amylase with the C domain or a portion thereof of another amylase. Preferably, the A and B domains of the amylase variants described herein have at least 75% identity, such as 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, with the A and B domains of the amylase of SEQ ID NO: 3.

[0167] Preferably, the A and B domains of the amylase variants described herein have at least 75% identity to SEQ ID NO:6, 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. This means that the amino acid sequences forming the A and B domains have at least 75% identity to SEQ ID NO:6, 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%, or at least "Sequence identity" means having 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.

[0168] Preferably, the C domain of the amylase variants described herein has at least 75% identity to the C domain of the amylase of SEQ ID NO: 5, 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 The C domain may have 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.

[0169] Preferably, the C domain of the amylase variants described herein has at least 75% identity 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 or 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.

[0170] In one embodiment of the invention, the amino acid sequences forming the A and B domains have at least 75% identity to the amino acid sequence of SEQ ID NO:6, 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 not 100%. The amino acid sequence forming the C domain has an identity of at least 75% with 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% sequence identity.

[0171] In one embodiment of the invention, the amino acid sequences forming the A and B domains have 100% identity to the amino acid sequence of SEQ ID NO:6 and the amino acid sequence forming the C domain has at least 75% identity to the amino acid sequence of 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%, or at least at least 99%, at least 99.5%, 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.

[0172] In one embodiment of the invention, the amino acid sequences forming the A and B domains have at least 75% identity to the amino acid sequence of SEQ ID NO: 6, 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 has 100% identity to SEQ ID NO:8.

[0173] In one embodiment, the present invention provides a hybrid derived from at least two different amylases (the hybrid comprises A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are 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 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 99.5%, at least 99.5%, at least 99.5%, 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 110.5%, at least 111.5%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118.5%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 1 8.5%, 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, The amino acid sequence of the C domain 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 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. 9.5%, 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) and introducing into the hybrid one or more amino acid modifications as described herein.

[0174] Amylase variants according to the present invention having amylase activity may have a sequence similar to the amino acid sequence set forth in SEQ ID NO: 1 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 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%, and comprising A and B domains and a C domain, wherein the amino acid sequences of the A and B domains are at least 75% identical to the amino acid sequence of SEQ ID NO:6, 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, and the amino acid sequence of the C domain 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% identical to the amino acid sequence of SEQ ID NO:8.5%, 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.

[0175] In one embodiment, the amylase variant of the present invention having amylase activity comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 1, 3, 4 or any of SEQ ID NOs: 15 to 41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, with one or more amino acid modifications as listed herein.

[0176] Preferably, the amylase variant according to the present invention having amylase activity comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 1, 3, 4 or any of SEQ ID NOs: 15 to 41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, with 1 to 6, preferably 2 to 6 or 3 to 6, for example 1, 2, 3, 4, 5, 6 or 6, amino acid substitutions selected from the group consisting of the amino acid substitution X4Q according to the numbering of SEQ ID NO: 3 and X25H, X176K, X186E, X251E, X405M and X482W according to the numbering of SEQ ID NO: 3, and preferably comprising a deletion of 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.

[0177] In another embodiment, the amylase variant according to the present invention having amylase activity comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 1, 3, 4 or any of SEQ ID NOs: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, with one or more amino acid modifications as mentioned herein, and has a length of 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, It further comprises 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, and preferably 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 according to the numbering of SEQ ID NO: 3 as described herein.

[0178] Preferably, the amylase variant according to the present invention having amylase activity comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 1, 3, 4 or any of SEQ ID NOs: 15 to 41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, with 1 to 6, preferably 2 to 6 or 3 to 6, for example 1, 2, 3, 4, 5 or 6 amino acid substitutions selected from the group consisting of the amino acid substitutions X4Q according to the numbering of SEQ ID NO: 3 and X25H, X176K, X186E, X251E, X405M and X482W according to the numbering of SEQ ID NO: 3, and 1 to 50, preferably Preferably, it further comprises 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, and preferably comprises a deletion of 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.

[0179] Preferably, the amylase variant according to the present invention having amylase activity comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 with 1 to 6, preferably 2 to 6 or 3 to 6, such as 1, 2, 3, 4, 5 or 6, amino acid substitutions selected from the group consisting of the amino acid substitutions X4Q according to the numbering of SEQ ID NO: 3 and X25H, X176K, X186E, X251E, X405M and X482W according to the numbering of SEQ ID NO: 3, and further comprises 1 to 10, preferably 1 to 5 conservative amino acid exchanges.

[0180] Conservative amino acid substitutions may occur throughout the entire sequence of the amylase variant. In one embodiment, such mutations do not involve functional domains of the amylase variant. In one embodiment, conservative mutations do not involve the catalytic center of the amylase variant.

[0181] In one embodiment, the amylase variants of the present invention preferably exhibit one or more improved properties compared to the parent amylase, preferably compared to the amylase as set forth in SEQ ID NO: 1, 3, 4 or any 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.

[0182] Preferably, the improved properties are expressed as an improvement factor (IF) of >1.0. Preferably, for improved thermal stability and improved cleaning performance, the improvement is expressed as an improvement factor. Preferably, the improvement factor is 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, or 2.0 or more. Preferably, the IF for cleaning performance is 1.1 or more, 1.2 or more, or 1.3 or more. Preferably, the IF for thermal stability is 1.1 or more, 1.2 or more, 1.3 or more, 1.5 or more, or 2.0 or more.

[0183] Instead, the improvement in amylase properties is expressed as a percentage improvement compared to the parent amylase. Preferably, the amylase variants of the invention exhibit improved properties of 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% compared to the parent amylase, preferably compared to the amylase as set forth in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1.

[0184] Alternatively, particularly with regard to improved stability, the improved amylase properties are expressed as residual activity after a stability challenge. Preferably, storage stability is expressed as residual activity after storage under the respective storage conditions, preferably after storage in a detergent composition (preferably 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 improved by 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% compared to the parent amylase, preferably compared to SEQ ID NO: 1 or SEQ ID NO: 3, preferably compared to the amylase as set forth in SEQ ID NO: 1 or the amylase as set forth in SEQ ID NO: 33. The residual activity compared to the parent amylase can also be converted into an improvement by forming a ratio of the residual activity of the variant to the residual activity of the parent amylase.

[0185] Preferably, the improved property is one or more properties selected from the group consisting of increased expression, activity, stability, thermal stability, 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 detergents, performance in laundry detergents, performance in ADW detergents. 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, cleaning performance, laundry detergent cleaning performance and / or ADW detergent cleaning performance, cleaning 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, stability in a detergent composition (preferably 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 property is improved expression, improved solubility, improved thermal stability, improved thermal stability in a detergent composition, improved stability during storage in a detergent composition, and / or improved cleaning performance. Preferably, the improved property is improved thermal stability, preferably improved thermal stability in a detergent composition, improved stability under storage in a detergent composition, and / or improved cleaning performance. Preferably, the improved property is improved thermal stability, improved thermal stability in a detergent composition, improved stability under storage conditions, preferably improved stability under storage in a detergent composition, preferably improved stability under storage in a laundry detergent and / or improved stability under storage in an ADW detergent, improved cleaning performance, preferably improved cleaning performance in a laundry detergent, improved cleaning performance in an ADW detergent, and / or cleaning 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 cleaning performance.Most preferably, the improved performance is preferably improved storage stability, preferably improved stability in detergent compositions, compared to the amylase as set forth in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1 or for the amylase as set forth in SEQ ID NO: 33.

[0186] Preferably, the present invention relates to an amylase variant of a parent amylase, said variant comprising: (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 3; (ii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to amino acid positions selected from the group consisting of 25, 176, 186, 251, 405, and 482 according to the numbering of SEQ ID NO: 3; (iii) any of SEQ ID NOs: 1, 3, 4 or SEQ ID NOs: 15 to 41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, preferably at least 91% identity to SEQ ID NO: 1, but less than 100% sequence identity (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); and (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 the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3). Includes:

[0187] Preferably, the present invention relates to an amylase variant of a parent amylase, said variant comprising: (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 3; (ii) amino acid substitutions at one or more, preferably two or more, more preferably three or more amino acid positions corresponding to amino acid positions selected from the group consisting of 25, 176, 186, 251, 405 and 482 according to the numbering of SEQ ID NO: 3; (iii) any of SEQ ID NOs: 1, 3, 4 or SEQ ID NOs: 15 to 41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 80%, preferably at least 91% identity to SEQ ID NO: 1, but less than 100% sequence identity (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); and (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 the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3). Includes:

[0188] Preferably, the amylase variant of the present invention comprises: (i) the amino acid substitution X4G according to the numbering of SEQ ID NO: 3; (ii) one or more, two or more, three or more, four or more, five or more, or all of the amino acid substitutions selected from the group consisting of X25H, X176K, X186E, X251E, X405M, and X482W according to the numbering of SEQ ID NO: 3; (iii) any of SEQ ID NOs: 1, 3, 4, or SEQ ID NOs: 15 to 41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, 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%, or 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 (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 the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3). Includes:

[0189] Preferably, the amylase variant of the present invention comprises: (i) the amino acid substitution X4G according to the numbering of SEQ ID NO: 3; (ii) one or more, two or more, three or more, four or more, five or more, or all of the amino acid substitutions selected from the group consisting of X25H, X176K, X186E, X251E, X405M, and X482W according to the numbering of SEQ ID NO: 3; (iii) SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1 with 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 (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 the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3). Includes:

[0190] Preferably, the amylase variant of the present invention comprises: (i) (following the numbering in SEQ ID NO: 3) X4Q+X25H+X176K+X186E, X4Q+X25H+X176K+X186E+X251E, X4Q+X25H+X176K+X186E+X405M, X4Q+X25H+X176K+X186E+X482W, X4Q+X25H+X176K+X186E+X251E+X405M, X4Q+X25H+X176K+X186E+X251E+X482W, X4Q+X25H+X176K+X186E+X405M+X482W, and X4Q+X25H+X176K+X186E+X251E+X405M+X482W a combination of amino acid substitutions selected from the group consisting of: (ii) SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1 with 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%, or 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 (iii) 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 the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3). Includes:

[0191] Preferably, the amylase variant of the present invention comprises: (i) the amino acid substitution X4G according to the numbering of SEQ ID NO: 3; (ii) one or more, two or more, three or more, four or more, five or more, or all of the amino acid substitutions selected from the group consisting of X25H, X176K, X186E, X251E, X405M, and X482W according to the numbering of SEQ ID NO: 3; (iii) SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1 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 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, (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 the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3). and (v) The amylase variant preferably exhibits one or more improved properties relative to said parent amylase, preferably relative to the parent amylase as set forth in SEQ ID NO: 1 or SEQ ID NO: 33, preferably relative to the parent amylase as set forth in SEQ ID NO: 1 or SEQ ID NO: 33, preferably the amylase variant has increased stability, thermostability, storage stability, storage stability in a detergent composition, cleaning performance, cleaning performance in a laundry detergent and / or cleaning performance in a dishwashing detergent, preferably the improved property is improved storage stability and / or cleaning performance, preferably improved cleaning performance on laundry, preferably the improved property is improved storage stability, preferably said improved property is expressed as an improvement factor (IF) > 1.0, preferably the improvement factor is 1.1 or greater, preferably 1.2 or greater, more preferably 1.3 or greater, preferably the amylase variant preferably has SEQ ID NO: 33 and / or the amylase variant preferably exhibits improved storage stability in a detergent composition, preferably compared to a reference amylase, preferably compared to an amylase having an amino acid sequence as set forth in SEQ ID NO: 33, and / or preferably exhibits improved cleaning performance on laundry, preferably at 40°C, compared to an amylase having an improvement of 1.1 or more, preferably 1.2 or more, more preferably 1.3 or more, after storage in a detergent composition as described herein, preferably at 37°C, preferably for 14, 28, 42 or 56 days, preferably 56 days, or at 40°C, preferably for 14, 24, 56 or 84 days, preferably 86 days, as preferably determined in ES1-C detergent as described herein, preferably containing an additional builder (preferably HEDP).

[0192] Particularly preferably, the amylase variant of the present invention is (i) (following the numbering in SEQ ID NO: 3) X4Q+X25H+X176K+X186E, X4Q+X25H+X176K+X186E+X251E, X4Q+X25H+X176K+X186E+X405M, X4Q+X25H+X176K+X186E+X482W, X4Q+X25H+X176K+X186E+X251E+X405M, X4Q+X25H+X176K+X186E+X251E+X482W, X4Q+X25H+X176K+X186E+X405M+X482W, and X4Q+X25H+X176K+X186E+X251E+X405M+X482W a combination of amino acid substitutions selected from the group consisting of: (ii) 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% sequence identity to SEQ ID NO:1, but less than 100%; and (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 amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3). Includes:

[0193] Particularly preferred are: (i) Amino acid substitution combinations (according to the numbering of SEQ ID NO: 3): X4Q+X25H+X176K+X186E+X251E+X405M+X482W, (ii) 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 (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 amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably the deletions D183* and G184* (according to the numbering of SEQ ID NO: 3). and amylase variants comprising:

[0194] In one embodiment, the amylase variant further comprises an amino acid substitution at position 439, preferably X439K, more preferably W439K (according to the numbering of SEQ ID NO: 3), preferably the combination of amino acid substitutions (according to the numbering of SEQ ID NO: 3) X4Q+X25H+X176K+X186E+X251E+X405M+X439K+X482W.

[0195] Therefore, preferred is an amylase variant comprising or consisting of the amino acid sequence set out in SEQ ID NO: 1 with the amino acid modifications X4Q+X25H+X176K+X186E+X251E+X405M+X439K+X482W according to the numbering of SEQ ID NO: 3.

[0196] However, most preferred is an amylase variant comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 1 with the amino acid modifications X4Q+X25H+X176K+X186E+X251E+X405M+X482W according to the numbering of SEQ ID NO: 3.

[0197] Nucleic Acid Constructs The present invention also refers to polynucleotides encoding the amylase variants of the present invention. Preferably, the polynucleotides are codon-optimized for improved expression in a particular host cell, preferably a Bacillus cell.

[0198] Therefore, the present invention provides (a) SEQ ID NO: 2 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% identity to a nucleic acid sequence (wherein the nucleic acid encodes an amylase variant having amylase activity as described herein), (b) a sequence identical to SEQ ID NO: 1 by 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%, or at least a nucleic acid sequence encoding a polypeptide having 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% identity, but less than 100% identity (wherein the nucleic acid encodes an amylase variant having amylase activity as described herein); (c) under high stringency conditions, preferably very high stringency conditions, (i) the coding sequence of SEQ ID NO: 1 or any amylase variant having amylase activity as described herein; or (ii) a polynucleotide represented by SEQ ID NO: 2 a polynucleotide that hybridizes to the complement of (d) a fragment of (a), (b), or (c), wherein the fragment encodes a polypeptide having amylase activity; or (e) a nucleic acid sequence that is completely complementary to any of (a) to (d); (f) a polynucleotide that differs from any of the nucleic acid sequences set forth in (a) through (e) solely due to the degeneracy of the genetic code; It also refers to a nucleic acid, preferably an isolated nucleic acid, a synthetic nucleic acid and / or a recombinant nucleic acid comprising:

[0199] The present invention also refers to a nucleic acid construct, preferably an expression cassette, comprising a polynucleotide described herein.

[0200] Typically, an expression cassette contains three elements: a promoter sequence, an open reading frame, and a 3' untranslated region that 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 within the coding sequence to increase the amount of mature mRNA that accumulates in the cytosol. An expression cassette can be part of a vector, or can be integrated into the genome of a host cell and replicated along with the genome of the host cell. Expression cassettes are usually capable of increasing or decreasing expression.

[0201] The present invention also refers 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.

[0202] As used herein, a vector may provide segments for the transcription and translation of an exogenous polynucleotide upon transformation into a host cell or host cell organelle. Such additional segments may include regulatory nucleotide sequences, one or more origins of replication required for its maintenance and / or replication in a particular cell type, one or more selectable markers, a polyadenylation signal, a multiple cloning site, or other suitable sites for insertion of the exogenous coding sequence. One example is when the vector is required to be maintained in a bacterial cell as an episomal genetic element (e.g., a plasmid or cosmid molecule). Non-limiting examples of suitable origins of replication include the f1-ori and colE1.

[0203] A vector can replicate in, for example, bacterial host cells as a plasmid without integration into the host cell genome, or it can integrate some or all of its DNA into the host cell genome, thereby bringing about the replication and expression of that DNA.

[0204] A polynucleotide encoding an amylase variant can be introduced into a vector by standard recombinant DNA techniques. Once in the vector, the polynucleotide containing the coding sequence can be suitable for introduction (transformed, transduced, transfected, etc.) into a host cell or host cell organelle. A cloning vector suitable for expression of the polynucleotide sequence in the host cell or host cell organelle can be selected.

[0205] host cell The present invention also refers 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 the host cell.

[0206] Polynucleotides encoding amylase variants as described herein can be transiently or stably introduced into host cells and can be maintained non-integrated, for example as a plasmid. Stable transformation is usually by chromosomal integration of the nucleic acid comprising the exogenous coding sequence or as an episome (a separate piece of nuclear DNA). Transient transformation is usually by non-chromosomal integration of the nucleic acid comprising the exogenous nucleic acid sequence or as an episome. Alternatively, polynucleotides encoding amylase variants as described herein can be integrated into the host genome.

[0207] Introduction of nucleic acids into host cells can be accomplished by, for example, but not limited to, protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168:111-115), use of 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), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169:5271-5278). Specific transformation protocols are known in the art for various types of host cells (see, for example, Hanahan, 1983, J. Mol. Biol. 166:557-580 for E. coli protoplast transformation).

[0208] Various host cells can be used to express the nucleic acid constructs described herein.The host cell comprising the gene constructs described herein can be obtained by one of the methods described herein for introducing polynucleotides into such host cells.The host cell of the present invention does not naturally express amylase variants.Therefore, the host cell is a recombinant host cell, and the nucleic acid constructs described herein are heterologous to the host cell.

[0209] In one embodiment, the host cell is a prokaryotic or 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.

[0210] In one embodiment, the host cell is a bacterial cell. The bacterial host cell can be any Gram-positive or Gram-negative bacterium. 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, and Gluconobacter. In certain embodiments, the bacterial host cell is an Escherichia coli cell. In one embodiment, the host cell is a bacterial cell. In certain embodiments, the host cell is of the genus Escherichia or Bacillus.

[0211] Preferably, the bacterial host cell is a Bacillus cell. The bacterial host cell can be any Bacillus cell. Bacillus cells useful in the practice of the present invention include 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 arginine ... Examples of suitable Bacillus species include, but are not limited to, Bacillus pumilus, Bacillus stearothermophilus, Bacillus methylotrophicus, Bacillus cereus, Bacillus 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, a Bacillus pumilus cell, or a Bacillus subtilis cell. Preferably, the bacterial host cell is a Bacillus licheniformis cell.

[0212] Alternatively, the bacterial host cell may be Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus bulgaricus, Lactobacillus reuteri, Escherichia coli, Staphylococcus aureus, Corynebacterium glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetacidophilum, or any of the following: acetoacidophilum, Corynebacterium callunae, Corynebacterium ammoniagenes, Corynebacterium thermoaminogenes, Corynebacterium melassecola, Corynebacterium effiziens, Corynebacterium efficiens, Corynebacterium deserti, Brevibacterium flavum, Brevibacterium lactofermentum lactofermentum, Brevibacterium divarecatum, Pseudomonas putida, Pseudomonas syringae, Streptomyces coelicolorcoelicolor, Streptomyces lividans, Streptomyces albus, Streptomyces avermitilis, Gluconobacter oxydans, Gluconobacter morbifer, Gluconobacter thailandicus, Acetobacter aceti, Clostridium acetobutylicum, Clostridium saccharobutylicum, Clostridium beijerinckii, Streptococcus equisimilis The bacterial strain may be Streptococcus equi subsp., Zooepidemicus, Basfia succiniciproducens, Streptococcus pyogenes, Streptococcus uberis, Streptococcus equi subsp., Zooepidemicus, or Basfia succiniciproducens.

[0213] Alternative and additional host cells include Aspergillus niger, Aspergillus oryzae, Hansenula polymorpha, Thermomyces lanuginosus, Fusarium oxysporum, Fusarium heterosporum, Pichia pastoris (also known as Komagataella phaffii), Myceliopthora thermophile (C1), Themothelomyces thermophila, Schizosaccharomyces pombe, and the like. pombe, Trichoderma, preferably Trichoderma reesei, and Saccharomyces, preferably Saccharomyces cerevisiae or Rhizomucor.

[0214] In another embodiment, the bacterial host cell may further contain modifications, such as deletions or disruptions, of other genes that may be undesirable for the production, recovery or application of the polypeptide of interest.

[0215] Production method Another embodiment of the present invention is a method for obtaining an amylase variant of a parent amylase, comprising the steps of: a) a parent amylase, preferably any one of SEQ ID NOs: 1, 3, 4 or any one of SEQ ID NOs: 15 to 41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1; (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 3, and (ii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to amino acid positions selected from the group consisting of 25, 176, 186, 251, 405, and 482 according to the numbering of SEQ ID NO:3; introducing 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, thereby providing an amylase variant of said parent amylase, said variant having at least 60% but less than 100% sequence identity with the amino acid sequence of any of SEQ ID NOs: 1, 3, 4 or SEQ ID NOs: 15-41, preferably the polypeptide of SEQ ID NO: 1, and said amylase variant having amylase activity, preferably having improved properties with respect to said parent. The method includes:

[0216] A preferred embodiment of the present invention is a method for obtaining an amylase variant of a parent amylase, comprising the steps of: a) a parent amylase, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 3, and (ii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to amino acid positions selected from the group consisting of 25, 176, 186, 251, 405, and 482 according to the numbering of SEQ ID NO:3; introducing 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, thereby providing an amylase variant of said parent amylase, said variant having at least 80% but less than 100% sequence identity with the amino acid sequence to the polypeptide of SEQ ID NO: 1, said amylase variant having amylase activity, preferably having improved properties relative to said parent. The method includes:

[0217] Methods for introducing amino acid modifications, such as 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, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0218] The resulting amylase variant can be produced on an industrial scale and then purified. Industrial production of enzymes is typically achieved by culturing host cells that express the enzyme (also known as fermentation). Suitable host cells are described herein. A nucleic acid sequence encoding an amylase variant 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.

[0219] Thus, in yet another embodiment, the present invention provides a method for producing an amylase variant, comprising the steps of: (a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding an amylase variant as described herein by introducing into the host cell a nucleic acid construct comprising a polynucleotide encoding an amylase variant as described herein; (b) culturing the recombinant host cell of step (a) under conditions conducive to expression of the polynucleotide; (c) optionally recovering the amylase variant encoded by the polynucleotide; The present invention relates to a method comprising:

[0220] Cultivation of the host cells is typically carried out in a suitable nutrient medium that allows the recombinant cells to grow and express the desired protein. At the end of fermentation, the fermentation broth is collected and may be further processed, the fermentation broth comprising a liquid fraction and a solid fraction. The enzyme of interest may be further purified from the fermentation broth.

[0221] The amylase variants described herein may or may not be secreted from the microbial cells (into the liquid fraction of the fermentation broth) (and thus be contained within the cells in the fermentation broth). Accordingly, the amylase variants can be recovered from the liquid fraction 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 secretory signal peptide added to the end of the amino acid sequence of the amylase variant. Recovery of the amylase variants can 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, harvesting, centrifugation, filtration, extraction, and precipitation. If the product of interest precipitates or crystallizes in the fermentation broth or is at least partially bound to particulate matter in the fermentation broth, additional processing steps may be required to release the protein of interest from the biomass or to solubilize the crystals and precipitates of the protein of interest. WO 0043502 A1, WO 2008110498 A1, and WO 2017097869 A1 describe methods for recovering proteins of interest that precipitate and / or crystallize from a fermentation broth during fermentation. When the desired protein is contained within the cells of the fermentation broth, it may be necessary to release the product of interest from the cells. Release from the cells can be achieved by, for example, but not limited to, cell lysis by techniques well known to those skilled in the art, such as lysozyme treatment, sonication, French press, or combinations thereof.

[0222] Amylase variants can be purified from the fermentation broth by methods known in the art. For example, amylase variants can be isolated from the fermentation broth by conventional procedures, including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. The isolated polypeptide can then be purified by a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoresis (e.g., preparative isoelectric focusing (IEF)), solubility differences (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, 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.

[0223] Preparation of amylase Purified solutions of the amylase variants described herein can be further processed to form amylase-containing compositions. Accordingly, compositions comprising the amylase variants described herein and at least one additional component are also claimed herein.

[0224] Accordingly, the present invention provides a method of making a composition, comprising the steps of: a) an amylase variant as described herein; and b) one or more components described herein; and Also reference is made to a method comprising the step of mixing

[0225] Further, therefore, the present invention provides a method for improving amylase stability in a composition, comprising the steps of: a) an amylase variant as described herein; and b) one or more components described herein; and Also reference is made to a method comprising the step of mixing

[0226] The composition may be a non-complex formulation, such as an amylase variant formulation, or a complex formulation, such as a detergent composition.

[0227] In one embodiment of the present invention, the amylase variant is formulated as an amylase variant formulation, preferably a concentrated amylase variant formulation. The amylase variant formulation can be either solid or liquid. The protein formulation can be obtained by 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 / 19444 A1 and WO 97 / 43482 A1.

[0228] In one embodiment, the amylase variant formulation, particularly a liquid enzyme formulation, additionally 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 lacks surfactants. The solvent can be water and / or an organic solvent. Aqueous amylase variant formulations of the present invention can comprise water in an amount greater than about 50%, greater than about 60%, greater than about 70%, or greater than about 80% by weight based on the total weight of the enzyme formulation. Amylase variant containing formulations of the present invention can comprise an organic solvent in an amount greater than 30%, greater than 40%, greater than about 50%, greater than about 60%, greater than about 70%, or greater than about 80% by weight based on the total weight of the enzyme formulation. The organic solvent can be a water-miscible solvent. The organic solvent can be one or more selected from the group consisting of glycerol, propanediol, polypropylene glycol, and polyethylene glycol.

[0229] In one embodiment, the amylase variant formulation includes at least one preservative. Preferably, the term "preservative" refers to a substance added to a liquid composition for preservation purposes, and more preferably, the term "preservative" excludes compounds known to have a preservative function that are present in the liquid composition formed during the production process. In one embodiment, the preservative is selected from the group consisting of 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol, formic acid in its acid form or salt form, and 4,4'-dichloro-2-hydroxydiphenyl ether. Typically, the liquid composition of the present invention includes at least one preservative in an amount of less than 10 ppm, such as an amount ranging from 2 ppm to 5 wt. % based on the total weight of the liquid composition. Alternatively, the amylase variant formulation does not include a preservative, meaning that the preservative is present in an amount of less than 1 ppm, preferably 0 ppm.

[0230] Preferably, the amylase variant formulations include an enzyme stabilization system. Preferably, the amylase variant formulations described herein include 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 compatible with the amylase.

[0231] Preferably, the enzyme stabilization system comprises at least one compound selected from the group consisting of a polyol (preferably 1,3-propanediol, ethylene glycol, glycerol, 1,2-propanediol 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, formate (preferably sodium formate), acetic acid, acetate or lactate), a borate, boric acid, a 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 compounds selected from the group consisting of salts, polyols, and short-chain carboxylic acids, and preferably one or more compounds selected from the group consisting of borate, boric acid, boronic acid (preferably 4-formylphenylboronic acid (4-FPBA)), peptide aldehyde, peptide acetal, and peptide aldehyde hydrosulfite adduct. In a preferred embodiment, the stabilization system, in the absence of a protease, comprises a protease inhibitor, preferably selected from borate, boric acid, boronic acid (preferably 4-FPBA), peptide aldehyde (preferably a peptide aldehyde such as Z-VAL-H or Z-GAY-H), peptide acetal, and peptide aldehyde hydrosulfite adduct, 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 comprise boron. Preferably, the amylase variant formulation comprises a calcium salt, preferably calcium chloride.

[0232] 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 lacks surfactants.

[0233] Accordingly, the present invention provides a method for producing an amylase variant preparation, preferably a concentrated amylase variant preparation, comprising the steps of: a) an amylase variant as described herein; and 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; Also reference is made to a method comprising the step of mixing

[0234] Further, therefore, the present invention provides a method for improving amylase stability in a formulation, comprising: a) an amylase variant as described herein; and 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; Also reference is made to a method comprising the step of mixing

[0235] Second enzyme In another embodiment, the 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 a protease, a second amylase, a lipase, a cellulase, a mannanase, a hemicellulase, a phospholipase, an esterase, a pectinase, a lactase, a peroxidase, a xylanase, a cutinase, a pectate lyase, a keratinase, a reductase, an oxidase, a phenoloxidase, a lipoxygenase, a ligninase, a pullulanase, a tannase, a pentosanase, a malanase, a beta-glucanase, an arabinosidase, a hyaluronidase, a chondroitinase, a laccase, a nuclease, a DNase, a phosphodiesterase, or a phytase. The second enzyme is selected from the group consisting of: α-galactosidase, β-galactosidase, α-glucosidase, β-gluc ...

[0236] The compositions of the invention may contain one enzyme or more than one enzyme of different types, for example, an amylase and a protease, or more than one enzyme of the same type, for example, two or more different proteases, or a mixture thereof, for example, an amylase and two different proteases.

[0237] Proteases A protease is an active protein that exerts "protease activity" or "proteolytic activity." Proteolytic activity relates to the rate of degradation of a protein by a protease or proteolytic enzyme over a given period of time.

[0238] Preferably, the second enzyme distinct from the amylase variant is a protease having at least 40-100% identity to the full-length polypeptide sequence of any 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 set forth in SEQ ID NOs: 10, 11, 12, 13 or 14, preferably SEQ ID NO: 10.

[0239] 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 has at least one amino acid sequence at the following positions: 3, 4, 9, 15, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111 and further comprising an amino acid substitution at one or more of: 7, 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 the BPN numbering), and have proteolytic activity. In one embodiment, such proteases are not mutated at positions Asp32, His64, and Ser221 (according to the 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 to SEQ ID NO: 10, and is further characterized by having the 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 has proteolytic activity. Most preferred are proteases that have at least 80% but less than 100% sequence identity with SEQ ID NO: 10, are characterized by having the amino acid glutamic acid (E) at position 101 (according to BPN numbering) and have proteolytic activity.The protease can 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 further 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), or (i) a combination of two or more amino acids from (a)-(h). A suitable protease may be at least 80% identical to SEQ ID NO: 10 and is characterized by comprising one amino acid (according to (a) to (h)) or a combination according to (i) together with amino acids 101E, 101D, 101N, 101Q, 101A, 101G or 101S (according to BPN numbering), and has proteolytic activity. In one embodiment, the protease is at least 80% identical to SEQ ID NO: 10 and is characterized by comprising the 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 further characterized by comprising S3T+V4I+S9R+A15T+V68A+D99S+R101S+A103S+I104V+N218D (according to BPN numbering), 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 further characterized by comprising R101E and S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61D,R, S87E, G97S, A98D,E,R, S106A,W, N117E, H120V,D,K,N, S125M, P129D,E 136Q, S144W, S161T, S163A,G, Y171L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D and L262N,Q,D (according to BPN numbering), and have proteolytic activity.

[0240] Lipase "Lipase," "lipolytic enzyme," and "lipid esterase" all refer to enzymes in EC class 3.1.1 ("carboxyl ester hydrolases"). Lipase refers to 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.

[0241] In one aspect of the invention, suitable lipases (component (b)) are selected from: lipases from the genus Humicola (synonym Thermomyces) as described in EP 258068, EP 305216, WO 92 / 05249 and WO 2009 / 109500, for example from H. lanuginosa (T. lanuginosus) or from H. insolens as described in WO 96 / 13580; lipases from Rhizomucor mihaii as described in WO 92 / 05249; Lipases derived from Pseudomonas species (some of which have now been renamed Burkholderia), such as P. alcaligenes or P. pseudoalcaligenes (EP 218272, WO 94 / 25578, WO 95 / 30744, WO 95 / 35381, WO 96 / 00292), P. cepacia (EP 331376), P. stutzeri (GB 1372034), P. fluorescens, Pseudomonas species sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (WO 96 / 12012), Pseudomonas mendocina (WO 95 / 14783), P. glumae (P.Lipases from Streptomyces glumae (WO 95 / 35381, WO 96 / 00292), Streptomyces griseus (WO 2011 / 150157) and S. pristinaespiralis (WO 2012 / 137147), GDSL-type Streptomyces lipases (WO 2010 / 065455), lipases from Thermobifida fusca as disclosed in WO 2011 / 084412, Geobacillus stearothermophilus as disclosed in WO 2011 / 084417, Lipases derived from Bacillus stearothermophilus, for example Bacillus lipases as disclosed in WO 00 / 60063, B. subtilis as disclosed in Dartois et al. (1992), Biochemica et Biophysica Acta, 1131, 253-360 or WO 2011 / 084599, B. stearothermophilus (JP 64-074992 A) or B. pumilus (B.lipase from Candida antarctica as disclosed in WO 94 / 01541, cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536, WO 88 / 09367), cutinase from Magnaporthe grisea (WO 2010 / 107560), cutinase from Fusarum solani pisi as disclosed in WO 90 / 09446, WO 00 / 34450 and WO 01 / 92502 pisi and cutinases from Humicola lanuginosa as disclosed in WO 00 / 34450 and WO 01 / 92502.

[0242] 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.

[0243] Commercially available lipase enzymes include, but are not limited to, those sold under the trade names Lipolase™, Lipex™, Lipolex™ and Lipoclean™ (Novozymes A / S), Lumafast (originally from Genencor), Preferenz L (DuPont) and Lipomax (Gist-Brocades / now DSM).

[0244] In one embodiment, the lipase is selected from fungal triacylglycerol lipases (EC class 3.1.1.3). The fungal triacylglycerol lipase may be selected from Thermomyces lanuginosus lipases. In one embodiment, the Thermomyces lanuginosa lipase is selected from the triacylglycerol lipases according to amino acids 1-269 of SEQ ID NO: 2 of U.S. Pat. No. 5,869,438 and variants thereof having lipolytic activity.

[0245] The Thermomyces lanuginosa lipase may be selected from variants 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 when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO:2 of U.S. Pat. No. 5,869,438.

[0246] The Thermomyces lanuginosa lipase may be selected from variants with lipolytic activity that contain only conservative mutations that do not belong to the functional domain of amino acids 1-269 of SEQ ID NO:2 of U.S. Patent No. 5,869,438. The lipase variant of this embodiment with lipolytic activity may 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 U.S. Patent No. 5,869,438.

[0247] The Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity comprising the following amino acid substitutions: T231R and N233R when compared to amino acids 1-269 of SEQ ID NO:2 of U.S. Patent No. 5,869,438. The lipase variant may further comprise one or more of the following amino acid exchanges: Q4V, V60S, A150G, L227G, P256K when compared to amino acids 1-269 of SEQ ID NO:2 of U.S. Patent No. 5,869,438.

[0248] The Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity comprising the 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 U.S. Pat. No. 5,869,438, and which are 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 U.S. Pat. No. 5,869,438.

[0249] The Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity comprising the amino acid substitutions T231R and N233R within amino acids 1-269 of SEQ ID NO:2 of U.S. Pat. No. 5,869,438, and which are 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 U.S. Pat. No. 5,869,438.

[0250] The Thermomyces lanuginosa lipase may be a variant of amino acids 1 to 269 of SEQ ID NO: 2 of U.S. Pat. No. 5,869,438 having lipolytic activity, wherein the variant of amino acids 1 to 269 of SEQ ID NO: 2 of U.S. Pat. No. 5,869,438 is characterized by containing the amino acid substitutions T231R and N233R.

[0251] The Thermomyces lanuginosa lipase may be selected from variants with lipolytic activity that preferably comprise at least one, preferably two or more, more preferably all of the following substitutions N11K, A18K, G23K, K24A, V77I, D130A, V154I, V187T, T189Q within the polypeptide sequence of amino acids 1 to 269 of SEQ ID NO: 1 of WO 2015 / 010009, and that are at least 95%, at least 96% or at least 97% similar when compared to the full-length polypeptide sequence of amino acids 1 to 269 of SEQ ID NO: 1 of WO 2015 / 010009.

[0252] amylase Amylases different from the amylases described herein include those 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).

[0253] The amylase may be derived from Bacillus licheniformis having SEQ ID NO: 2 as described in WO 95 / 10603, and at least 95% variants thereof. Suitable variants are described in WO 95 / 10603 and contain 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, and have amylolytic activity. The variant is described in SEQ ID NO: 4 of WO 94 / 02597, WO 94 / 018314, WO 97 / 043424 and WO 99 / 019467.

[0254] The amylase may further be derived from B. stearothermophilus having SEQ ID NO: 6 as disclosed in WO 02 / 10355, or may be an amylase optionally having a C-terminal truncation over the wild-type sequence. Suitable variants of SEQ ID NO: 6 include those containing deletions at 179, and / or 181, and / or 182 and / or a substitution at position 193.

[0255] The amylase may further be derived from Bacillus sp. 707 having SEQ ID NO: 6 as disclosed in WO 99 / 19467 and at least 95% variants thereof. Preferred variants of SEQ ID 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.

[0256] The amylase may further be derived 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.

[0257] The amylase may further be derived from Bacillus sp. DSM 12649 having SEQ ID NO: 4 as disclosed in WO 00 / 22103 and at least 95% variants thereto.

[0258] The amylase may further be derived from Bacillus sp. A 7-7 (DSM 12368) having an amino acid sequence that is at least 95% identical to SEQ ID NO:2 as disclosed in WO 02 / 10356, in particular spanning the region from amino acid 32 to 516 according to SEQ ID NO:2.

[0259] The amylase may further be derived from Bacillus strain TS-23 having SEQ ID NO: 2 as disclosed in WO 2009 / 061380 and variants thereof.

[0260] The amylase may further be derived from Cytophaga sp. having SEQ ID NO: 1 as disclosed in WO 2013 / 184577 and at least 95% variants thereto.

[0261] The amylase may further be derived from Bacillus megaterium DSM 90 having SEQ ID NO: 1 as disclosed in WO 2010 / 104675 and at least 95% variants thereof.

[0262] The amylase may further be derived from a Bacillus sp. comprising amino acids 1-485 of SEQ ID NO: 2 as described in WO 00 / 60060, and at least 95% variants thereof.

[0263] The amylase may further be derived from Bacillus amyloliquefaciens or a variant thereof, preferably selected from the amylase according to SEQ ID NO: 3 as described in WO 2016 / 092009.

[0264] 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 in said SEQ ID NO: 12.

[0265] The amylase may have SEQ ID NO: 6 as described in WO 2011 / 098531 or an amylase variant comprising a substitution at one or more positions within SEQ ID NO: 6 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].

[0266] The amylase may have SEQ ID NO: 1 as described in WO 2013 / 001078 or an amylase variant comprising modifications at two or more (several) positions corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476 and G477 within SEQ ID NO: 1.

[0267] The amylase may have SEQ ID NO:2 as described in WO 2013 / 001087 or an amylase variant comprising a deletion of positions 181+182 or 182+183 or 183+184 within SEQ ID NO:2, optionally with one or more modifications at any of the positions corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476 and G477 within SEQ ID NO:2.

[0268] The amylase may be a hybrid alpha-amylase derived from the amylases mentioned above, for example as described in WO 2006 / 066594.

[0269] The hybrid amylase may be according to WO2014 / 183920 having A and B domains at least 90% identical to SEQ ID NO: 2 of WO2014 / 183920 and a C domain at least 90% identical to SEQ ID NO: 6 of WO2014 / 183920, wherein the hybrid amylase has amylolytic activity, preferably the hybrid alpha-amylase is at least 95% identical to SEQ ID NO: 23 of WO2014 / 183920 and has amylolytic activity.

[0270] The hybrid amylase may be according to WO 2014 / 183921 having A and B domains at least 75% identical to 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 a C domain at least 90% identical to SEQ ID NO:6 of WO 2014 / 183921, the hybrid amylase having amylolytic activity, preferably a hybrid alpha-amylase at least 95% identical to SEQ ID NO:30 as disclosed in WO 2014 / 183921 and having amylolytic activity, the hybrid amylase having A and B domains from an alpha amylase from Bacillus sp. A 7-7 (DSM 12368) and a C domain at least 90% identical to SEQ ID NO:6 of Bacillus cereus. The hybrid amylase may be according to WO2021 / 032881, comprising a C domain derived from an alpha-amylase derived from Bacillus cereus, preferably wherein 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 as disclosed in WO2021 / 032881), more preferably wherein the hybrid amylase is at least 80% identical to SEQ ID NO: 54 as disclosed in WO2021 / 032881.

[0271] In one embodiment, the at least one amylase is selected from commercially available amylases, including, but not limited to, products sold under the trade names Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™, Amplify™, Amplify Prime™ (Novozymes A / S) and Rapidase™, Purastar™, Powerase™, Effectenz™ (M100 from DuPont), Preferenz™ (S1000, S110 and F1000, DuPont), PrimaGreen™ (ALL, DuPont), Optisize™ (DuPont).

[0272] Mannanase As used herein, a "mannanase" is an enzyme selected from the group of mannan-degrading enzymes. The mannan-degrading enzymes may be selected from β-mannosidases (EC 3.2.1.25), endo-1,4-β-mannosidases (EC 3.2.1.78), and 1,4-β-mannobiosidases (EC 3.2.1.100). Preferably, the mannan-degrading enzymes are selected from the group of endo-1,4-β-mannosidases (EC 3.2.1.78), a group of enzymes that may be referred to herein as endo-β-1,4-D-mannanases, β-mannanases, or mannanases.

[0273] The mannanase may be selected from alkaline mannanases of family 5 or 26 (i.e., GH5 or GH26). The term "alkaline mannanase" is intended to encompass mannanases that have an enzymatic activity of at least 40% of its maximum activity at a given pH in the range of 7 to 12, preferably 7.5 to 10.5.

[0274] Mannanases are disclosed in Japanese Patent Application Laid-Open No. 0304706 (beta-mannanase derived from Bacillus sp.), Japanese Patent Application Laid-Open No. 63056289 (alkali- and thermostable beta-mannanase), Japanese Patent Application Laid-Open No. 63036774 (Bacillus microorganisms FERM P-8856 producing beta-mannanase and beta-mannosidase at alkaline pH), Japanese Patent Application Laid-Open No. 08051975 (alkaline beta-mannanase derived from alkaliphilic Bacillus sp. AM-001), and International Publication No. 97 / 11164 (Bacillus amyloliquefaciens). amyloliquefaciens], WO 91 / 18974 [mannanases active at extremes of pH and temperature], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 2014 / 100018 [endo-(3-mannanase 1 cloned from Bacillus circulans or Bacillus lentus strain CMG1240 (Bleman1, see U.S. Pat. No. 5,476,775)]. Suitable mannanases are described in WO 99 / 064619.

[0275] The mannanase may be selected from mannanases derived from Trichoderma organisms, such as those disclosed in WO 93 / 24622.

[0276] The mannanase may be selected from commercially available mannanases such as Mannaway® (Novozymes A / S) or Preferenz® (M100) (DuPont).

[0277] cellulase "Cellulase" is an enzyme capable of hydrolyzing cellulose. Cellulases can be selected from cellobiohydrolases (1,4-PD-glucan cellobiohydrolases, EC 3.2.1.91), endo-ss-1,4-glucanases (EC 3.2.1.4), and ss-glucosidases (EC 3.2.1.21). Endoglucanases in EC class 3.2.1.4 can be named endoglucanases, endo-1,4-ss-D-glucan 4-glucanohydrolases, endo-1,4-beta-glucanases, carboxymethylcellulases, and beta-1,4-glucanases.

[0278] Endoglucanases can be classified by amino acid sequence similarity (Henrissat, B. UniProt accessed 10 / 26 / 2011) under family 5, which contains more than 20 endoglucanases in EC 3.2.1.4. T.-M. Enveri, “Microbial Cellulases” WM Fogarty, Microbial Enzymes and Biotechnology, Applied Science Publishers, p. 183-224 (1983); Methods in Enzymology, (1988) Vol. 160, p. 200-391 (edited by Wood, WA and Kellogg, ST); Beguin, P., “Molecular Biology of Cellulose Degradation”, Annu.Rev.Microbiol.(1990), Vol.44, pp.219248; Begun, P. and Aubert, JP., “The biological degradation of cellulose”, FEMS Microbiology Reviews 13(1994) p.25-58; Henrissat, B., “Cellulases and their interaction with See also "Cellulose", Cellulose (1994), Vol. 1, pp. 169-196.

[0279] Preferably, the at least one cellulase is selected from glycosyl hydrolase family 7 (GH7, pfam00840), which is preferably selected from endoglucanases (EC 3.2.1.4).

[0280] Preferably, alkaline cellulase is used, and "alkaline cellulase" is intended to encompass cellulase that has enzymatic activity at a given pH in the range of 7 to 12, preferably 7.5 to 10.5.

[0281] In one embodiment, the cellulase is selected from cellulases that comprise a cellulose binding domain. In another embodiment, the cellulase comprises a catalytic domain but does not comprise a cellulose binding domain.

[0282] In one embodiment, the at least one endoglucanase in the EC class 3.2.1.4 is Bacillus species such as Bacillus sp. CBS 670.93 and CBS 669.93; Melanocarpus, such as Melanocarpus albomyces, as disclosed in WO 97 / 14804; Clostridium species, for example Clostridium thermocellum, Humicola, such as Humicola insolens (DSM 1800), as disclosed in EP 0 495 257, EP 0 531 315, EP 0 531 372, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,776,757, WO 89 / 09259, WO 91 / 17244, WO 94 / 07998 (the human variant of the sequence 43 kd shown in FIG. 1), WO 95 / 24471, WO 96 / 11262 and WO 98 / 12307; Fusarium species, such as Fusarium oxysporum, for example strain J79 (DSM2672), as disclosed in EP 0 495 257, EP 0 531 315, EP 0 531 372, U.S. Pat. No. 5 648 263, U.S. Pat. No. 5 776 757, WO 89 / 09259, WO 91 / 17244, WO 95 / 24471 and WO 96 / 11262, Thielavia, such as Thielavia terrestris or Myceliophthora thermophila strain CBS 11765 as disclosed in EP 0 531 315, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,776,757, 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, Trichoderma species, such as Trichoderma reesei, Trichoderma longibrachiatum or Trichoderma harzianum, as disclosed in EP 1 305 432, EP 1 240 525, WO 92 / 06165, WO 94 / 21801, WO 94 / 26880, WO 95 / 02043, WO 95 / 24471 and WO 02 / 099091, Aspergillus, such as Aspergillus aculeatus, as disclosed in WO 93 / 17244; Erwinia species, such as Erwinia chrysanthermi, as described by M.H. Boyer et al. in European Journal of Biochemistry, vol. 162, 311-316 (1987); 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). Acremonium genus such as Acremonium furatum, Cellvibrio, such as 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; Cephalosporium species, such as Cephalosporium sp. RYM-202, as disclosed in WO 96 / 11262 It comes from.

[0283] Suitable cellulases also include cellulolytic variants of the cellulases described above. In one embodiment, the cellulase variants include variants having at least 40-100% identity compared to the full-length polypeptide sequence of the parent enzyme as disclosed above. In one embodiment, the cellulolytic variant cellulase is 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.

[0284] The cellulase can be a Humicola insolens DSM 1800 cellulase complex having endoglucanase, cellobiohydrolase and beta-glucosidase activities.

[0285] The cellulase may preferably be Humicola insolens DSM 1800 endoglucanase (EC 3.2.1.4) having a polypeptide sequence according to positions 21 to 435 of SEQ ID NO: 2 as disclosed in WO 2018 / 224544, or a variant at least 95% identical thereto.

[0286] The cellulase may preferably be a Humicola insolens endoglucanase (EC 3.2.1.4) having 43 kD according to the polypeptide sequence as disclosed in Figure 1a of WO 94 / 07998 ("43 kD hum") or a variant thereof preferably at least 90% identical thereto, preferably one disclosed in WO 94 / 07998.

[0287] The cellulase can be a Bacillus sp. cellulase (EC 3.2.1.4) selected from polypeptides at least 80% similar to and / or identical to the amino acid sequence of positions 1 to 773 of SEQ ID NO: 2 of WO 2004 / 053039 or catalytically active fragments thereof. In one embodiment, the cellulase is a mature polypeptide at least 95% identical to SEQ ID NO: 1 of WO 2018 / 224544.

[0288] The cellulase can be a Thielavia terrestris cellulase (EC 3.2.1.4) having a polypeptide or catalytically active fragment thereof that is at least 80% similar and / or identical to the amino acid sequence of positions 1 to 299 of SEQ ID NO: 4 of WO 2004 / 053039. In one embodiment, the cellulase is a mature polypeptide that is at least 95% identical to SEQ ID NO: 4 of WO 2018 / 224544.

[0289] The cellulase may preferably be a mature Sordaria fimicola cellulase having a polypeptide sequence according to SEQ ID NO: 5 of WO 2018 / 224544 or a variant at least 95% identical thereto.

[0290] The at least one cellulase may be selected from Renozyme®, Celluzyme®, Celluclean®, Endolase® and Carezyme® (Novozymes A / S), Clazinase™, and Puradax HA™ (Genencor Int. Inc.) and KAC-500(B)™ (Kao Corporation).

[0291] Detergent Composition In one embodiment, the present invention relates to the use of an amylase variant in a detergent composition. Accordingly, the present invention also relates to a detergent composition comprising an amylase variant as described herein and one or more detergent components.

[0292] Accordingly, the present invention provides a method of making a detergent composition, comprising the steps of: a) an amylase variant as described herein; and b) one or more detergent components described herein; and Also reference is made to a method comprising the step of mixing

[0293] Further, the present invention therefore provides a method of making a detergent composition with improved amylase stability and / or providing a detergent composition with improved cleaning performance, comprising the steps of: a) an amylase variant as described herein; and b) one or more detergent components described herein; and Also reference is made to a method comprising the step of mixing

[0294] The one or more detergent components may be selected from the group consisting of additional enzymes different from the amylase variant, enzyme stabilizing systems, surfactants, antifoaming agents, builders, polymers, bleaching systems (bleaches), rheology modifiers, hydrotropes, softeners, drying agents, brighteners, buffers, preservatives, anticorrosion additives, dyes and fragrances.

[0295] Preferably, at least one component of the detergent is selected from the group consisting of surfactants, builders, polymers, preservatives and a second enzyme different from the amylase variant.

[0296] Preferably, one or more of the detergent components, preferably surfactants and / or builders, are biodegradable and / or bio-based.

[0297] A detergent component may have more than one function in the final application of the detergent composition; therefore, any detergent component referred to herein with respect to a particular function may also have another function in the final application of the detergent composition. The function of a particular detergent component in the final application of the detergent composition typically depends on its amount in the detergent composition, i.e., the effective amount of the detergent component. The detergent components vary in type and / or amount in the detergent composition depending on the desired application, such as washing white fabrics, colored fabrics, and wool. The components selected further depend on the physical form of the detergent composition (liquid, solid, gel, provided in a pouch or as a tablet, etc.). For example, the components selected for laundry formulations further depend on local customs, which themselves relate to aspects such as the wash temperature used, washing machine configuration (horizontal axis machine vs. vertical axis machine), water consumption per wash cycle, and geographical features such as average water hardness.

[0298] In one embodiment, the detergent composition is a blend of three or more detergent components, at least one component effective in stain removal, at least one component effective in providing optimal cleaning conditions, and at least one component effective in maintaining the physical characteristics of the detergent.

[0299] The detergent composition can be a liquid or solid detergent composition or a combination of liquid and solid detergent compositions. The liquid detergent composition is preferably a gel detergent composition. The solid detergent composition can be a soap bar or a powder detergent composition, preferably a powder detergent composition, which can be compressed into tablets.

[0300] 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 the 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) composition.

[0301] The detergent pouch can be of any form, shape, and material suitable for holding the composition, e.g., preventing release of the composition from the pouch prior to contact with water. The pouch is made of a water-soluble film that encloses an internal volume. The internal volume can be divided into compartments of the pouch. Preferred films are polymeric materials, preferably polymers that can be formed into films or sheets, such as polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC). The pouch can contain solid laundry detergent compositions or portions of components and / or liquid detergent compositions or portions of components separated by a water-soluble film. The compartment for the liquid components can be different in composition from the compartment containing the solids (see, e.g., U.S. Patent Application Publication No. 2009 / 0011970).

[0302] Preferably, the amylase variant according to the present invention may be added to the detergent composition in an amount corresponding to 0.002 to 6 mg of active enzyme variant per gram of detergent composition, preferably 0.005 to 5 mg / g, 0.005 to 3 mg / g, 0.01 to 2 mg / g or 0.05 to 2 mg / g.

[0303] In one embodiment the detergent composition has a pH in the range of 5 to 12, preferably in the range of 6 to 11, more preferably in a range selected from 6 to 10, 7 to 9 and 7.5 to 8.5. In one embodiment the formulation is a detergent composition, preferably a liquid detergent composition.

[0304] In one embodiment, the detergent composition according to the present invention comprises one or more surfactants. Depending on their ionic charge, surfactants are called nonionic, anionic, cationic or amphoteric.

[0305] The detergent compositions of the present invention may contain one or more surfactants, which may be anionic, cationic, nonionic, semi-polar, zwitterionic, or mixtures thereof. In preferred embodiments, the detergent compositions of the present invention contain at least one surfactant. In certain embodiments, the detergent compositions of the present invention contain a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant is typically present at a level of about 0.1 to 60% by weight, e.g., 1 to 40%, 3 to 20%, or 3 to 10% by weight. The surfactant is selected based on the desired cleaning application and includes any conventional surfactant known in the art. Any surfactant known in the art for use in detergents may be utilized. 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. Further useful examples are disclosed in earlier editions of the same publications known to those skilled in the art.

[0306] When included therein, the detergent will typically contain about 1-40% by weight, e.g., 5-30%, 5-15%, or 20-25% by weight, of anionic surfactants. Non-limiting examples of anionic surfactants include sulfates and sulfonates, particularly linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkenesulfonates, alkane-2,3-diylbis(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, and the like. phosphates (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 glycerol esters, alpha-sulfofatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonates (MES), alkyl- or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, di- and monoesters of sulfosuccinic acid or soaps and combinations thereof.

[0307] If included, the detergent will typically contain from about 0 to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyldimethylethanolamine quats (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

[0308] When included therein, the detergent will typically contain about 0.2 to 40% by weight of anionic surfactant, for example 0.5 to 30% by weight, especially 1 to 20% by weight, 3 to 10% by weight, 3 to 5% by weight or 8 to 12% by weight. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFAs), alkoxylated fatty acid alkyl esters, such as ethoxylated fatty acid alkyl esters and / or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), N-acyl N-alkyl derivatives of polyhydroxyalkyl fatty acid amides or glucosamine (glucosamides, GA or fatty acid glucamides, FAGA), and products available under the trade names SPAN and TWEEN®, and combinations thereof.

[0309] When included therein, the detergent will typically contain from about 0 to about 10% by weight of semi-polar surfactants. Non-limiting examples of semi-polar surfactants include amine oxides (AOs) such as alkyl dimethyl amine oxide, N-(cocoalkyl)-N,N-dimethyl amine oxide, and N-(tallow-alkyl)-N,N-bis-(2-hydroxy-ethyl) amine oxide, fatty acid alkanolamides, and ethoxylated fatty acid alkanolamides, and combinations thereof.

[0310] If included, the detergent will typically contain from about 0 to about 10% by weight of zwitterionic surfactants. Non-limiting examples of zwitterionic surfactants include betaines, alkyldimethylbetaines, sulfobetaines, and combinations thereof.

[0311] The detergent compositions according to the present invention may comprise one or more compounds selected from complexing agents (chelants, sequestrants), precipitating agents and ion-exchange compounds capable of forming water-soluble complexes with calcium and magnesium. Such compounds may be referred to herein as "builders" or "building agents", although this is not meant to limit such compounds to this function in the final application of the detergent composition.

[0312] In one embodiment, the detergent compositions of the present invention contain at least one builder selected from non-phosphate builders, such as sodium gluconate, sodium citrate, sodium silicate, sodium carbonate, sodium phosphonate, sodium aminocarboxylate, sodium polycarboxylate, sodium polysulfonate, and sodium polyphosphonate. In one embodiment, the detergent compositions of the present invention contain a strong metal ion-trapping builder. Preferably, the detergent compositions of the present invention are phosphate-free, meaning essentially free of phosphate builders. As used herein, "essentially phosphate-free" should be understood to mean that the phosphate and polyphosphate content, determined by gravimetric measurement, is in the range of 10 ppm to 1% by weight in total, with reference to the respective detergent compositions of the present invention. In another preferred embodiment, the detergent compositions contain a phosphonate, preferably DTPMP and / or HEDP.

[0313] In one embodiment, the detergent composition of the present invention comprises at least one "citrate" selected from monoalkali and dialkali metal salts of citric acid, particularly the monosodium and preferably trisodium salts, ammonium or substituted ammonium salts of citric acid, and such citric acids. The citrate may be used as an anhydrous compound or a hydrate, such as sodium citrate dihydrate. The citrate may be present in a total amount ranging from 0% to about 20% by weight, from about 0.5% to about 10% by weight, or from 1 to 5% by weight, based on the total weight of the detergent composition. In one embodiment, the detergent composition of the present invention comprises a total amount of citrate ranging from about 1 to 3% by weight, based on the total weight of the detergent composition.

[0314] The detergent compositions of the present invention may comprise one or more silicates. In the context of the present invention, "silicates" refers in particular to aluminosilicates (i.e., Na), such as sodium disilicate and sodium metasilicate, and sodium aluminosilicates such as Zeolite A. 12 (AlO2) 12 (SiO2) 12 *27H2O) and layered silicates, especially those of the formula alpha-Na2Si2O5, beta-Na2Si2O5 and delta-Na2Si2O5.

[0315] The detergent compositions of the present invention may contain one or more carbonate salts. The term "carbonate" includes alkali metal carbonates and alkali metal bicarbonates, with sodium salts being preferred. Sodium carbonate (Na2CO3) is particularly preferred.

[0316] The detergent compositions of the present invention may contain one or more phosphate salts. "Phosphonates" include 2-phosphonium butane-1,2,4-tricarboxylic acid (PBTC), ethylenediaminetetra(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(methylenephosphonate), 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, CH (28-x) N3Na x O 15 P5(x=7), hexamethylenediamine(tetramethylenephosphonate), potassium salt, C 10 H (28-x) N2K x O 12Salts thereof may also be suitable, including, but not limited to, P4 (x=6) and bis(hexamethylene)triamine(pentamethylenephosphonic acid), (HO2)POCH2N[(CH2)2N[CH2PO(OH)2]2]2.

[0317] The detergent composition of the present invention may contain one or more aminocarboxylates. Non-limiting examples of suitable "aminocarboxylates" include diethanolglycine (DEG), dimethylglycine (DMG), nitrilotriacetic acid (NTA), N-hydroxyethylaminodiacetic acid, ethylenediaminetetraacetic acid (EDTA), N-(2hydroxyethyl)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. Further preferred 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-sulfomethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfomethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), Alpha-alanine-N,N-diacetic acid (alpha-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), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA), as well as alkali metal or ammonium salts thereof, are preferred. MGDA or EDDS are preferred. The term "ammonium salt" in this context refers to a salt with at least one cation having a permanently or temporarily quaternized nitrogen atom. Examples of cations having at least one permanently quaternized nitrogen atom include tetramethylammonium, tetraethylammonium, dimethyldiethylammonium, and nC. 10 -C 20Examples of cations having at least one nitrogen atom that is temporarily quaternized include 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 + and ammonia.

[0318] In one embodiment, the detergent composition of the present invention comprises two or more builders. Preferably, the detergent composition of the present invention contains less than 0.2 wt. % of nitrilotriacetic acid (NTA) or 0.01 to 0.1 wt. % of NTA, based on the total weight of the detergent composition.

[0319] 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 their corresponding salts, such as alkali (e.g., sodium) salts, in an amount in the range of 0.1 wt % to 25.0 wt %, in the range of 1.0 wt % to 18.0 wt %, in the range of 3.0 wt % to 15.0 wt %, in the range of 3.0 wt % to 10.0 wt %, or in the range of 5.0 wt % to 8.0 wt %, based on the total weight of the detergent composition.

[0320] The detergent compositions of the present invention may contain one or more hydrotropes. The one or more hydrotropes may be selected from organic solvents known in the art, such as ethanol, isopropanol, ethylene glycol, 1,2-propylene glycol, and other organic solvents that are water-miscible under standard, unrestricted conditions. In one embodiment, the detergent compositions of the present invention contain 1,2-propylene glycol in a total amount ranging from 5 to 10 wt. %, preferably about 6 wt. %, based on the total weight of the detergent composition. Further non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0321] In one embodiment, the detergent composition includes at least one preservative. Preferably, the term "preservative" refers to a substance added to a liquid composition for preservation purposes, and more preferably, the term "preservative" excludes compounds known to have a preservative function that are present in the liquid composition formed during the manufacturing process. In one embodiment, the preservative is selected from the group consisting of 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol, formic acid in its acid form or as a salt thereof, and 4,4'-dichloro-2-hydroxydiphenyl ether. Typically, the liquid composition of the present invention includes at least one preservative in an amount of less than 10 ppm, such as an amount ranging from 2 ppm to 5 wt. % based on the total weight of the liquid composition. Alternatively, the detergent composition is preservative-free, meaning that the preservative is present in an amount of less than 1 ppm, preferably less than 0 ppm.

[0322] 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 a protease, a second amylase, a lipase, a cellulase, a mannanase, a hemicellulase, a phospholipase, an esterase, a pectinase, a lactase, a peroxidase, a xylanase, a cutinase, a pectate lyase, a keratinase, a reductase, an oxidase, a phenoloxidase, a lipoxygenase, a ligninase, a pullulanase, a tannase, a pentosanase, a malanase, a beta-glucanase, an arabinosidase, a hyaluronidase, a chondroitinase, a laccase, a nuclease, a DNase, a phosphodiesterase, a phytase. The second enzyme is selected from the group consisting of: α-galactosidase, β-galactosidase, α-glucosidase, β-gluc ...

[0323] Particularly preferred additional enzymes are disclosed elsewhere herein, the disclosures of which are also incorporated by reference into this section of the specification.

[0324] The compositions of the present invention may contain one type of enzyme or two or more enzymes of different types, for example, an amylase and a protease, or two or more enzymes of the same type, for example, two or more different proteases, or a mixture thereof, for example, an amylase and two different proteases.

[0325] The detergent composition may contain a water-soluble source of calcium and / or magnesium ions. In one embodiment, the detergent composition comprises an enzyme stabilizing system as described herein. Preferably, especially in the case of liquid detergent compositions, the detergent composition may comprise at least one protease inhibitor as described herein, preferably selected from a boronic acid derivative, preferably 4-FPBA, and a peptide aldehyde, preferably Z-VAL-H or Z-GAY-H. Preferably, the detergent composition does not contain boron.

[0326] In one embodiment, the present invention provides a method for providing a detergent composition, preferably a liquid detergent composition, more preferably a liquid laundry detergent composition, comprising in one or more steps: (a) at least one amylase variant according to the present invention (preferably the amylase is provided within an amylase variant formulation as described herein); (b) at least one detergent component selected from surfactants, builders, polymers, preservatives, and a second enzyme different from the amylase variant, preferably present in an amount effective in cleaning performance and / or maintaining the physical characteristics of the detergent; The present invention relates to a method comprising the step of mixing

[0327] In one embodiment, the present invention provides a) an amylase variant as described herein; b) one or more surfactants, preferably at a concentration of 0.2 to 65%, preferably 0.2 to 40%, c) one or more builders, preferably at a concentration of 0.01 to 25%, and d) optionally, one or more additional compounds selected from the group consisting of additional enzymes different from the amylases under a) above, antifoaming agents, polymers, bleaching systems (bleaches), rheology modifiers, hydrotropes, softeners, drying agents, brighteners, buffers, preservatives, anticorrosion additives, dyes, and fragrances. and preferably the detergent composition is a liquid, powder, pouch or capsule detergent composition.

[0328] Preferably, the detergent composition, preferably powder detergent composition, of the present invention comprises in addition to an amylase variant as described herein one or more compounds selected from the group consisting of alcohol ethoxylate 7EO, coco fatty acid C12-18, C12-C14-fatty alcohol ether sulfate (1 to 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.

[0329] Preferably, the detergent composition, preferably powder detergent composition, of the present invention comprises, in addition to the amylase variant as described herein: b) one or more surfactants selected from the group consisting of alcohol ethoxylate 7EO, coco 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 to 65%; c) one or more builders selected from the group consisting of HEDP, MGDA, GLDA and DTPMP, preferably at a concentration of 0.01 to 25%, and 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. Includes:

[0330] Preferably, the detergent compositions, preferably liquid detergent compositions, of the present invention comprise, in addition to an amylase variant as described herein, one or more compounds selected from the group consisting of alcohol ethoxylate 7EO, coco fatty acid C12-18, C12-C14-fatty alcohol ether sulfate (1-3EO, preferably 2EO), linear alkylbenzenesulfonic acid, sulfonic acid, 1,2 propanediol, triethanolamine, monoethanolamine, NaOH, glycerol, ethanol, sodium citrate and polymers.

[0331] Preferably, the detergent compositions, preferably liquid detergent compositions, of the present invention comprise, in addition to the amylase variants as described herein: b) one or more surfactants selected from the group consisting of alcohol ethoxylate 7EO, coco 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 to 65%; c) one or more builders selected from the group consisting of HEDP, MGDA, GLDA and DTPMP, preferably at a concentration of 0.01 to 25%, and d) one or more compounds selected from the group consisting of sulfonic acid, 1,2 propanediol, triethanolamine, monoethanolamine, NaOH, glycerol, ethanol, sodium citrate, and polymers. Includes:

[0332] 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): A formulation comprising 0.05% to 1.0% of an amylase variant described herein. 8% to 15% anionic cleansing surfactants (such as alkyl benzene sulfonates, alkyl ethoxylated sulfates, and mixtures), 0.5% to 4% of a non-ionic cleansing surfactant (such as alkyl ethoxylated alcohol), 0-4% cationic detergent surfactants (e.g., quaternary ammonium compounds), 0% to 4% of other cleansing surfactants (such as zwitterionic cleansing surfactants, amphoteric surfactants, and mixtures thereof), 1% to 4% carboxylic acid polymers (such as copolymers of maleic and acrylic acids), 0.5% to 4% polyethylene glycol polymers (e.g., polyethylene glycol polymers containing polyvinyl acetate side chains), 0.1-2% polyester soil release polymer (such as Repel-o-tex and / or Texcare polymers); 0.5% to 2% cellulose polymers (such as carboxymethylcellulose, methylcellulose, and combinations thereof); 0% to 4% of other polymers (amine polymers, dye transfer inhibitor polymers, hexamethylenediamine derivative polymers and their mixtures, etc.), 0% to 4% by weight of zeolite builder and phosphate builder (such as zeolite 4A and / or sodium tripolyphosphate); 0% to 3% of other builders (such as sodium citrate and / or citric acid), 15% to 30% carbonate (e.g., sodium carbonate and / or sodium bicarbonate), 0% to 10% silicates (e.g., sodium silicate), 10% to 40% fillers (such as sodium sulfate and / or biofillers), a source of 10%–20% available oxygen (e.g., sodium percarbonate); 2% to 8% bleach activator (such as tetraacetylethylenediamine (TAED) and / or nonanoyloxybenzenesulfonate (NOBS)), 0% to 0.1% bleach catalyst (such as an oxaziridinium-based bleach catalyst and / or a transition metal bleach catalyst); 0% to 10% of other bleaching agents (such as reducing bleaches and / or preformed peracids), 0.2% to 1% of a chelant (such as ethylenediamine-N'N'-disuccinic acid (EDDS) and / or hydroxyethanediphosphonic acid (HEDP)), 0% to 0.1% photobleaching agent (such as zinc and / or aluminum sulfonated phthalocyanine), 0% to 1% tinting agent (such as Direct Violet 99, Acid Red 52, Acid Blue 80, Direct Violet 9, Solvent Violet 13, and any combination thereof), 0.1% to 0.4% of a brightener (such as Brightener 15 and / or Brightener 49), 0% to 4% fabric softener (such as montmorillonite clay and / or polydimethylsiloxane (PDMS)); 0% to 1% flocculant (e.g., polyethylene oxide), 0% to 0.1% foam inhibitors (such as silicones and / or fatty acids), 0.1% to 1% fragrance (such as fragrance microcapsules, spray fragrance, starch-encapsulated fragrance harmonizer, fragrance-loaded zeolite, and any combination thereof); 0% to 1% aesthetic agents (such as colored soap rings and / or colored speckles / noodles), and optionally, about 0.05% to about 0.2% by weight of a protease (such as Savinase, Coronase, Ovozyme, Kannase, Liquanase, Polarzyme, Purafect, Purafast, Properase, Excellase, FN3, FN4, Effectenz P, Preferenz P, Progress Uno, Progress Excel, Blaze, or Excellenz P); Optionally, about 0.05% to about 0.2% by weight of an additional amylase different from the amylase variants described herein (such as Termamyl®, Termamyl Ultra®, Natalase®, Optisize HT Plus®, Purastar, Powerase®, Stainzyme®, Preferenz S, Effectenz S, Amplify, Amplify Prime, Achieve alpha, Excellentz S, and any combination thereof); optionally, 0.05% to 0.2% cellulase (such as Carezyme, Celluclean, Puradax, Biotouch, Whitezyme, Revitalenz, and combinations thereof); optionally, 0.05% to 0.2% lipase (such as Lipex, Lipolex, Lipoclean, Preferenz L, and any combination thereof); Optionally, 0.05% to 0.2% of other enzymes (such as xyloglucanase, cutinase, pectate lyase (such as Xpect), mannanase (such as Mannanway, Mannastar, Marvellenz, Effectenz M, Preferenz M, Preferenz F and combinations thereof), bleaching enzymes and combinations thereof).

[0333] 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): A formulation comprising 0.05% to 1.0% of an amylase variant described herein. about 0.5% by weight to about 1.5% by weight of a carboxyl group-containing polymer (comprising about 60% by weight to about 70% by weight of a monomer (A) based on acrylic acid and about 30% by weight to about 40% by weight of a sulfonic acid group-containing monomer (B), and having an average molecular weight in the range of about 23,000 to about 50,000, preferably about 25,000 to 38,000 as described in WO2014032269); about 8% to about 15% by weight of an anionic cleansing surfactant (such as alkyl benzene sulfonates, alkyl ethoxylated sulfates, and mixtures thereof); about 0.5% to 4% by weight of a non-ionic cleansing surfactant (e.g., alkyl ethoxylated alcohol); about 0% to about 4% by weight of a cationic cleansing surfactant (such as a quaternary ammonium compound); about 0% to 4% by weight of other cleansing surfactants (such as zwitterionic cleansing surfactants, amphoteric surfactants, and mixtures thereof); about 1% to about 4% by weight of a carboxylic acid polymer (such as a copolymer of maleic acid and acrylic acid); about 0% to about 4% by weight of a polyethylene glycol polymer (such as a polyethylene glycol polymer containing polyvinyl acetate side chains); about 0.1% to about 2% by weight of a polyester soil release polymer (such as Repel-O-Tex® and / or Texcare® polymers); about 0.5% to about 2% by weight of a cellulose polymer (such as carboxymethyl cellulose, methyl cellulose, and combinations thereof); about 0% to about 4% by weight of other polymers (such as amine polymers, dye transfer inhibitor polymers, hexamethylenediamine derivative polymers, and mixtures thereof); about 0% to about 4% by weight of a zeolite builder and a phosphate builder (such as Zeolite 4A and / or sodium tripolyphosphate); about 0% to about 3% by weight of other builders (such as sodium citrate and / or citric acid); about 15% by weight to about 30% by weight of a carbonate (e.g., sodium carbonate and / or sodium bicarbonate); about 0% to about 10% by weight of a silicate (such as sodium silicate); about 10% to about 40% by weight of a filler (such as sodium sulfate and / or a biofiller); about 10% to about 20% by weight of a source of available oxygen (e.g., sodium percarbonate); about 2% to about 8% by weight of a bleach activator (such as tetraacetylethylenediamine (TAED) and / or nonanoyloxybenzenesulfonate (NOBS)); about 0% to about 0.1% by weight of a bleach catalyst (such as an oxaziridinium-based bleach catalyst and / or a transition metal bleach catalyst); about 0% to about 10% by weight of other bleaching agents (such as reduced bleaches and / or preformed peracids); about 0.2% to about 1% by weight of a chelant (such as ethylenediamine-N'N'-disuccinic acid (EDDS) and / or hydroxyethanediphosphonic acid (HEDP)); about 0% to about 0.1% by weight of a photobleaching agent (such as zinc and / or aluminum sulfonated phthalocyanine); about 0% to about 0.5% by weight of a hueing agent (such as Direct Violet 99, Acid Red 52, Acid Blue 80, Direct Violet 9, Solvent Violet 13, and any combination thereof); about 0.1% to about 0.4% by weight of a brightener (such as Brightener 15 and / or Brightener 49); 0% to 15% by weight of a fabric softener (such as montmorillonite clay and / or polydimethylsiloxane (PDMS)); 0% to 1% by weight of a flocculant (e.g., polyethylene oxide); 0% to 0.1% by weight of a foam suppressor (such as a silicone and / or a fatty acid); 0.1% to 1% by weight of a fragrance (such as fragrance microcapsules, spray fragrance, starch-encapsulated fragrance harmonizer, fragrance-loaded zeolite, and any combination thereof); 0% to 1% by weight of aesthetic agents (such as colored soap rings and / or colored speckles / noodles); optionally, about 0.05% to about 0.2% by weight of a protease (such as Savinase, Coronase, Ovozyme, Kannase, Liquanase, Polarzyme, Purafect, Purafast, Properase, Excellase, FN3, FN4, Effectenz P, Preferenz P, Progress Uno, Progress Excel, Blaze, or Excellenz P); Optionally, about 0.05% to about 0.2% by weight of an additional amylase different from the amylase variants described herein (such as Termamyl®, Termamyl Ultra®, Natalase®, Optisize HT Plus®, Purastar, Powerase®, Stainzyme®, Preferenz S, Effectenz S, Amplify, Amplify Prime, Achieve alpha, or Excellentenz S); optionally, about 0.05% to 0.5% by weight of cellulase (typically, such as Carezyme®, Celluzyme®, Puradax, Celluclean®, Biotouch, Whitezyme, Revitalenz, and combinations thereof, having an enzymatic activity of about 10 to 50 mg active enzyme / g); optionally, about 0.2% to about 1% by weight of a lipase (typically, such as Lipex®, Lipolex®, Lipoclean®, Preferenz L, and any combination thereof, having an enzymatic activity of about 10 mg to about 50 mg of active enzyme / g); Optionally, 0% to 2% by weight of other enzymes (typically xyloglucanases (e.g., Whitezyme®), cutinases, pectate lyases (e.g., Xpect), mannanases (e.g., Mannanway, Mannastar, Marvellenz, Effectenz M, Preferenz M, Preferenz F, and combinations thereof), bleaching enzymes, etc., typically having an enzymatic activity of about 10 mg to about 50 mg of active enzyme / g.

[0334] Further preferred detergent compositions comprise the components listed below (all percentages are w / w): water, alcohol ethoxy sulfate, alcohol ethoxylate, amino oxide, citric acid, C12-18 palm kernel capped fatty acid, ethanol, 1,2 propanediol, sodium formate, calcium chloride, sodium hydroxide, silicone emulsion, trans-sulfated EHDQ, amylase variant as described herein, - sodium linear alkylbenzene sulfonate 8.8%, ethoxylated fatty alcohol C12-18 (7EO) 4.7%, sodium soap 3.2%, antifoam DC2-4248S 3.9%, sodium aluminum silicate zeolite 4A 28.3%, sodium carbonate 11.6%, sodium salt of copolymer derived from 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, 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, amylase variant as described herein, - 5-15% anionic surfactants, <5% nonionic surfactants, phosphonates, soaps, enzymes, optical brighteners, benzisothiazolinones, methylisothiazolinones, fragrances, alpha-isomethyl ionone, citronellol, geraniol, linalool, amylase variants as described herein, - Water, Sodium Dodecyl Benzene Sulfonate, C14-C15 Pareth-7, Sodium Citrate, Propylene Glycol, Sodium Palm Kernel Oil, Sodium Laureth Sulfate, MEA Dodecyl Benzene Sulfonate, Sulfated Ethoxylated Hexamethylenediamine Quaternized, Sodium Cumene Sulfonate, Fragrance, PEG / Vinyl Acetate Copolymer, Sodium Formate, Hydrogenated Castor Oil, Sodium Diethylenetriamine Pentamethylene Phosphonate, PEG / PPG-10 / 2 Propylheptyl Ether, Buthiophenyl Methylpropional, Polyvinylpyridine-N-Oxide, Sorbitol, Glycerin, Ethanolamine, Sodium Hydroxide, Alpha-Isomethylonone, Calcium Chloride, Geraniol, Linalool, Citronellol, Tripropylene Glycol, Benzisothiazolinone, Dimethicone, Sodium Acetate, Cellulase, Colorant, Glyceryl Stearate, Hydroxyethylcellulose, Silica, Amylase Variant as described herein, - Water, Sodium Laureth Sulfate, Propylene Glycol, C14-C15 Pareth-7, Sodium Citrate, Sodium Palm Kernel Fatty Acid, Alcohol, Sodium Formate, Sulfated Ethoxylated Hexamethylenediamine Quaternized, Sodium Hydroxide, Fragrance, Polyvinylpyridine-N-Oxide, Sorbitol, Calcium Chloride, Glycerin, Sodium Acetate, Colorant, Cellulase, Amylase Variant as described herein, - Water, Sodium Laureth Sulfate, Propylene Glycol, C14-C15 Pareth-7, Sodium Citrate, Sodium Palm Kernel Fatty Acid, Alcohol, Sodium Formate, Sulfated Ethoxylated Hexamethylenediamine Quaternized, Sodium Hydroxide, Fragrance, Sorbitol, Calcium Chloride, Glycerin, Sodium Acetate, Colorant, Cellulase, Amylase Variant as described herein, - Water, Sodium Laureth Sulfate, Propylene Glycol, C14-C15 Pareth-7, Sodium Citrate, Sodium Palm Kernel Fatty Acid, Alcohol, Sodium Formate, Sulfated Ethoxylated Hexamethylenediamine Quaternized, Sodium Hydroxide, Sorbitol, Calcium Chloride, Glycerin, Sodium Acetate, Cellulase, Silica, Amylase Variant as described herein, - Water, Sodium Dodecyl Benzene Sulfonate, C14-C15 Pareth-7, Sodium Citrate, Propylene Glycol, Sodium Palm Kernel Oil, Sodium Laureth Sulfate, MEA Dodecyl Benzene Sulfonate, Sulfated Ethoxylated Hexamethylenediamine Quaternized, Sodium Cumene Sulfonate, Fragrance, PEG / Vinyl Acetate Copolymer, Sodium Formate, C12-C14 Pareth-7, hydrogenated castor oil, sodium diethylenetriamine pentamethylenephosphonate, PEG / PPG-10 / 2 propylheptyl ether, buthiophenyl methylpropional, fluorescent brightener, sorbitol, glycerin, ethanolamine, sodium hydroxide, alpha-isomethylonone, calcium chloride, geraniol, linalool, citronellol, tripropylene glycol, sodium chloride, benzisothiazolinone, dimethicone, sodium acetate, cellulase, colorant, glyceryl stearate, hydroxyethylcellulose, silica, amylase variant as described herein, - 15-30% anionic surfactants, non-ionic surfactants, 5-15% soap, <5% polycarboxylates, fragrances, phosphates, optical brighteners, amylase variants as described herein, - 15-30% anionic surfactant, 5-15% nonionic surfactant, soap, benzisothiazolinone, methylisothiazolinone, fragrance, amylase variant as described herein, 11% LAS, 2% AS / AEOS, 2% soap, 3% AEO, 15.15% sodium carbonate, 3% sodium silicate, 18.75% zeolite, 0.15% chelant, 2% sodium citrate, 1.65% AA / MA copolymer, 2.5% CMC and 0.5% SRP, an amylase variant as described herein, 16.5% LAS, 15% zeolite, 12% sodium disilicate, 20% sodium carbonate, 1% socalan, 35.5% sodium sulfate, amylase variant as described herein, - 15-30% anionic surfactants, <5% nonionic surfactants, phosphonates, polycarboxylates, zeolites, enzymes, fragrances, hexyl cinnamal, amylase variants as described herein, - 15-30% of the following: anionic surfactants, oxygen-based bleaches and zeolites, less than 5% of the following: nonionic surfactants, phosphonates, polycarboxylates, soaps, further ingredients: fragrances, hexyl cinnamal, benzyl salicylate, linalool, optical brighteners, enzymes and citronellol, amylase variants as described herein, 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, dipropylethyltetramine, sodium hydroxide, sodium formate, calcium formate, dimethicone, Liquitint™, hydrogenated castor oil, fragrance, amylase variant as described herein, linear alkylbenzene sulfonate, propylene glycol, citric acid, sodium hydroxide, ethanolamine, ethanol, alcohol sulfate, polyethyleneimine ethoxylate, sodium fatty acids, diquaternium ethoxysulfate, diethylene glycol, laureth-9, alkyldimethylamine oxide, fragrance, disodium diaminostilbene disulfonate, DTPA, sodium formate, calcium formate, polyethylene glycol 4000, mannanase, Liquitint™ Blue, dimethicone, amylase variant as described herein, - water, sodium alcohol ethoxy sulfate, propylene glycol, ethanol, sodium linear alkylbenzene sulfonate, 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, disulfonate, dimethicone, benzisothiazolinone, amylase variant as described herein, - 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 distyrylbiphenyl, disulfonate, calcium formate, polyethylene glycol 4000, mannanase, pectinase, Liquitint™ Blue, dimethicone, amylase variant as described herein, water, alcohol ethoxy sulfate, propylene glycol, sodium fatty acids, lauryltrimonium chloride, ethanol, sodium hydroxide, sodium cumene sulfonate, citric acid, ethanolamine, diethylene glycol, silicone polyether, fragrance, polyethylene-imine ethoxylate, amylase, laureth-9, DTPA, polyacrylamide quaternium chloride, disodium diaminostilbene disulfonate, sodium formate, Liquitint™ Orange, dipropylethyltetraamine, dimethicone, cellulase, amylase variant as described herein, water, sodium alcohol ethoxy sulfate, sodium alkyl sulfate, MEA citrate, linear alkylbenzene sulfonate, MEA salt, propylene glycol, diethylene glycol, polyethyleneimine ethoxylate, ethanol, sodium fatty acids, ethanolamine, lauramine oxide, laureth-9, DTPA, sodium cumene sulfonate, sodium formate, calcium formate, linear alkylbenzene sulfonate, sodium salt, alcohol sulfate, sodium hydroxide, diquaternium ethoxy sulfate, fragrance, amylase, mannanase, pectinase, disodium diaminostilbene disulfonate, benzisothiazolinone, Liquitint™ Blue, dimethicone, dipropylethyltetraamine, amylase variant as described herein, - water, sodium alcohol ethoxy sulfate, MEA citrate, sodium alkyl sulfate, alcohol ethoxylate, linear alkylbenzene sulfonate, MEA salt, sodium fatty acid, polyethyleneimine ethoxylate, diethylene glycol, propylene glycol, diquaternium ethoxy sulfate, polyethyleneimine, ethoxylate propoxylate, ethanol, sodium cumene sulfonate, fragrance, DTPA, disodium diaminostilbene disulfonate, mannanase, cellulase, sodium formate, calcium formate, lauramine oxide, Liquitint™ Blue, dimethicone / polydimethylsilicone, amylase variant as described herein, water, alcohol ethoxysulfate, linear alkylbenzene sulfonate, alcohol ethoxylate, citric acid, ethanolamine, sodium fatty acids, diethylene glycol, propylene glycol, sodium hydroxide, polyethyleneimine ethoxylate, silicone polyether, ethanol, amylase, sodium cumene sulfate, diquaternium ethoxysulfate, laureth-9, fragrance, DTPA, disodium diaminostilbene disulfonate, disodium distyrylbiphenyl disulfonate, sodium formate, calcium formate, mannanase, Liquitint™ orange, dimethicone, polyacrylamide quaternium chloride, cellulase, dipropylethyltetraamine, amylase variant as described herein, - Water, alcohol ethoxy sulfate, diethylene glycol, monoethanolamine citrate, Sodium formate, propylene glycol, linear alkylbenzene sulfonate, ethanolamine, ethanol, polyethyleneimine ethoxylate, benzisothiazoline, calcium formate, citric acid, sodium diethylenetriaminepentaacetate, dimethicone, diquaternium ethoxysulfate, disodium diaminostilbene disulfonate, laureth-9, mannanase, sodium cumene sulfonate, sodium fatty acids, amylase variant as described herein, - water, alcohol ethoxy sulfate, MEA citrate, alcohol sulfate, alcohol ethoxylate, linear alkylbenzene sulfonate MEA, sodium fatty acids, polyethyleneimine ethoxylate, diethylene glycol, propylene glycol, diquaternium ethoxy sulfate, polyethyleneimine ethoxylate propoxylate, ethanol, sodium cumene sulfonate, fragrance, DTPA, disodium diaminostilbenedisulfonate, mannanase, cellulase, sodium formate, calcium formate, lauramine oxide, Liquitint™ Blue, dimethicone, amylase variant as described herein, water, sodium alcohol ethoxy sulfate, MEA citrate, linear alkylbenzene sulfonate: sodium salt, alcohol ethoxylate, linear alkylbenzene sulfonate: MEA salt, sodium fatty acid, polyethyleneimine ethoxylate, diethylene glycol, propylene glycol, diquaternium ethoxy sulfate, polyethyleneimine ethoxylate propoxylate, ethanol, sodium cumene sulfonate, citric acid, DTPA, disodium diaminostilbenedisulfonate, sodium formate, calcium formate, dimethicone, amylase variant as described herein, - water, alcohol ethoxylate sulfate, linear alkylbenzene sulfonate sodium / Mea salt, propylene glycol, diethylene glycol, sodium formate, ethanol, sodium fatty acids, fragrance, lauramine oxide, DTPA, polyethyleneamine ethoxylate, calcium formate, disodium diaminostilbene disulfonate, dimethicone, tetramine, Liquitint™ Blue, amylase variant as described herein, 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 diethylenetriaminepentaacetate, disodium distyrylbiphenyldisulfonate, calcium formate, mannanase, exiloglucanase, sodium formate, hydrogenated castor oil, dyes, subtilisin, benzisothiazoline, fragrance, amylase variant as described herein, deionized water, dipropylene glycol butyl ether, sodium alkyl sulfate, hydrogen peroxide, ethanol, magnesium sulfate, alkyldimethylamine oxide, citric acid, sodium hydroxide, trimethoxybenzoic acid, fragrance, amylase variant as described herein, water, alkyl ethoxylate, linear alkyl benzene sulfonate, hydrogen peroxide, diquaternium ethoxysulfate, ethanolamine, disodium distyryl biphenyl disulfonate, tetrabutyl ethylidine bisphenol, F&DC Yellow 3, fragrance, amylase variant as described herein, sodium percarbonate, sodium sulfate, sodium carbonate, sodium aluminosilicate, nonanoyloxybenzenesulfonate, sodium polyacrylate, water, sodium alkylbenzenesulfonate, DTPA, polyethylene glycol, sodium palmitate, amylase, modified starch, FD&C Blue 1, fragrance, amylase variant as described herein, - water, alkyl ethoxylate, MEA borate, linear alkyl benzene sulfonate, propylene glycol, diquaternium ethoxysulfate, calcium chloride enzyme, ethanolamine, benzisothiazolinone, sodium citrate, sodium hydroxide, fragrance, amylase variant as described herein, water, alkylamine oxide, dipropylene glycol phenyl ether, hydrogen peroxide, citric acid, ethylenediamine, disuccinic acid sodium salt, sodium alkyl sulfate, fragrance, amylase variant as described herein, sodium bicarbonate, sodium carbonate, sodium percarbonate, alcohol ethoxylate, sodium chloride, maleic acid / acrylic acid copolymer, nonanoyloxybenzenesulfonate, sodium sulfate, colorants, diethylenetriaminepentaacetic acid sodium salt, hydrated aluminosilicate (zeolite), polyethylene glycol, sodium alkylbenzenesulfonate, sodium palmitate, starch, water, fragrance, amylase variant as described herein, - Polyvinyl alcohol pouch film (with compressed liquid and powder portions): Liquid component: dipropylene glycol, diquaternium ethoxysulfate, water, glycerin, Liquitint® Orange, amylase variant as described herein, - Active Ingredients: Sodium percarbonate, nonanoyloxybenzenesulfonate, sodium carbonate, sodium sulfate, sodium aluminosilicate, sodium polyacrylate, sodium alkylbenzenesulfonate, maleic / acrylic acid copolymer, water, polyethylene glycol, sodium palmitate, modified starch, glycerin, DTPA, fragrance, amylase variant as described herein, - water, sodium alcohol ethoxy sulfate, linear alkylbenzene sulfonate, sodium / MEA salt, MEA citrate, propylene glycol, polyethyleneimine, ethoxylate, ethanol, diethylene glycol, polyethyleneimine propoxyethoxylate, sodium fatty acids, sodium cumene sulfonate, DTPA, fragrance, disodium diaminostilbene disulfonate, calcium formate, sodium formate, gluconase, dimethicone, Liquitint™ Blue, mannanase, amylase variant as described herein, sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzenesulfonate, bentonite, water, sodium percarbonate, sodium polyacrylate, silicates, alkyl sulfates, nonanoyloxybenzenesulfonate, DTPA, polyethylene glycol 4000, silicones, ethoxylates, fragrance, polyethylene oxide, palmitic acid, disodium diaminostilbene disulfonate, Liquitint™ Red, FD&C Blue 1, cellulase, amylase variant as described herein, water, sodium alcohol ethoxy sulfate, MEA citrate, linear alkylbenzene sulfonate: sodium / MEA salt, propylene glycol, polyethyleneimine ethoxylate, ethanol, diethylene glycol, polyethyleneimine, propoxyethoxylate, diquaternium ethoxy sulfate, alcohol sulfate, dimethicone, fragrance, sodium fatty acids, DTPA, sodium bisulfite, disodium diaminostilbene disulfonate, glucanase, castor oil, calcium formate, MEA, styrene acrylic acid copolymer, sodium formate, Liquitint™ Blue, amylase variant as described herein, - water, sodium alcohol ethoxy sulfate, MEA citrate, linear alkylbenzene sulfonate: sodium / MEA salt, propylene glycol, ethanol, diethylene glycol, polyethyleneimine propoxyethoxylate, polyethyleneimine ethoxylate, alcohol sulfate, dimethicone, fragrance, sodium fatty acids, DTPA, sodium bisulfite, disodium diaminostilbene disulfonate, castor oil, calcium formate, MEA, styrene acrylic acid copolymer, propanaminium propanamide, gluconase, sodium formate, Liquitint™ Blue, amylase variant as described herein, water, sodium alcohol ethoxy sulfate, MEA citrate, linear alkylbenzene sulfonate: sodium / MEA salt, propylene glycol, polyethyleneimine ethoxylate, ethanol, diethylene glycol, polyethyleneimine propoxyethoxylate, diquaternium ethoxy sulfate, alcohol sulfate, dimethicone, fragrance, sodium fatty acids, DTPA, sodium bisulfite, disodium diaminostilbene disulfonate, glucanase, castor oil, calcium formate, MEA, styrene acrylic acid copolymer, propanaminium propanamide, sodium formate, Liquitint™ Blue, amylase variant as described herein, sodium carbonate, sodium aluminosilicate, alcohol sulfate, sodium sulfate, linear alkylbenzene sulfonate, water, sodium polyacrylate, silicates, ethoxylates, sodium percarbonate, polyethylene glycol 4000, disodium diaminostilbene disulfonate, silicones, cellulase, amylase variants as described herein, sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzenesulfonate, alkyl sulfate, sodium percarbonate, water, sodium polyacrylate, silicates, nonanoyloxybenzenesulfonate, ethoxylates, polyethylene glycol 4000, fragrance, DTPA, disodium diaminostilbene disulfonate, palmitic acid, silicone, cellulase, amylase variant as described herein, sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzenesulfonate, water, nonanoyloxybenzenesulfonate, alkyl sulfate, sodium polyacrylate, silicates, sodium percarbonate, ethoxylates, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, disodium diaminostilbene disulfonate, silicone, cellulase, amylase variant as described herein, sodium carbonate, sodium aluminosilicate, sodium sulfate, sodium percarbonate, alkyl sulfate, linear alkylbenzene sulfonate, 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 sulfate, amylase variant as described herein, sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzenesulfonate, sodium percarbonate, nonanoyloxybenzenesulfonate, alkyl sulfate, water, silicone, 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, sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzenesulfonate, sodium percarbonate, alkyl sulfate, water, sodium polyacrylate, silicate, nonanoyloxybenzenesulfonate, ethoxylate, polyethylene glycol 4000, DTPA, fragrance, cellulase, disodium diaminostilbene disulfonate, silicone, FD&C Blue 1, amylase variant as described herein, - water, sodium alcohol ethoxy sulfate, MEA citrate, linear alkylbenzene sulfonate, sodium salt, linear alkylbenzene sulfonate:MEA salt, alcohol ethoxylate, sodium fatty acid, propylene glycol, diethylene glycol, polyethyleneimine ethoxylate propoxylate, diquaternium ethoxy sulfate, ethanol, sodium cumene sulfonate, fragrance, DTPA, sodium bisulfate, disodium diaminostilbene disulfonate, mannanase, cellulase, sodium formate, calcium formate, lauramine oxide, Liquitint™ Blue, dimethicone / polydimethylsilicone, amylase variant as described herein, - water, sodium alcohol ethoxy sulfate, linear alkylbenzene sulfonate: sodium / MEA salt, MEA citrate, propylene glycol, polyethyleneimine ethoxylate, fragrance, ethanol, diethylene glycol, polyethyleneimine propoxyethoxylate, alcohol sulfate, sodium fatty acids, DTPA, disodium diaminostilbene disulfonate, MEA, mannanase, gluconase, sodium formate, dimethicone, Liquitint™ Blue, tetramine, amylase variant as described herein, - water, sodium alcohol ethoxy sulfate, MEA citrate, linear alkylbenzene sulfonate, sodium salt, linear alkylbenzene sulfonate:MEA salt, alcohol ethoxylate, sodium fatty acid, propylene glycol, diethylene glycol, polyethyleneimine ethoxylate propoxylate, diquaternium ethoxy sulfate, ethanol, sodium cumene sulfonate, fragrance, DTPA, sodium bisulfate, disodium diaminostilbene disulfonate, mannanase, cellulase, sodium formate, calcium formate, lauramine oxide, Liquitint™ Blue, dimethicone / polydimethylsilicone, amylase variant as described herein, sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzenesulfonate, sodium percarbonate, nonanoyloxybenzenesulfonate, alkyl sulfate, water, silicone, 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 -Water, dodecylbenzenesulfonate, laureth-11, peg-75 lanolin, propylene glycol, alcohol denat., potassium soyate, potassium hydroxide, disodium cocoamphodiacetate, ethylenediamine triacetate, cocosalklylacetamide, fragrance, zinc ricinoleate, sodium chloride, benzisothiazolinone, methylisothiazolinone, ci 16255, benzyl alcohol, amylase variant as described herein.

[0335] Preferably, the above-listed compositions containing a protease further contain 4-FPBA and / or a peptide aldehyde protease inhibitor, most preferably Z-GAY or Z-VAL.

[0336] The amylase variants described herein may be included in one of the following detergent compositions:

[0337] [Table 5]

[0338] [Table 6]

[0339] [Table 7]

[0340] [Table 8]

[0341] [Table 9]

[0342] [Table 10]

[0343] [Table 11]

[0344] How to use The present invention also relates to the use of the amylase variants as described herein in cleaning processes such as laundry or hard surface cleaning, preferably for home care or I&I cleaning, including manual and automatic dishwashing and medical device cleaning. Accordingly, the present invention also refers to the use of amylase variants as described herein to provide detergent compositions with improved enzyme stability, preferably amylase stability, and / or to provide detergent compositions with improved cleaning performance, preferably on amylase-sensitive stains.

[0345] Thus, the present invention also refers to a method of 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 ingredient, preferably a surfactant and / or builder.

[0346] Furthermore, the present invention also refers to a method for improving amylase stability in a detergent composition and / or improving the cleaning performance of a detergent composition, preferably on amylase-sensitive stains, comprising the step of incorporating an amylase variant as described herein into the detergent composition.

[0347] Preferred Embodiments Particularly preferred herein are the following: 1. An amylase variant of a parent amylase, (i) amino acid substitutions at two or more, three or more, four or more, five or more, six or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 4, 25, 176, 186, 251, 405, and 482 according to the numbering of SEQ ID NO: 3; and (ii) any of SEQ ID NOs: 1, 3, 4, or 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, preferably at least 91% but less than 100% sequence identity to SEQ ID NO: 1 (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) amylase variants, including

[0348] 2. (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 3; (ii) amino acid substitutions at one or more, two or more, three or more, four or more, five or more, or all of the amino acid positions corresponding to amino acid positions selected from the group consisting of 25, 176, 186, 251, 405, and 482 according to the numbering of SEQ ID NO: 3; and (iii) any of SEQ ID NOs: 1, 3, 4, or 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, preferably at least 91% but less than 100% sequence identity to SEQ ID NO: 1 (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) 2. The amylase variant according to embodiment 1, comprising:

[0349] 3. The amylase variant according to any of the preceding embodiments, comprising an amino acid substitution at position 25. 4. An amylase variant according to any of the preceding embodiments, wherein said amino acid substitution at position 4 is X4Q or an amino acid substitution similar to X4Q.

[0350] 5. The amylase variant according to any of the preceding embodiments, wherein the amino acid substitution at position 25 is X25H, the amino acid substitution at position 176 is X176K, the amino acid substitution at position 186 is X186E, the amino acid substitution at position 251 is X251E, the amino acid substitution at position 405 is X405M, and the amino acid substitution at position 482 is X482W, or an amino acid substitution similar to these substitutions.

[0351] 6. The amylase variant according to any of the preceding embodiments, wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1.

[0352] 7. In one embodiment, the amylase variant of the present invention (i) the amino acid substitution X4G according to the numbering of SEQ ID NO: 3; (ii) one or more, two or more, three or more, four or more, five or more, or all of the amino acid substitutions selected from the group consisting of X25H, X176K, X186E, X251E, X405M, and X482W according to the numbering of SEQ ID NO: 3; and (iii) any of SEQ ID NOs: 1, 3, 4, or 15 to 41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 60%, preferably at least 91% but less than 100% sequence identity to SEQ ID NO: 1 Includes:

[0353] 8. An amylase variant according to any of the preceding embodiments, having at least 80%, preferably 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% sequence identity to SEQ ID NO:1, but less than 100%.

[0354] 9.a) substitutions at one or more positions selected from 25, 176 and 186, preferably one or more substitutions selected from X25H, H176K and X186E, or b) a substitution at one or more positions selected from 251, 405 and 482, preferably one or more substitutions selected from X251E, X405M and X482W, or c) substitutions at one or more positions selected from 25, 176 and 186, preferably one or more substitutions selected from X25H, X176K and X186E, and substitutions at one or more positions selected from 251, 405 and 482, preferably one or more substitutions selected from X251E, X405M and X482W 2. The amylase variant according to any of the preceding embodiments, comprising:

[0355] 10. The amylase variant according to any of the preceding embodiments, comprising two or all of the amino acid substitutions selected from the group consisting of: X25H, X176K and X186E.

[0356] 11.X4Q+X176K+X186E, X4Q+X25H+X176K+X186E, X4Q+X25H+X176K+X186E+X251E, X4Q+X25H+X176K+X186E+X405M, X4Q+X25H+X176K+X186E+X482W, X4Q+X25H+X176K+X186E+X251E+X405M, X4Q+X25H+X176K+X186E+X251E+X482W, X4Q+X25H+X176K+X186E+X405M+X482W, and X4Q+X25H+X176K+X186E+X251E+X405M+X482W

[0023] The amylase variant according to any of the preceding embodiments, comprising a combination of amino acid substitutions selected from the group consisting of:

[0357] 12. The amylase variant according to any of the preceding embodiments, comprising the amino acid substitutions X4Q+X25H+X176K+X186E+X251E+X405M+X482W (according to the numbering of SEQ ID NO: 3).

[0358] 13. The amylase variant according to 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 of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably D183* and G184*, wherein the numbering is according to SEQ ID NO: 3.

[0359] 14. An amylase variant according to any of the preceding embodiments, having 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 of SEQ ID NOs: 1, 3 or 4, preferably SEQ ID NO: 1.

[0360] 15. An amylase variant according to any of the preceding embodiments, comprising 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 wherein 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.

[0361] 16. An amylase variant according to any of the preceding embodiments, comprising or consisting of the amino acid sequence set forth in any of SEQ ID NOs: 1, 3, 4 or any of SEQ ID NOs: 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1, with 1 to 6, preferably 2 to 6 or 3 to 6, for example 1, 2, 3, 4, 5 or 6, amino acid substitutions selected from the group consisting of the amino acid substitutions X4Q and X25H, X176K, X186E, X251E, X405M and X482W and preferably 1 to 10, preferably 1 to 5 further 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.

[0362] 17. The amylase variant according to any of the preceding embodiments, further comprising 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 involve the catalytic center of the amylase variant.

[0363] 18. An amylase variant according to any of the previous embodiments, comprising or consisting of an amino acid sequence as set forth in any of SEQ ID NO: 1, with the modifications X4Q+X25H+X176K+X186E+X251E+X405M+X482W according to the numbering of SEQ ID NO: 3 and optionally 1 to 10, preferably 1 to 5, further conservative amino acid substitutions.

[0364] 19. The amylase variant according to any of the previous embodiments, wherein the substitutions are selected from G4Q, N25H, R176K, G186E, T251E, L405M and Y482W (according to the numbering of SEQ ID NO: 3).

[0365] 20. The amylase variant preferably exhibits one or more improved properties relative to said parent amylase, preferably relative to SEQ ID NO: 1 or SEQ ID NO: 3, preferably relative to the parent amylase set forth in SEQ ID NO: 1 or the amylase set forth in SEQ ID NO: 33, preferably the amylase variant has increased stability, thermostability, storage stability, storage stability in a detergent composition, cleaning performance, cleaning performance in a laundry detergent and / or cleaning performance in a dishwashing detergent, preferably the improved property is improved storage stability and / or improved cleaning performance, preferably improved cleaning performance on laundry, most preferably the improved property is improved storage stability, preferably said improved property is expressed as an improvement factor (IF) > 1.0, preferably the improvement factor is 1.1 or greater, preferably 1.2 or greater, more preferably 1.3 or greater, preferably the amylase variant exhibits one or more improved properties 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 set forth in SEQ ID NO: 1 or SEQ ID NO: 3 3, and preferably has an improvement of at least 1.1, preferably at least 1.2, more preferably at least 1.3 after storage in a detergent composition as described herein for 14, 28, 42 or 56 days, preferably for 56 days, or at 40°C for 14, 24, 56 or 84 days, preferably for 84 days, as preferably determined in ES1-C detergent as described herein containing an additional builder (preferably HEDP); and / or preferably the amylase variant shows improved cleaning performance on laundry, preferably at 40°C, relative to an amylase having an amino acid sequence as set forth in SEQ ID NO: 33.

[0366] 21. A polynucleotide encoding an amylase variant according to any of the preceding embodiments. 22. A formulation comprising an amylase variant according to any one of embodiments 1 to 20 and at least one additional component, preferably a solvent.

[0367] 23. A formulation according to embodiment 22, wherein the composition comprises one or more second enzymes different from the amylase variants referred to in any of the preceding claims, preferably 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.

[0368] 24. A formulation according to embodiment 22 or 23, comprising an enzyme stabilization system, preferably comprising a calcium salt, and optionally a protease inhibitor if no protease is present. 25. A detergent composition comprising an amylase variant according to any one of embodiments 1 to 20 or a formulation according to any of embodiments 22 to 24, preferably a laundry detergent composition or a hard surface cleaning detergent composition, most preferably a laundry detergent composition.

[0369] 26. A detergent composition according to embodiment 25, comprising one or more surfactants and / or one or more builders, preferably one or more strong metal ion-scavenging builders. 27. The detergent composition according to any of embodiments 25 or 26, comprising a component selected from the group consisting of an enzyme stabilization system, a surfactant, an antifoaming agent, a builder, a polymer, a bleaching system (bleach), a rheology modifier, a hydrotrope, a softener, a drying agent, a brightener, a buffer, a suds control agent, a preservative, an anticorrosion additive, a dye, and a fragrance.

[0370] 28. The detergent composition according to any of embodiments 25 to 27, comprising a builder, wherein the builder is selected from MDGA, GLDA, DTPMP, HEDP and EDDS, preferably MDGA or EDDS. 29. The detergent composition according to any of embodiment 28, wherein the builder is a non-phosphate builder.

[0371] 30. The detergent composition according to any of embodiments 25-29, comprising a surfactant, wherein the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, and combinations thereof. 31. The detergent composition according to any of embodiments 25-30, which is devoid of anionic surfactants.

[0372] 32. The detergent composition according to any of embodiments 25-31, wherein the surfactant and / or builder is biodegradable and / or bio-based. 33. The detergent composition according to any of embodiments 25-32, which is a liquid or solid. 34. The detergent composition according to any of embodiments 25-33, in the form of a pouch.

[0373] 35. The detergent composition according to any of embodiments 25-34, which is a liquid laundry detergent composition. 36. The detergent composition according to any of embodiments 25-35, which does not contain boron.

[0374] 37. The detergent composition according to any of embodiments 25-36, which does not contain a preservative. 38. A method for cleaning, comprising the step of contacting a textile or a hard surface, preferably a textile, with an amylase variant according to any of embodiments 1 to 20 or a formulation according to any of embodiments 22 to 24 or a detergent composition according to any of embodiments 25 to 37, preferably at low temperature, preferably 30°C or 40°C, preferably 30°C.

[0375] 39. Use of an amylase variant according to any of embodiments 1 to 20, or a formulation according to any of embodiments 22 to 24, or a detergent composition according to any of embodiments 25 to 37, for improving amylase stability in a detergent composition, preferably for an amylase as set forth in SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 1 or an amylase as set forth in SEQ ID NO: 33, preferably on amylase-sensitive stains, preferably at 30°C or 40°C, preferably 40°C, as preferably determined in an ES1-C detergent as described herein, preferably containing an additional builder (preferably HEDP). [Example]

[0376] Common methods: Amylase expression The amylase-encoding gene was cloned into a Gram-positive expression vector containing a promoter sequence, a sequence encoding a secretory signal peptide, and a ribosome binding site using a standard restriction ligation protocol. Following the reaction, the plasmid construction mixture was transformed into B. subtilis PY79 using an established natural competent transformation method. Successful transformants were selected by plating on LB agar plates supplemented with 20 μg / ml kanamycin sulfate and incubating overnight at 37°C. After overnight selection, individual colonies were obtained and subsequently grown in rich medium (e.g., LB broth) with 20 μg / ml kanamycin sulfate overnight at 37°C with shaking at 250 rpm. The following morning, cells were pelleted by centrifugation, and plasmid DNA was isolated by alkaline lysis using a Macherey-Nagel NucleoSpin kit. The isolated DNA could then be transformed into electrocompetent Bacillus licheniformis cells.

[0377] B. licheniformis cells were made electrocompetent using standard Gram-positive electrocompetent cell preparation techniques. As an example, cells were prepared by growing a B. licheniformis strain in an osmolyte-rich, rich medium (e.g., LB broth with 0.5 M D-sorbitol) and harvesting the cells during early exponential growth phase. The cells were harvested by chilling on ice and pelleting by centrifugation. After harvest, the cells were washed to remove salts using an osmolyte-rich wash buffer (e.g., 10% glycerol with 0.5 M D-sorbitol and 0.5 M D-mannitol) with three cycles of suspension and centrifugation pelleting. Finally, the cells were concentrated by resuspending in wash buffer at 1–10% of the original culture volume.

[0378] Once prepared, the plasmid DNA was added to electrocompetent B. licheniformis cells in a 0.2 cm Bio-Rad electroporation cuvette. Cells were electroporated using a Bio-Rad Gene Pulser Xcell according to the manufacturer's instructions. Cells were immediately rescued after shock by the addition of 1 ml of osmolyte-rich medium. After 2 hours of recovery at 37°C, successful transformants were selected by plating on LB agar plates supplemented with 20 μg / ml kanamycin sulfate and incubating overnight at 37°C.

[0379] Amylase expression and protein quantification Single colonies of the expression strains were picked into 60 μL of rich medium (e.g., LB broth) supplemented with 20 μg / mL kanamycin sulfate in a 96-well plate (GE Life Sciences part 28403943). The cultures were grown at 37°C for 16 hours, after which 15 μL of the culture was used to inoculate 600 μL of a defined glucose-mineral medium and 20 μg / mL kanamycin sulfate in a 96-well plate. The cultures were grown at 37°C for 48 hours, after which the supernatant was collected by pelleting the cells by centrifugation and removing the remaining culture medium. Larger quantities were made by combining supernatants from the same clone. The expression level of alpha-amylase activity (concentration in mg / mL) was determined by LabChip (LC) analysis to allow for comparable dosage in laundry detergents.

[0380] Amylase activity measurement Alpha-amylase activity can be determined by a method utilizing ethylidene-4-nitrophenyl-alpha-D-maltoheptaoside (EPS) as a substrate. D-Maltoheptaoside is a block oligosaccharide that can be cleaved by endo-amylase. After cleavage, the alpha-glucosidase included in the kit digests the substrate, liberating free PNP molecules with a yellow color, which can be measured by visible spectrophotometry at 405 nm. Kits containing the EPS substrate and alpha-glucosidase are 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 per mg of enzyme) of the target alpha-amylase under a given set of conditions. Residual activity was calculated by dividing the activity after storage time by the activity of the sample at time zero. The residual activity was compared to the residual activity of a reference given in the examples below.

[0381] Example 1: Storage stability in Formil detergent at 37°C for 56 days. To further assess whether the improved thermal stability also translates into increased stability in detergents, the amylases were formulated into a commercial liquid detergent (Formil) and tested for their residual activity after 56 days of storage at 37°C.

[0382] Storage in liquid laundry detergent was achieved by incubating amylase in Formil liquid detergent (Lidl, Germany). To inactivate all endogenous enzymes in the detergent, the detergent was incubated at 85°C for 2-3 hours. Inactivation was demonstrated when amylase activity was undetectable.

[0383] [Table 12]

[0384] Both amylase samples were then diluted with 50 mM HEPES pH 8.0 to obtain equivalent active ingredient levels. The detergent was dosed to a final amylase concentration of 0.12 mg / g and further supplemented with protease (0.5% protease formulation containing protease having SEQ ID NO: 10 with the R101E mutation (according to the numbering of SEQ ID NO: 11)). The mixtures were incubated at 37°C, and after the designated time points, amylase activity was measured using the amylase detection method described above.

[0385] [Table 13]

[0386] As can be derived from the table above, the amylase variants of the present invention are more stable than the reference amylase.

[0387] Example 2: Storage stability in builter detergents up to 84 days at 40°C.

[0388] [Table 14]

[0389] Amylases were formulated into a liquid model detergent (ES1-C) and tested for their residual activity after 84 days of storage at 40°C. The final dosage was 0.1 g / L amylase. To assess the stability of amylase in the presence of strong chelators, HEDP and / or CaCl2 were supplemented as indicated in the table. Additional Ca 2+ was not replenished by the enzyme.

[0390] [Table 15]

[0391] After the designated time points, samples were taken and the residual activity was measured using EPS as a substrate as shown above. The residual activity was calculated by dividing the activity after the storage time by the activity of the sample at time zero. The residual activity was compared to the residual activity of a reference amylase (SEQ ID NO: 33).

[0392] [Table 16]

[0393] As can be derived from the table above, the amylase variants of the present invention are more stable than the reference amylase.

[0394] Example 3: Improved cleaning performance of amylases Determination of washing performance was carried out using a Launder-o-meter. Several soiled swatches were washed in a liquid laundry formulation at 40°C with a cotton ballast fabric and 20 steel balls. After washing, the fabrics were rinsed, spun, and air-dried. Washing performance was determined by measuring the CIELab values of the soiled fabrics before and after washing using a MACH5 multi-area color measurement. For data evaluation, ΔE was calculated between unwashed stains and washed stains, and ΔΔE was calculated between detergents with and without enzymes. Data represent the average of two separate experiments.

[0395] [Table 17]

[0396] [Table 18]

[0397] As can be derived from the table above, the amylase variants of the present invention provide better cleaning results compared to the reference amylase.

[0398] Determination of washing performance in a full-scale washing machine: Multiple stain monitors were washed in a full-scale washing machine in the presence of cotton ballast fabric at 40°C in liquid laundry formulations with different amylases. Each stain was washed in quadruplicate (two units with two strips per stain per unit). After washing, the stain monitors were allowed to air dry. Wash performance was determined by measuring the CIELab values of the stained fabrics before and after washing using a MACH5 multi-area color measurement. For data evaluation, mean values were determined, and ΔY was calculated between unwashed and washed stains, and ΔΔY was calculated between detergents with and without enzymes.

[0399] [Table 19]

[0400] [Table 20]

[0401] As can be derived from the table above, 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, (i) comprises an amino acid substitution at position 4 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 amino acid positions selected from the group consisting of 25, 176, 186, 251, 405, and 482 according to the numbering of SEQ ID NO: 3; and (iii) An amylase variant having at least 60% but less than 100% sequence identity with any of SEQ ID NOs: 1, 3, 4 or 15-41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO:

1.

2. The amylase variant of claim 1, comprising an amino acid substitution at position 25.

3. The amylase variant of claim 1 or 2, wherein the amino acid substitution at position 4 is X4Q.

4. 4. The amylase variant of claim 1, wherein the amino acid substitution at position 25 is X25H, the amino acid substitution at position 176 is X176K, the amino acid substitution at position 186 is X186E, the amino acid substitution at position 251 is X251E, the amino acid substitution at position 405 is X405M, and the amino acid substitution at position 482 is X482W.

5. a) substitutions at one or more positions selected from 25, 176 and 186, preferably one or more substitutions selected from X25H, H176K and X186E, or b) substitutions at one or more positions selected from 251, 405 and 482, preferably one or more substitutions selected from X251E, X405M and X482W, or c) substitutions at one or more positions selected from 25, 176 and 186, preferably one or more substitutions selected from X25H, X176K and X186E, and substitutions at one or more positions selected from 251, 405 and 482, preferably one or more substitutions selected from X251E, X405M and X482W The amylase variant according to any one of claims 1 to 4, comprising:

6. X4Q+X176K+X186E, X4Q+X25H+X176K+X186E, X4Q+X25H+X176K+X186E, X4Q+X25H+X176K+X186E+X251E, X4Q+X25H+X176K+X186E+X405M, X4Q+X25H+X176K+X186E+X482W, X4Q+X25H+X176K+X186E+X251E+X405M, X4Q+X25H+X176K+X186E+X251E+X482W, X4Q + X25H + X176K + X186E + X405M + X482W, and X4Q+X25H+X176K+X186E+X251E+X405M+X482W The amylase variant according to any one of claims 1 to 5, comprising a combination of amino acid substitutions selected from the group consisting of:

7. 7. The amylase variant according to any one of claims 1 to 6, further comprising a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, 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, wherein the numbering is according to the amino acid sequence set forth in SEQ ID NO:

3.

8. 8. The amylase variant according to any one of claims 1 to 7, which 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 of SEQ ID NOs: 1, 3, 4 or any of SEQ ID NOs: 15 to 41, preferably SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO:

1.

9. 9. An amylase variant according to any one of claims 1 to 8, which exhibits improved storage stability and / or improved cleaning performance, preferably exhibiting improved storage stability in a detergent composition.

10. A polynucleotide encoding the amylase variant of any one of claims 1 to 9.

11. A composition comprising an amylase variant according to any one of claims 1 to 9 and at least one additional component.

12. 12. The composition of claim 11, comprising one or more second enzymes different from the amylase variant of any one of claims 1 to 11, preferably 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.

13. 13. A composition according to claim 11 or 12 which is a detergent composition, preferably a laundry detergent composition or a hard surface cleaning detergent composition.

14. A composition according to any one of claims 11 to 13, comprising one or more surfactants and / or one or more builders, preferably one or more strong metal ion-scavenging builders.

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