Enzyme composition comprising a protease, a mannanase and / or a cellulase

Abstract

The present invention provides novel enzyme compositions, more specifically, enzyme compositions containing at least two enzymes selected from the group consisting of proteases, mannanases, and cellulases, as well as methods for producing and using these enzyme compositions.

Description

[Technical Field]

[0001] The present invention provides novel enzyme compositions, more specifically, enzyme compositions containing at least two enzymes selected from the group consisting of proteases, mannanases, and cellulases, as well as methods for producing and using these enzyme compositions. [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 incorporated into cleaning formulations to increase cleaning efficiency and reduce energy consumption in the washing process.

[0003] The most commonly used enzymes in detergents are proteases and amylases, with mannanases and cellulases being less commonly used. Proteases, amylases, mannanases, and cellulases are all hydrolases that differ in the substrates they can degrade. Proteases degrade proteins, amylases degrade starches, mannanases degrade galactomannans, and cellulases degrade cellulose.

[0004] However, successful cleaning often depends on the effectiveness of removing stains composed of various soil types. Thus, successful cleaning depends on the effective removal of stains containing complex mixtures of proteins, starches, galactomannans, cellulose, and other types of partially undefined soils. The inventors of the present application have identified specific enzyme combinations that are particularly suitable for use in removing such complex stains. Summary of the Invention [Means for solving the problem]

[0005] The present invention is directed to compositions comprising two or more enzymes selected from different enzyme classes (also referred to herein as "enzyme blends"). Specifically, the present invention is directed to compositions comprising at least two enzymes selected from the group consisting of proteases, mannanases, and cellulases. The present invention is also directed to methods for producing the enzyme compositions.

[0006] The use of the specific enzyme compositions of the present invention provides beneficial cleaning effects when cleaning objects, and therefore the present invention is also directed to the use of the enzyme compositions for cleaning objects and to detergent compositions comprising the enzyme compositions. DETAILED DESCRIPTION OF THE INVENTION

[0007] 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.

[0008] While the present invention has been described with reference to specific embodiments, this description is not intended to be construed in a limiting sense.

[0009] definition Unless otherwise noted, terms used herein are to be understood according to conventional usage by those of ordinary skill in the art.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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 denote 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 inconsistent time interval between the steps, i.e., the steps may be performed simultaneously, or there may be a time interval of seconds, minutes, hours, days, weeks, months, or years between such steps.

[0014] Throughout this application, various publications are referenced. The entire disclosures of these publications and the references cited therein in their entireties are hereby incorporated by reference in order to more fully describe the state of the art to which this invention pertains.

[0015] 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.

[0016] A "variant" enzyme differs from a "parent" enzyme by specific amino acid substitutions, preferably at one or more amino acid positions.

[0017] In describing the variants of the present invention, abbreviations for single amino acids are used according to the IUPAC accepted one-letter or three-letter amino acid abbreviations.

[0018] "Amino acid modification," as used herein, refers to an amino acid substitution, deletion, or insertion.

[0019] 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." Substitutions 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").

[0020] 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."

[0021] An "insertion" is described by adding the number of the position in the amino acid sequence after the original amino acid, followed by the original amino acid and the additional amino acid. For example, the insertion of a lysine next to a glycine at position 180 would be designated as "Gly180GlyLys" or "G180GK." If two or more amino acid residues are inserted, for example, a Lys and an Ala after Gly180, this would be designated as "Gly180GlyLysAla" or "G195GKA."

[0022] 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 the substitution 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 (e.g., "R170Y G195E" or "R170Y,G195E", respectively). When different modifications can be introduced at a certain position, the different modifications are separated by commas, for example, "Arg170Tyr,Glu" and "R170T,E" represent the substitution 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 parentheses, for example, as "Arg170[Tyr,Gly]" or Arg170{Tyr,Gly} or in short R170[Y,G] or R170{Y,G}.

[0023] 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 that is found in nature (i.e., without any human intervention).

[0024] The term "introduction of at least two negative charges" into a specific amino acid sequence refers to an increase in the net charge of the specific amino acid sequence by at least two negative charges. Such an increase in the net charge of a specific amino acid sequence by at least two negative charges can be achieved by modifying the amino acid sequence and can be achieved by one or more amino acid sequence modifications selected from the group consisting of substitution, deletion, and insertion, preferably one or more amino acid substitutions. An increase in the net charge of a specific amino acid sequence by at least two negative charges can be achieved by removing a positive charge or introducing a negative charge, or a combination thereof. Four amino acids, namely aspartic acid (Asp, D), glutamic acid (Glu, E), lysine (Lys, K), and arginine (Arg, R), have side chains that can carry a charge at neutral pH. At pH 7.0, two of the following residues (acidic side chains): aspartic acid (Asp, D) and glutamic acid (Glu, E) have negative charges, and two of the following residues (basic side chains): lysine (Lys, K) and arginine (Arg, R) have positive charges. Therefore, the introduction of at least two negative charges into an amino acid sequence can be achieved, for example, by substituting glutamic acid for arginine, substituting two uncharged leucine residues for two glutamic acid residues, inserting two aspartic acid residues, or deleting two lysine residues. The introduction of at least two negative charges by modifying an amino acid sequence is preferably evaluated under conditions typically encountered in washing steps, preferably at pH 6-11, preferably at pH 7-9, more preferably at pH 7.5-8.5, even more preferably at pH 7.0-8.0, and most preferably at pH 7.0 or 8.0.

[0025] The term "heterologous" (or exogenous or foreign or recombinant or non-native or non-naturally) polypeptide is defined herein as a polypeptide that is not native to the host cell, a polypeptide that is native to the host cell but has been modified by structural alterations to the native polypeptide, e.g., deletions, substitutions and / or insertions, or a polypeptide that is native to the host cell but whose expression is quantitatively altered or whose expression is directed from a different genomic location than in the native host cell as a result of recombinant DNA techniques, e.g., manipulation of the host cell's DNA by a stronger promoter.

[0026] Similarly, the term "heterologous" (or exogenous or foreign or recombinant or non-native or non-naturally) polynucleotide refers to a polynucleotide that is not native to a host cell; a polynucleotide that is native to a host cell but that has been modified by structural alterations, e.g., deletions, substitutions, and / or insertions, to the native polypeptide; a polypeptide that is native to a host cell but whose expression has been quantitatively altered as a result of manipulation of the regulatory elements of the polynucleotide by recombinant DNA techniques, e.g., a stronger promoter; or a polynucleotide that is native to a host cell but that is not integrated into its native genetic environment as a result of genetic manipulation by recombinant DNA techniques. In reference to two or more polynucleotide sequences or two or more amino acid sequences, the term "heterologous" is used to characterize the fact that the two or more polynucleotide sequences or 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 that has been introduced by man using genetic techniques. With respect to polynucleotides, "recombinant" includes those produced using genetic / recombinant DNA techniques. (a) the sequence of a polynucleotide or a portion thereof; or (b) one or more gene control sequences, such as, but not limited to, a promoter, operably linked to the polynucleotide; or (c) Both a) and b) is not present 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 sequences and variant polypeptide sequences can be defined by their sequence identity when compared with parent sequences. Sequence identity is usually expressed as "sequence identity %" or "identity %". For calculating 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, and is usually generated by using a mathematical method called an alignment algorithm.

[0030] According to the present invention, the alignment is generated 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 percentage is calculated as follows: the number of identical residues is divided by the length of the alignment region representing each of the sequences of the present invention over its entire length, and multiplied by 100: identity percentage = (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, a pairwise alignment is made 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 other sequences to which the sequence of the present invention is compared are also removed for pairwise alignment. After aligning the two sequences, the second step is to determine the identity value 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)), using the program's default parameters (gap open = 10.0, gap extension = 0.5, and matrix = EDNAFULL).

[0033] Variant polypeptides can also be defined by their sequence similarity when compared to another sequence. Sequence similarity is usually provided as "% sequence similarity" or "% similarity". % 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, which also applies to the determination of % similarity according to the present invention, and are also according to the BLOSUM62 matrix, which is one of the most used amino acid similarity matrices for database searching and sequence alignment, e.g., as used by the program "NEEDLE": 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, Amino acid Y is similar to amino acids F, H, and W

[0034] For calculating sequence similarity, the first step is to generate a sequence alignment as described above. After aligning two sequences, the second step is to determine the identity value from the generated alignment. For this purpose, the similarity percentage is calculated as follows: the number of identical residues + the number of similar residues is divided by the length of the alignment region that represents the sequence of the present invention over its entire length, and multiplied by 100: identity percentage = [(identical residues + similar residues) / length of the alignment region that represents the sequence of the present invention over its entire length] * 100.

[0035] As used herein, a "fragment" or "subsequence" refers to a portion of a polynucleotide sequence or an amino acid sequence. The term "functional fragment" refers to any nucleic acid sequence or amino acid sequence that contains only a 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%, 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 contiguous nucleic acids or amino acids compared to the original nucleic acid sequence or original amino acid sequence, respectively.

[0036] 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 a promoter) as described herein.

[0037] 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 in a given cell. The term "vector," as used herein, encompasses any type of cloning vehicle, including, but not limited to, a plasmid, a phagemid, a viral vector (e.g., a phage), a bacteriophage, a baculovirus, a cosmid, a fosmid, an artificial chromosome, or 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 host cell of origin or destination.

[0038] The terms "introduction of a polynucleotide" or "transformation of a polynucleotide," as referred to herein, encompass the transfer of an exogenous polynucleotide into a host cell, regardless of the transfer method used. That is, the term "transformation of a polynucleotide," as used herein, is independent of vectors, shuttle systems, or host cells, and not only relates to polynucleotide transfer methods of transformation known in the art (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.

[0039] A polynucleotide that encodes 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.

[0040] 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 one other 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."

[0041] 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, non-complex protein formulations contain proteins at higher concentrations than complex formulations, e.g., detergent compositions. Thus, preferably, non-complex protein formulations are concentrated protein variant formulations. Preferably, non-complex protein formulations contain 2-120 mg / g of active enzyme, while complex formulations, such as detergent compositions, contain 0.002-6 mg / g of active enzyme.

[0042] "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.

[0043] 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.

[0044] 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 at time zero (100%) under defined conditions and at a specific time point (x%) thereafter. 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 itself or in a formulation.

[0045] An "enzyme inhibitor," as used herein, is a compound that slows or stops enzyme activity. Enzyme inhibitors often also stabilize the three-dimensional structure of the enzyme. Thus, enzyme inhibitors usually also act as "enzyme stabilizers."

[0046] "pH stability" refers to the ability of an enzyme to exhibit enzymatic activity after exposure to a particular pH value.

[0047] 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.

[0048] 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 after storage in a detergent composition (preferably in Model ES1-C detergent) at a temperature of 40°C or 50°C for 28 days, more preferably 56 days.

[0049] 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 interchangeably with laundry and hard surface cleaning. Cleaning performance is compared under appropriate wash conditions. The term "appropriate wash conditions" is used herein to indicate 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 a lower amount of enzyme is required by weight to achieve the same end result as in the corresponding control conditions.

[0050] 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.

[0051] "Detergent composition," "detergent formulation," "cleaning formulation," or "detergent" refers to a composition designed to clean 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 type of chemical that may 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 ingredients" includes the amount of a particular component that provides effective stain removal and effective cleaning conditions (e.g., pH, temperature, water hardness), effectively provides optical benefits (e.g., optical brightening, dye transfer prevention, color care), and effectively facilitates processing (maintaining physical properties during processing, storage, and use, e.g., rheology modifiers, hydrotropes, drying agents).

[0052] 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 relation to 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.

[0053] 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" (textiles made by weaving, knitting, or felting fibers) or "garment" (any article of clothing made from textiles) as used herein also include the broader term woven fabric.

[0054] The term "fiber" includes natural fibers, synthetic fibers and mixtures thereof. Examples of natural fibers are those of plant origin (such as flax, jute and cotton) or animal origin containing 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. Fibers can be single filaments or part of textile products such as knitwear, woven fabrics or nonwoven fabrics.

[0055] The term "hard surface cleaning" relates to both household and industrial hard surface cleaning, and refers to the process of treating hard surfaces with a solution containing the detergent composition of the present invention.Hard surfaces can include any hard surfaces in households or industries, such as floors, furniture, walls, sanitary ceramics, glass, medical equipment, metal surfaces including cutlery and tableware.Specific forms of hard surface cleaning are dishwashing, manual dishwashing (MDW) or automatic dishwashing (ADW).

[0056] The term "dishwashing" refers to any form of washing dishes, for example, by manual or automatic dishwashing, including, but not limited to, cleaning all forms of ceramics such as plates, cups, glasses, bowls, all forms of cutlery such as spoons, knives, forks and serving utensils, as well as ceramic, plastics such as melamine, metal, china, glass and acrylic.

[0057] 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.

[0058] 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 cleaning methods and mechanical / automatic cleaning methods. Manual cleaning is used when there is no mechanical unit or when the medical device to be cleaned is too fragile or difficult to clean with a mechanical unit. Mechanical / automatic cleaning methods remove soils and microorganisms through an automatic washing and rinsing process, which includes ultrasonic cleaning and washing.

[0059] In the field of cleaning, the term "stain" is typically used in relation to laundry, e.g., cleaning for textiles, fabrics or fibers, while the term "soil" is typically used in relation to hard surface cleaning, e.g., cleaning dishes and cutlery. However, in this specification, the terms "stain" and "soil" are used interchangeably.

[0060] As used herein, "metal ion-scavenging builders" differ from precipitating builders in that a modest amount of precipitate is formed when the builder is used in an amount sufficient to combine with all calcium ions in an aqueous solution having an initial neutral pH and a hardness (German hardness) of 7° dH. "Strong builders" have a logarithmic stability constant (Log K) of the cation / chelator complex greater than 4, particularly greater than 5, 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.

[0061] A "complex stain" (also referred to herein as a "mixed stain") is a stain that contains two or more soil components selected from fat, protein, starch, mannan, cellulose, and preferably the stain contains a thickener such as galactomannan or locust bean gum. An example of a complex stain is a salad dressing stain. Additional food stains such as chocolate pudding or baby food are considered to be complex stains.

[0062] As used herein, "synergy" or "synergistic effect" refers to the interaction of two or more enzymes that produces a combined effect, e.g., cleaning performance, that is greater than the sum of the individual effects.

[0063] As used herein, the term "low temperature" refers to a temperature range of 40°C or less, preferably 10 to 40°C, more preferably 20 to 40°C, and more preferably 20 to 35°C.

[0064] Detailed Description The present invention provides novel enzyme compositions, more specifically, enzyme compositions containing two or more enzymes selected from the group consisting of proteases, mannanases, and cellulases, as well as methods for producing and using these enzyme compositions.

[0065] In particular, the inventors have found that in such enzyme compositions: aa. A protease variant of a parent protease, (i) an amino acid sequence comprising at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16; and (ii) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NOs: 16 to 34, preferably SEQ ID NO: 16 a protease variant comprising bb. A mannanase variant of a parent mannanase, (i) one or more amino acid substitutions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379 according to the numbering of SEQ ID NO: 35, and (ii) an amino acid sequence that is at least 60% but less than 100% identical to the sequence of positions 31 to 490 of SEQ ID NO: 35 mannanase variants comprising: cc. A cellulase variant comprising an amino acid sequence at least 60% identical to SEQ ID NO: 42 It has been found that the use of at least one enzyme selected from the group consisting of:

[0066] composition In one embodiment, the present invention is directed to an enzyme composition.

[0067] In particular, the present invention provides a composition comprising two or more enzymes selected from the group consisting of proteases, mannanases, and cellulases, a. Proteases are I. Protease variants of a parent protease, (i) an amino acid sequence comprising at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16; and (ii) an amino acid sequence that is at least 60% but less than 100% identical to any of SEQ ID NOs: 16 to 34, preferably SEQ ID NO: 16 or a protease variant comprising II. Protease variants of a parent protease, (i) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 3, 4, 9, 19, 22, 43, 62, 63, 76, 87, 99, 101, 103, 104, 116, 118, 128, 129, 130, 156, 160, 167, 170, 182, 185, 188, 194, 205, 213, 217, 218, 232, 245, 256, 259, 262, and 271 according to the numbering of SEQ ID NO: 17, and an amino acid sequence having at least 60% but less than 100% sequence identity to SEQ ID NO: 22; or (ii) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 24, 53, 76, 78, 101, 128, and 217 according to the numbering of SEQ ID NO: 17, and having at least 60% but less than 100% sequence identity to SEQ ID NO: 17; or (iii) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 9, 89, 130, 133, 144, 159, 183, 189, 217, 224, 238, 243, 252, 259, 270, and 271 according to the numbering of SEQ ID NO: 19, and having at least 60% but less than 100% sequence identity to SEQ ID NO: 19; or (iv) an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 22, SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 31 Protease variants including is selected from B. Mannanase I. Mannanase variants of a parent mannanase, (i) one or more amino acid substitutions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379 according to the numbering of SEQ ID NO: 35, and (ii) an amino acid sequence that is at least 60% but less than 100% identical to the sequence of positions 31 to 490 of SEQ ID NO: 35 or a mannanase variant comprising II. Mannanase variants of a parent mannanase, (i) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 39, and an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 24, 123, and 256 according to the numbering of SEQ ID NO: 39; or (ii) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 38, and an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 10, 19, 30, 38, 59, 60, 67, 97, 103, 129, 143, 167, 168, 184, 225, 228, 235, 244, 258, 261, and 298 according to the numbering of SEQ ID NO: 38; or (iii) an amino acid sequence that is at least 60% identical to SEQ ID NO: 40; or (iv) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 40, as well as an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 30, 81, 93, 95, 118, 138, 139, 200, 202, 210, 260, 283, 288, 294, and 295 according to the numbering of SEQ ID NO: 40. Mannanase variants containing is selected from C. Cellulase I. A cellulase variant comprising an amino acid sequence that is at least 60% identical to SEQ ID NO: 42, or II. Cellulase variants comprising an amino acid sequence at least 60% identical to SEQ ID NO: 46, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 44, or SEQ ID NO: 47 is selected from At least one enzyme aa. A protease variant of a parent protease, (i) an amino acid sequence comprising at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16; and (ii) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NOs: 16 to 34, preferably SEQ ID NO: 16 a protease variant comprising bb. A mannanase variant of a parent mannanase, (i) one or more amino acid substitutions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379 according to the numbering of SEQ ID NO: 35, and (ii) an amino acid sequence that is at least 60% but less than 100% identical to the sequence of positions 31 to 490 of SEQ ID NO: 35 mannanase variants comprising: cc. A cellulase variant comprising an amino acid sequence at least 60% identical to SEQ ID NO: 42 The present invention relates to a composition of matter selected from the group consisting of:

[0068] In a preferred embodiment, the present invention provides an enzyme composition comprising two or more enzymes selected from the group consisting of proteases, mannanases, and cellulases, a. Proteases are I. Protease variants of a parent protease, (i) an amino acid sequence comprising at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16; and (ii) an amino acid sequence that is at least 60% but less than 100% identical to any of SEQ ID NOs: 16 to 34, preferably SEQ ID NO: 16 Protease variants including is selected from B. Mannanase I. Mannanase variants of a parent mannanase, (i) one or more amino acid substitutions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379 according to the numbering of SEQ ID NO: 35, and (ii) an amino acid sequence that is at least 60% but less than 100% identical to the sequence of positions 31 to 490 of SEQ ID NO: 35 Mannanase variants containing selected from, and C. Cellulase I. Cellulase variants comprising an amino acid sequence at least 60% identical to SEQ ID NO: 42 The present invention relates to an enzyme composition selected from the group consisting of:

[0069] In one embodiment, the composition comprises a protease and a mannanase as described herein. In one embodiment, the composition comprises a protease and a cellulase as described herein. In one embodiment, the composition comprises a mannanase and a cellulase as described herein. Preferably, the composition comprises a protease and a mannanase as described herein. More preferably, the present invention is directed to a composition comprising a protease, a mannanase, and a cellulase as described herein. In one embodiment, the composition further comprises an amylase. Thus, in another embodiment, the composition comprises a protease, a mannanase, a cellulase, and an amylase as described herein.

[0070] Preferably, the composition comprises a protease and a mannanase, a protease and a cellulase, a mannanase and a cellulase, or a protease, a mannanase and a cellulase, wherein the protease, the cellulase and the mannanase are aa. A protease variant of a parent protease, (i) an amino acid sequence comprising at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16; and (ii) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NOs: 16 to 34, preferably SEQ ID NO: 16 a protease variant comprising bb. A mannanase variant of a parent mannanase, (i) one or more amino acid substitutions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379 according to the numbering of SEQ ID NO: 35, and (ii) an amino acid sequence that is at least 60% but less than 100% identical to the sequence of positions 31 to 490 of SEQ ID NO: 35 mannanase variants comprising: cc. A cellulase variant comprising an amino acid sequence at least 60% identical to SEQ ID NO: 42 is selected from.

[0071] In one embodiment, the present invention is directed to an enzyme composition comprising two or more enzymes selected from the group consisting of proteases, mannanases, and cellulases, wherein the composition comprises two or more different proteases, mannanases, and / or cellulases described herein, such as anti-graying cellulases and anti-pilling cellulases.

[0072] Proteases In one embodiment, the enzyme composition of the present invention comprises a protease. The proteases and protease variants described herein possess protease activity (also referred to herein as "proteolytic activity"). This property relates to the hydrolytic activity of a protease (proteolysis, which refers to the hydrolysis of peptide bonds connecting amino acids in a polypeptide chain) on protein-containing substrates, such as casein, hemoglobin, and BSA. Quantitatively, proteolytic activity relates to the rate of protein degradation by a protease or proteolytic enzyme over a given period of time. Methods for assaying proteolytic activity are well known in the literature (see, e.g., Gupta et al. (2002), Appl. Microbiol. Biotechnol. 60:381-395).

[0073] For example, proteolytic activity can be determined using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, abbreviated AAPF, see, e.g., DelMar et al. (1979), Analytical Biochem 99, 316-320) as a substrate. pNA is cleaved from the substrate molecule by proteolytic cleavage, resulting in the release of yellow-colored free pNA, which can be quantified by measuring the optical density at 405 nm (OD405).

[0074] The protease of the enzyme composition is - a protease variant of a parent protease, an amino acid sequence which, compared to SEQ ID NO: 16, comprises at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17 and an amino acid sequence which is at least 60% but less than 100% identical to SEQ ID NO: 16 to 34, preferably SEQ ID NO: 16; or a protease variant comprising - a protease variant of a parent protease, (i) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 3, 4, 9, 19, 22, 43, 62, 63, 76, 87, 99, 101, 103, 104, 116, 118, 128, 129, 130, 156, 160, 167, 170, 182, 185, 188, 194, 205, 213, 217, 218, 232, 245, 256, 259, 262, and 271 according to the numbering of SEQ ID NO: 17, and an amino acid sequence which has at least 60% but less than 100% sequence identity to SEQ ID NO: 22; or (ii) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 24, 53, 76, 78, 101, 128, and 217 according to the numbering of SEQ ID NO: 17, and an amino acid sequence that has at least 60% but less than 100% sequence identity to SEQ ID NO: 17; or (iii) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 9, 89, 130, 133, 144, 159, 183, 189, 217, 224, 238, 243, 252, 259, 270, and 271 according to the numbering of SEQ ID NO: 19, and an amino acid sequence which has at least 60% but less than 100% sequence identity to SEQ ID NO: 19; or (iv) an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 22, SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 31 Protease variants including is selected from.

[0075] Preferably, the protease of the enzyme composition is a protease variant of a parent protease, wherein the protease variant comprises an amino acid sequence that comprises at least two additional negative charges in the loop region of residues 98-104 according to the numbering of SEQ ID NO: 17 compared to SEQ ID NO: 16, and an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 16-34, preferably SEQ ID NO: 16.

[0076] Preferably, the protease is a variant protease of a parent protease, which is preferably a subtilisin protease.

[0077] Preferably, the parent protease of the protease variant is a protease having at least 60% sequence identity to any of SEQ ID NOs: 16 to 34, and more preferably, the parent protease of the protease variant is a protease set forth in any of SEQ ID NOs: 16 to 34. Most preferably, the parent protease of the protease variant is the protease set forth in SEQ ID NO: 16.

[0078] Preferably, in this embodiment, the amino acid residues in the parent protease at the above-mentioned positions (ie, X) correspond to the amino acid residues shown in SEQ ID NO:16 at each position according to the numbering of SEQ ID NO:17.

[0079] The variant protease comprises at least two additional negative charges in the loop region from residues 98 to 104 according to the numbering of SEQ ID NO: 17 (BPN' numbering, i.e., positions are numbered according to their correspondence with the amino acid sequence of B. amyloliquefaciens subtilisin BPN' established as SEQ ID NO: 17) compared to SEQ ID NO: 16. Preferably, the protease variant comprises the amino acids aspartic acid, histidine, and serine as the catalytic triad, and preferably, the protease is a subtilisin protease.

[0080] Preferably, at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17 are obtained by one or more amino acid modifications selected from the group consisting of substitutions, deletions and insertions, preferably substitutions or insertions, preferably substitutions.

[0081] Preferably, in the protease, the at least two additional negative charges compared to SEQ ID NO: 16 in the loop region of residues 98 to 104 are caused by one or more amino acid substitutions at amino acid positions according to the numbering of SEQ ID NO: 17 selected from the group consisting of 98, 99, 100, 101, 102, 103 and 104, preferably an amino acid substitution at position 101.

[0082] Preferably, at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17 are obtained by one or more amino acid modifications selected from the group consisting of X99E, X101D and X101E, preferably D99E, R101D and R101E.

[0083] In a preferred embodiment, the protease comprises an amino acid sequence comprising the amino acid substitution R101E or R101D as compared to SEQ ID NO: 16, according to the numbering of SEQ ID NO: 17. In another embodiment, the at least two additional negative charges in the loop region of residues 98-104 as compared to SEQ ID NO: 16 are not caused by the amino acid substitution X101E or X101D. Most preferably, the protease comprises an amino acid sequence comprising the amino acid substitution R101E as compared to SEQ ID NO: 16, according to the numbering of SEQ ID NO: 17.

[0084] In a preferred embodiment, loop sequences 98 to 104 have two additional negative charges of the following sequences ADGEGAI, ADGDGAI, ADGDGSV, ADGEGSV, AADGSGSV, AADGEGSV or ASEGEGSV, which have longer sequences with insertions within the loop sequence compared to SEQ ID NO: 16.

[0085] In a further preferred embodiment, the amino acid sequence of the protease comprises an alanine (103A) at position 103 and an isoleucine (104I) at position 104 according to the numbering of SEQ ID NO: 17 compared to SEQ ID NO: 16, more preferably 101R, 104I and 103A.

[0086] In a further preferred embodiment, the amino acid sequence of the protease does not contain additional amino acid residues in the loop region at positions 98 to 104 according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16. Preferably, the amino acid sequence of the protease does not contain additional amino acid residues at positions 42 to 43, 51 to 55, 155 to 165, 187 to 189, 217 to 218 or 218 to 219 according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16.

[0087] In a preferred embodiment, the protease comprises one or more further substitutions at one or more positions selected from the group consisting of 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 according to the numbering of SEQ ID NO:17 compared to SEQ ID NO:16.

[0088] Preferably, the protease further comprises one or more amino acid substitutions selected from the group consisting of X3T, X4I, X156D, X205I and X262E according to the numbering of SEQ ID NO:17.

[0089] Preferably, the protease further comprises one or more amino acid substitutions selected from the group consisting of S3T, V4I, S156D, V205I and L262E according to the numbering of SEQ ID NO:17.

[0090] Preferably, the protease further comprises one or more, preferably all, amino acid substitutions selected from the group consisting of X3T, X4I and X205I according to the numbering of SEQ ID NO:17.

[0091] Preferably, the protease further comprises one or more, preferably all, amino acid substitutions selected from the group consisting of S156D and L262E according to the numbering of SEQ ID NO:17.

[0092] Preferably, the protease is 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%, at least 99.5%, but less than 100% sequence identity.

[0093] Preferably, the protease has at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100% sequence identity to SEQ ID NO:16.

[0094] Preferably, the protease has a sequence similar to SEQ ID NO: 16 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%. %, 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%, at least 99.5%, but less than 100% sequence identity with SEQ ID NO: 16, and comprising at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17 compared to SEQ ID NO: 16.

[0095] Preferably, the protease has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, but less than 100%, sequence identity to SEQ ID NO: 16 and comprises at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17 compared to SEQ ID NO: 16.

[0096] Preferably, the protease has a sequence similar to SEQ ID NO: 16 by at least 80%, 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 comprises the amino acid substitution X101D or X101E, preferably X101E, preferably R101E, according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16.

[0097] Preferably, the protease comprises at least 80%, at least 85%, at least 90%, at least 95%, 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 with SEQ ID NO: 16 and comprises the amino acid substitution X101E, preferably R101E, according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16.

[0098] Preferably, the variant protease comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and the amino acid sequence of the protease comprises at least two additional negative charges in the loop region of residues 98-104 according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16, and the protease comprises at least 3, 4, 9, 15, 24, 27, 33, 36, 45, 55, 57, 58, 59, 61, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 200, 202, 203, 204, 205, and 274, 285, 290, 303, 304, 306, 117, 118, 120, 123, 124, 128, 129, 130, 131, 136, 137, 143, 154, 156, 160, 161, 163, 167, 170, 171, 172, 185, 194, 195, 199, 205, 206, 209, 217, 218, 222, 224, 232, 235, 236, 238, 244, 245, 248, 252, 261, 262 and 274.

[0099] In another embodiment of the invention, the protease as described herein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, wherein the amino acid sequence of the protease comprises, compared to SEQ ID NO: 16, at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, the amino acid substitution X101E or X101D, preferably X101E, according to the numbering of SEQ ID NO: 17. a. Threonine or Serine at position 3 (X3T or X3S), b. isoleucine or valine at position 4 (X4I or X4V); c. serine, alanine, threonine, or arginine at position 63 (X63S, X63A, X63T, or X63R); d. threonine, aspartic acid, or glutamic acid at position 156 (X156T, X156D, or X156E); e. serine or proline at position 194 (X194S or X194P), f. Serine, valine, or methionine at position 199 (X199S, X199V, or X199M) g. Isoleucine or valine at position 205 (X205I or X205V), and h. Aspartic acid, glutamic acid, glutamine, glycine, or leucine at position 217 (X217D, X217E, X217Q, X217G, or X217L) and further comprising at least one amino acid residue selected from the group consisting of:

[0100] Preferably, the protease has a sequence similar to SEQ ID NO: 16 by at least 80%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%. %, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100%, sequence identity to SEQ ID NO: 16 and comprising the amino acid substitutions X101D or X101E, preferably X101E, and one or more of the amino acid substitutions X3T, X4I and X205I, preferably S3T, V4I and V205I, according to the numbering of SEQ ID NO: 17. More preferably, the protease has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100%, sequence identity with SEQ ID NO: 16 and comprises the amino acid substitution R101D or R101E, preferably R101E, according to the numbering of SEQ ID NO: 17, and the amino acid substitutions S3T, V4I and V205I compared to SEQ ID NO: 16.

[0101] In another embodiment, the protease comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 16, and the protease comprises any of the following amino acids as compared to SEQ ID NO: 16: X101E or X101D according to the numbering of SEQ ID NO: 17, preferably X101E, X156D, X262E, X137H, X3T, X45E,D,Q, X55N, X58W,Y,L, X59D,M,N,T, X61D,R, X87 and one or more substitutions selected from the group consisting of X106A,W, X117E, X120V,D,K,N, X125M, X129D, X136Q, X144W, X161T, X163A,G, X171L, X172S, X185Q, X199M, X209W, X222Q, X238H, X244T, X261T,D and X262N,Q,D.

[0102] In one embodiment, the protease comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and the protease comprises, relative to SEQ ID NO: 16, the amino acid substitutions X101E or X101D according to the numbering of SEQ ID NO: 17, preferably X101E, and one, preferably both, of a substitution selected from the group consisting of X156D and X262E.

[0103] In another embodiment, the protease comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, wherein the protease has the following R amino acid substitutions relative to SEQ ID NO: 16: R101E or R101D, preferably R101E, and any of the following substitutions according to the numbering of SEQ ID NO: 17: 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, E136Q, S144W, S161T, S163A,G, Y171L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D and L262N,Q,D.

[0104] In one embodiment, the protease comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and the protease comprises, relative to SEQ ID NO: 16, the amino acid substitutions X101E or R101D, preferably R101E, according to the numbering of SEQ ID NO: 17, and one, preferably both, of a substitution selected from the group consisting of S156D and L262E.

[0105] More preferably, the protease has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100%, sequence identity with SEQ ID NO: 16 and comprises the amino acid substitution R101D or R101E, preferably R101E, and the amino acid substitutions S156D and L262E according to the numbering of SEQ ID NO: 16.

[0106] A protease having the above substitutions is shown, for example, in SEQ ID NO:28.

[0107] Thus, in one embodiment, the protease variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least The amino acid sequence may be 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%, at least 99.5% or 100% identical.

[0108] In another embodiment, the protease comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and the protease comprises, relative to SEQ ID NO: 16, the amino acid substitutions R101E according to the numbering of SEQ ID NO: 17, and one or more, preferably both, of S156D and L262E, and optionally at least one further mutation selected from I104T, H120D, Q137H, S141H, R145H and S163G.

[0109] Preferably, the protease has at least 80% sequence identity with SEQ ID NO: 16 and comprises at least two additional negative charges in the loop region of residues 98-104 according to the numbering of SEQ ID NO: 17 compared to SEQ ID NO: 16, and the protease comprises one or more conservative amino acid exchanges described herein compared to SEQ ID NO: 16. Preferably, the protease comprises at least one, at least two, at least three, at least four, at least five, at least six, at least 10, at least 15, at least 20, at least 30, or at least 40 conservative amino acid exchanges compared to SEQ ID NO: 16.

[0110] Preferably, compared to SEQ ID NO: 16, the proteases described herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid exchanges, preferably in addition to the substitutions X101D or X101E, preferably X101E and preferably the amino acid substitutions X3T, X4I and X205I or the amino acid substitutions X156D and X262E, in addition to modifications resulting in at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17.

[0111] Preferably, the protease has at least 80% sequence identity with SEQ ID NO: 16 and contains at least two additional negative charges in the loop region of residues 98-104 according to the numbering of SEQ ID NO: 17 compared to SEQ ID NO: 16, with the remaining differences in amino acid sequence compared to SEQ ID NO: 16 being due to conservative amino acid exchanges as described herein.

[0112] Preferably, the protease is a protease variant comprising the amino acid substitution X101E according to the numbering of SEQ ID NO: 17, optional amino acid substitutions X3T, X4I and X205I or amino acid substitutions X156D and X262E and an amino acid sequence that is at least 80% but less than 100% identical to SEQ ID NO: 16.

[0113] Most preferably, the protease is a protease variant comprising the amino acid substitution X101E according to the numbering of SEQ ID NO:17 and an amino acid sequence that is at least 80% but less than 100% identical to SEQ ID NO:16.

[0114] Most preferred is a protease variant comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16, with the amino acid substitution R101E according to the numbering of SEQ ID NO: 17, i.e. the only amino acid exchange compared to SEQ ID NO: 16.

[0115] Instead, a protease variant comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16 with the amino acid substitutions R101E, S3T, V4I and V205I according to the numbering of SEQ ID NO: 17 is preferred.

[0116] Instead, a protease variant comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16 with the amino acid substitutions R101E, S156D and L262E according to the numbering of SEQ ID NO: 17 is preferred.

[0117] In one embodiment, the protease variant of the enzyme composition comprises: (i) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 3, 4, 9, 19, 22, 43, 62, 63, 76, 87, 99, 101, 103, 104, 116, 118, 128, 129, 130, 156, 160, 167, 170, 182, 185, 188, 194, 205, 213, 217, 218, 232, 245, 256, 259, 262, and 271 according to the numbering of SEQ ID NO: 17, and an amino acid sequence having at least 60% but less than 100% sequence identity to SEQ ID NO: 22; or (ii) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 24, 53, 76, 78, 101, 128, and 217 according to the numbering of SEQ ID NO: 17, and having at least 60% but less than 100% sequence identity to SEQ ID NO: 17; or (iii) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 9, 89, 130, 133, 144, 159, 183, 189, 217, 224, 238, 243, 252, 259, 270, and 271 according to the numbering of SEQ ID NO: 19, and having at least 60% but less than 100% sequence identity to SEQ ID NO: 19; or (iv) an amino acid sequence having at least 60% sequence identity to SEQ ID NO: 22, SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 31 Includes:

[0118] In one embodiment, the protease variant has at least 60%, 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 and amino acid sequences having 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 an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 3, 4, 9, 19, 22, 43, 62, 63, 76, 87, 99, 101, 103, 104, 116, 118, 128, 129, 130, 156, 160, 167, 170, 182, 185, 188, 194, 205, 213, 217, 218, 232, 245, 256, 259, 262 and 271 according to the numbering of SEQ ID NO:17.

[0119] Preferably, the protease variant has at least 60%, 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 the sequences X3T, X4I, X9D / E / R, X19L, X22R / A, X43R, X62D, X63D / E, X76D, X87N, X99A / D, X101M / R / E, X103A, X104I, X116L, X118V, X128 according to the numbering of SEQ ID NO: 17 X129Q, X130A, X156D / E, X160D / S, X167A, X170S, X182E / D, X185E / D, X188D, X194P, X205I, X213A, X217E / D / Q, X218D, X232V, X245R, X256D / E, X259D / E, X262D / E, and X271F.

[0120] Preferably, the protease variant has at least 60%, 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 S3T, V4I, S9D / E / R, R19L, T22R / A, N43R, N62D, G63D / E, N76D, S87N, S99A / D, S101M / R / E, S103A, V104I, N116L, G118V, S128 according to the numbering of SEQ ID NO: 17 The amino acid sequence comprises an amino acid sequence having one or more amino acid substitutions selected from the group consisting of L, P129Q, S130A, S156D / E, G160D / S, Y167A, R170S, Q182E / D, N185E / D, S188D, A194P, V205I, T213A, L217E / D / Q, N218D, A232V, N245R, S256D / E, S259D / E, L262D / E, and E271F.

[0121] In a preferred embodiment, said protease variant further comprises a D and E insertion immediately adjacent to the C-terminus of position 99 according to the numbering of SEQ ID NO:17.

[0122] Proteases having such substitutions are shown, for example, in SEQ ID NO:24, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:29.

[0123] Thus, in one embodiment, the protease variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96.5%, at least 97%, at least 98.5%, at least 9 ... The amino acid sequence may be 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%, at least 99.5% or 100% identical.

[0124] In one embodiment, the protease variant comprises an amino acid sequence that is 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%, at least 99.5% or 100% identical to any of SEQ ID NO:24, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:29.

[0125] In one embodiment, the protease variant comprises an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO:24.

[0126] In one embodiment, the amino acid sequence comprises an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO:23.

[0127] In one embodiment, the protease variant comprises an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO:26.

[0128] In one embodiment, the amino acid sequence comprises an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO:27.

[0129] In one embodiment, the amino acid sequence comprises an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO:29.

[0130] In another embodiment, the protease variant has an identical sequence to SEQ ID NO: 17 by at least 60%, 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 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. 2.5%, 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 an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 24, 53, 76, 78, 101, 128 and 217 according to the numbering of SEQ ID NO:17.

[0131] Preferably, the protease variant has at least 60%, 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%, 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 an amino acid sequence having one or more amino acid substitutions selected from the group consisting of X24G / R, X53G, X76D, X78N, X101N, X128A / S and X217Q / L / E / D according to the numbering of SEQ ID NO: 17.

[0132] Preferably, the protease variant has at least 60%, 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%, 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 an amino acid sequence having one or more amino acid substitutions selected from the group consisting of S24G / R, S53G, N76D, S78N, S101N, G128A / S and Y217Q / L / E / D according to the numbering of SEQ ID NO: 17.

[0133] Proteases having such substitutions are shown, for example, in SEQ ID NOs: 32, 33 and 34.

[0134] Thus, in one embodiment, the protease variant has a similar structure to any of SEQ ID NOs: 32, 33 or 34 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 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 ... 0.5%, 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%, at least 99.5% or 100% identical.

[0135] In one embodiment, the protease variant comprises an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO:32.

[0136] In one embodiment, the protease variant comprises an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO:33.

[0137] In one embodiment, the protease variant comprises an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO:34.

[0138] In one embodiment, the protease variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 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 9 sequences according to the numbering of SEQ ID NO: 19. , 89, 130, 133, 144, 159, 183, 189, 217, 224, 238, 243, 252, 259, 270 and 271, preferably one or more, preferably all, substitutions selected from the group consisting of X159T, X183S, X238N, X243N, X259N and X270A, more preferably X9T, X130D, X133A, X144K, X159T, X183S, X189T , X217M, X224A, X238N, X243N, X252T, X259N, X270A and X271E, and more preferably one or more, preferably all, substitutions selected from the group consisting of X9T, X89A, X130D, X133A, X144K, X159T, X183S, X189T, X217M, X224A, X238N, X243N, X252T, X259N, X270A and X271E.

[0139] In one embodiment, the protease variant has an identical amino acid sequence to SEQ ID NO:19 by 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%, at least an amino acid sequence having at least 99.5% but less than 100% sequence identity with one or more, preferably all, substitutions selected from the group consisting of X159T, X183S, X238N, X243N, X259N and X270A according to the numbering of SEQ ID NO: 19, more preferably X9T, X130D, X133A, X144K, X159T, X183S, X189T, X217M, X224A, X238N, X243N, X259N and X270A; X238N, X243N, X252T, X259N, X270A and X271E, more preferably one or more, and preferably all, substitutions selected from the group consisting of X9T, X89A, X130D, X133A, X144K, X159T, X183S, X189T, X217M, X224A, X238N, X243N, X252T, X259N, X270A and X271E.

[0140] In one embodiment, the protease variant has an identical amino acid sequence to SEQ ID NO:19 by 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%, at least an amino acid sequence having at least 99.5% but less than 100% sequence identity and one or more, preferably all, substitutions selected from the group consisting of F159T, N183S, Y238N, T243N, D259N and V270A according to the numbering of SEQ ID NO: 19, more preferably P9T, N130D, T133A, N144K, F159T, N183S, S189T, Y217M, S224A, Y238N, T243N, N270A, and more preferably all of the substitutions selected from the group consisting of P9T, S89A, N130D, T133A, N144K, F159T, N183S, S189T, Y217M, S224A, Y238N, T243N, N252T, D259N, V270A and Q271E.

[0141] In one embodiment, the protease variant has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, or at least 99%, or at least 99.5 ... or 100% sequence identity.

[0142] Preferably, the protease variant has a sequence similar to or 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%, or at least 90% of any of the amino acid sequences set forth in SEQ ID NO:22, SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:31. 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%, at least 99.5% or 100% sequence identity.

[0143] Preferably, the protease variant has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to any of the amino acid sequences set forth in SEQ ID NO:22, SEQ ID NO:21, SEQ ID NO:30, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:31.

[0144] In one embodiment, the protease according to the invention preferably has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO:22.

[0145] In one embodiment, the protease variant has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO:21.

[0146] In one embodiment, the protease variant has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO:30.

[0147] In one embodiment, the protease variant has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO:20.

[0148] In one embodiment, the protease variant has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO:25.

[0149] In one embodiment, the protease variant has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO: 18.

[0150] In one embodiment, the protease according to the invention preferably has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO: 19.

[0151] In one embodiment, the protease variant has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO:31.

[0152] Mannanase In one embodiment, the enzyme composition of the present invention comprises a mannanase. The mannanase and mannanase variants described herein have mannanase activity. The "mannanase" described herein is an enzyme selected from the group of (galacto)mannan-degrading enzymes. The (galacto)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 enzyme is selected from the group of endo-1,4-mannanases, preferably 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. Endo-1,4-mannanases randomly hydrolyze 1,4-linkages within the mannan backbone to release mannooligosaccharides (MOS).

[0153] 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.

[0154] The mannanase of the enzyme composition is - mannanase variants of a parent mannanase, comprising one or more amino acid substitutions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379 according to the numbering of SEQ ID NO: 35, and an amino acid sequence that is at least 60% but less than 100% identical to the sequence according to positions 31 to 490 of SEQ ID NO: 35, and mannanase variants of a parent mannanase, (i) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 39, and an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 24, 123, and 256 according to the numbering of SEQ ID NO: 39; or (ii) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 38, and an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 10, 19, 30, 38, 59, 60, 67, 97, 103, 129, 143, 167, 168, 184, 225, 228, 235, 244, 258, 261, and 298 according to the numbering of SEQ ID NO: 38; or (iii) an amino acid sequence that is at least 60% identical to SEQ ID NO: 40; or (iv) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 40, as well as an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 30, 81, 93, 95, 118, 138, 139, 200, 202, 210, 260, 283, 288, 294, and 295 according to the numbering of SEQ ID NO: 40. Mannanase variants containing is selected from.

[0155] Preferably, the mannanase of the enzyme composition is a mannanase variant of the parent mannanase, and the mannanase variant comprises an amino acid substitution at one or more amino acid positions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379 according to the numbering of SEQ ID NO: 35.

[0156] Preferably, the mannanase of the enzyme composition is a mannanase variant of a parent mannanase, wherein the mannanase variant comprises one or more amino acid substitutions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379 according to the numbering of SEQ ID NO: 35 and an amino acid sequence having at least 60% but less than 100% sequence identity to any of SEQ ID NOs: 35 to 37.

[0157] Preferably, the parent mannanase of the mannanase variant is a mannanase having at least 60% sequence identity to any one of SEQ ID NOs: 35 to 37, and more preferably, the parent mannanase of the mannanase variant is a mannanase set forth in any one of SEQ ID NOs: 35 to 37. Most preferably, the parent mannanase of the mannanase variant is the mannanase set forth in SEQ ID NO: 35.

[0158] Preferably, in this embodiment, the amino acid residues in the parent protease at the above-mentioned positions (ie, X) correspond to the amino acid residues set forth in SEQ ID NO:35 at each position according to the numbering of SEQ ID NO:35.

[0159] Preferably, the mannanase variants are selected from the group consisting of X86N, X89V / L, X96D, X101T / V, X103Y / E / A, X107N, X108G, X109Q / A, X112N, X119Y / H / T, X122S, X124E / C / D, X126E, X127A, X129M / L / F, X231Q / K / L / P / Y, X233V, X235H / R / L / Q / N / Y, The antibody contains one or more amino acid substitutions selected from the group consisting of X244I / V / N, X254W, X255Y / H / R, X264Q / V, X270T, X272I, X273T, X274E / C / Q, X281L, X286E / L / Q / A, X289F / M / H, X290A, X296H / F / Y, X301E / C / T, X309L, X312F / Y, X314P, X317T, and X319D / E.

[0160] Preferably, the mannanase variants are selected from the group consisting of D86N, Q89V / L, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, The compound contains one or more amino acid substitutions selected from the group consisting of D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T, and A319D / E.

[0161] Preferably, the mannanase further comprises an amino acid substitution at one or more amino acid positions selected from the group consisting of 328, 329, 330, 331, 344, 359, 374, 416 and 432 according to the numbering of SEQ ID NO:35.

[0162] Preferably, the mannanase variant further comprises one or more amino acid substitutions selected from the group consisting of X328I / Q / V, X329L / S / V / T, X330P / D / T, X331A / Q, X344Q / F / T, X359R / Y / C / Q, X374G / V / A / R / N / P, X416W and X432P / N / L / R / S / T / G / H / I according to the numbering of SEQ ID NO: 35.

[0163] Preferably, the mannanase variant further comprises one or more amino acid substitutions selected from the group consisting of D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I according to the numbering of SEQ ID NO: 35.

[0164] Preferably, the mannanase variant comprises an amino acid substitution at one or more amino acid positions selected from the group consisting of 31, 89, 96, 119, 264, 289, 312, 348, 349, 352 and 379 according to the numbering of SEQ ID NO:35.

[0165] Preferably, the mannanase variant comprises amino acid substitutions at positions 89 and 96 according to the numbering of SEQ ID NO: 35 and at one or more amino acid positions selected from the group consisting of 31, 119, 264, 289 and 312.

[0166] Preferably, the mannanase variant comprises an amino acid substitution at one or more amino acid positions selected from the group consisting of 89, 96, 119, 264, 289, 312, 348, 349 and 352 according to the numbering of SEQ ID NO:35.

[0167] Preferably, the mannanase variant comprises one or more amino acid substitutions selected from the group consisting of X31V, X89V, X96D, X119Y / H / T, X264Q / V, X289F / M / H, X312F / Y, X348S / R / N / M / G, X349T / S / D / G, X352N / G and X379V according to the numbering of SEQ ID NO: 35.

[0168] Preferably, the mannanase variant comprises at least one of the amino acid substitutions X89V and X96D according to the numbering of SEQ ID NO: 35 and one or more amino acid substitutions selected from X31V, X119Y / H / T, X264Q / V, X289F / M / H and X312F / Y.

[0169] Preferably, the mannanase variant comprises one or more amino acid substitutions selected from the group consisting of A31V, Q89V, N96D, A119Y / H / T, E264Q / V, W289F / M / H, N312F / Y, T348S / R / N / M / G, E349T / S / D / G, S352N / G, D379V according to the numbering of SEQ ID NO: 35.

[0170] Preferably, the mannanase comprises at least one of the amino acid substitutions Q89V and N96D according to the numbering of SEQ ID NO: 35 and one or more amino acid substitutions selected from A31V, A119Y / H / T, E264Q / V, W289F / M / H and N312F / Y.

[0171] More preferably, the mannanase variant comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions X89V, X96D, X119Y / H / T, X264Q / V, X289F / M / H, X312F / Y, X348S / R / N / M / G, X349T / S / D / G and X352N / G according to the numbering of SEQ ID NO: 35.

[0172] More preferably, the mannanase variant comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions Q89V, N96D, A119Y / H / T, E264Q / V, W289F / M / H, N312F / Y, T348S / R / N / M / G, E349T / S / D / G and S352N / G according to the numbering of SEQ ID NO: 35.

[0173] More preferably, the mannanase variant comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions X89V, X96D, X119Y / H / T, X264Q / V, X289F / M / H, X312F / Y, X348S / R / N / M / G, X349T / S / D / G and X352N / G according to the numbering of SEQ ID NO: 35.

[0174] More preferably, the mannanase variant comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions Q89V, N96D, A119Y / H / T, E264Q / V, W289F / M / H, N312F / Y, T348S / R / N / M / G, E349T / S / D / G and S352N / G according to the numbering of SEQ ID NO: 35.

[0175] More preferably, the mannanase variant comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions X89V, X96D, X119H, X264Q, X289M, X312Y, X348N, X349G and X352G according to the numbering of SEQ ID NO: 35.

[0176] More preferably, the mannanase variant comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions Q89V, N96D, A119H, E264Q, W289M, N312Y, T348N, E349G and S352G according to the numbering of SEQ ID NO: 35.

[0177] More preferably, the mannanase variant comprises the amino acid substitutions X89V, X96D, X119H, X264Q, X289M and X312Y according to the numbering of SEQ ID NO: 35.

[0178] More preferably, the mannanase variant comprises the amino acid substitutions Q89V, N96D, A119H, E264Q, W289M and N312Y according to the numbering of SEQ ID NO: 35.

[0179] Most preferably, the mannanase variant comprises the amino acid substitutions X89V, X96D, X119H, X264Q, X289M, X312Y, X348N, X349G and X352G according to the numbering of SEQ ID NO: 35.

[0180] Most preferably, the mannanase variant comprises the amino acid substitutions Q89V, N96D, A119H, E264Q, W289M, N312Y, T348N, E349G and S352G according to the numbering of SEQ ID NO: 35.

[0181] Preferably, the mannanase variant further comprises an amino acid substitution at one or more amino acid positions selected from the group consisting of 341, 346, 348, 349, 352, 356 and 379 according to the numbering of SEQ ID NO:35.

[0182] Preferably, the mannanase variant further comprises an amino acid substitution at one or more amino acid positions selected from the group consisting of X341F, X346T, X348S / R / N / M / G, X349T / S / G / D, X352N / G, X356Y / V / T / Q / H / C and X379V according to the numbering of SEQ ID NO: 35.

[0183] Preferably, the mannanase variant further comprises an amino acid substitution at one or more amino acid positions selected from the group consisting of N341F, F346T, T348S / R / N / M / G, E349T / S / G / D, S352N / G, G356Y / V / T / Q / H / C and D379V according to the numbering of SEQ ID NO: 35.

[0184] Preferably, the mannanase variant has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, or at least a small fraction of any of the amino acid sequences shown in SEQ ID NOs: 35 to 37 or a sequence according to positions 31 to 490 of SEQ ID NO: 35, preferably a sequence according to positions 31 to 490 of SEQ ID NO: 35. The amino acid sequence may be 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%, at least 99.5%, but less than 100% identical.

[0185] More preferably, the mannanase variant has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least It comprises an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical.

[0186] Most preferably, the mannanase variant comprises an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to the sequence according to positions 31 to 490 of SEQ ID NO: 35.

[0187] Preferably, the mannanase of the enzyme composition is a mannanase variant of a parent mannanase, and the mannanase variant is selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 35 Amino acid substitutions at one or more amino acid positions selected from the group consisting of 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379 and a sequence corresponding to any of the amino acid sequences shown in SEQ ID NOs: 35 to 37 or positions 31 to 490 of SEQ ID NO: 35, preferably positions 31 to 490 of SEQ ID NO: 35, which differ from the sequence corresponding to positions 31 to 490 of SEQ ID NO: 35 by at least 65%, at least 70%, at least 75%, 5%, 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%, at least 99.5%, but less than 100% identical.

[0188] Preferably, the mannanase of the enzyme composition is a mannanase variant of the parent mannanase, wherein the mannanase variant has an amino acid substitution at one or more amino acid positions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379 according to the numbering of SEQ ID NO: 35, and has at least 85%, ... Also includes an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical.

[0189] Preferably, the mannanase variants are selected from the group consisting of X86N, X89V / L, X96D, X101T / V, X103Y / E / A, X107N, X108G, X109Q / A, X112N, X119Y / H / T, X122S, X124E / C / D, X126E, X127A, X129M / L / F, X231Q / K / L / P / Y, X233V, X235H / R / L / Q / N / Y, X244I / V / N, X254W, X255Y / H / R, X256I / V / N, X257I / V / N, X258I / V / N, X259I / V / N, X260I / V / N, X261I / V / N, X262I / V / N, X263I / V / N, X264I / V / N, X265I / V / N, X266I / V / N, X267I / V / N, X268I / V / N, X269I / V / N, X270I / V / N, X271I / V / N, X272I / V / N, X273I / V / N, X274I / V / N, X275I / V / N, X276I / V / N, X277I / V / N, X278I / V / N, X279I / V / N, X280I / V / N, X281I / V / N, X282I / V / N, X283I / V / N, X284I / V / N, X285I / V / N, X286I / V / N, X287I / V / N, X288I one or more amino acid substitutions selected from the group consisting of 264Q / V, X270T, X272I, X273T, X274E / C / Q, X281L, X286E / L / Q / A, X289F / M / H, X290A, X296H / F / Y, X301E / C / T, X309L, X312F / Y, X314P, X317T and X319D / E, any of the amino acid sequences shown in SEQ ID NOs: 35 to 37 or a sequence according to positions 31 to 490 of SEQ ID NO: 35, preferably The present invention also includes 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%, 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%, at least 99.5%, but less than 100% identical to a sequence from positions 31 to 490.

[0190] Preferably, the mannanase variants are selected from the group consisting of X86N, X89V / L, X96D, X101T / V, X103Y / E / A, X107N, X108G, X109Q / A, X112N, X119Y / H / T, X122S, X124E / C / D, X126E, X127A, X129M / L / F, X231Q / K / L / P / Y, X233V, X235H / R / L / Q / N according to the numbering of SEQ ID NO: 35. one or more amino acids selected from the group consisting of X244I / V / N, X254W, X255Y / H / R, X264Q / V, X270T, X272I, X273T, X274E / C / Q, X281L, X286E / L / Q / A, X289F / M / H, X290A, X296H / F / Y, X301E / C / T, X309L, X312F / Y, X314P, X317T, and X319D / E substitutions and an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to a sequence according to positions 31 to 490 of SEQ ID NO: 35.

[0191] Preferably, the mannanase variants are selected from the group consisting of X86N, X89V / L, X96D, X101T / V, X103Y / E / A, X107N, X108G, X109Q / A, X112N, X119Y / H / T, X122S, X124E / C / D, X126E, X127A, X129M / L / F, X231Q / K / L / P / Y, X233V, X235H / R / L / Q / N according to the numbering of SEQ ID NO: 35. one or more amino acids selected from the group consisting of X244I / V / N, X254W, X255Y / H / R, X264Q / V, X270T, X272I, X273T, X274E / C / Q, X281L, X286E / L / Q / A, X289F / M / H, X290A, X296H / F / Y, X301E / C / T, X309L, X312F / Y, X314P, X317T, and X319D / E substitutions and an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to a sequence according to positions 31 to 490 of SEQ ID NO: 35.

[0192] Preferably, the mannanase variant has one or more amino acid substitutions selected from the group consisting of X31V, X89V, X96D, X119Y / H / T, X264Q / V, X289F / M / H, X312F / Y, X348S / R / N / M / G, X349T / S / D / G, X352N / G and X379V according to the numbering of SEQ ID NO: 35, and has a similar structure to the sequence according to positions 31 to 490 of SEQ ID NO: 35 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 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119 ... 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%, at least 99.5%, but less than 100% identical.

[0193] More preferably, the mannanase variant has at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions X89V, X96D, X119Y / H / T, X264Q / V, X289F / M / H, X312F / Y, X348S / R / N / M / G, X349T / S / D / G and X352N / G according to the numbering of SEQ ID NO: 35 and a sequence according to positions 31 to 490 of SEQ ID NO: 35 that is at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, 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 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119 ... 0.5%, 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%, at least 99.5%, but less than 100% identical.

[0194] More preferably, the mannanase variant has at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions X89V, X96D, X119H, X264Q, X289M, X312Y, X348N, X349G and X352G according to the numbering of SEQ ID NO: 35 and a sequence according to positions 31 to 490 of SEQ ID NO: 35 that is 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 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 1121%, at least 1122%, at least 1131%, at least 1132%, at least 1141%, at least 1142%, at least 1151%, at least 1152%, at least 1161%, at least 1162%, at least 1171%, at least 1172, at least 1181%, at least 1182, at least 1191, at least 1192, at least 1193, at least 1194, at least 1195, at least 1196, at least 1197, at least 11 The amino acid sequence may be 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%, at least 99.5%, but less than 100% identical.

[0195] More preferably, the mannanase variant has amino acid substitutions X89V, X96D, X119H, X264Q, X289M and X312Y according to the numbering of SEQ ID NO: 35 and an amino acid sequence of positions 31 to 490 of SEQ ID NO: 35 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 and / or a sequence of amino acids that is 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.

[0196] Most preferably, the mannanase variant has amino acid substitutions X89V, X96D, X119H, X264Q, X289M, X312Y, X348N, X349G, and X352G according to the numbering of SEQ ID NO: 35 and the amino acid sequence of positions 31 to 490 of SEQ ID NO: 35 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 Any of the above amino acid sequences may be 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%, at least 99.5%, but less than 100% identical.

[0197] Most preferred is a mannanase variant comprising or consisting of an amino acid sequence according to positions 31 to 490 of SEQ ID NO: 35, having amino acid substitutions Q89V, N96D, A119H, E264Q, W289M, N312Y, T348N, E349G and S352G according to the numbering of SEQ ID NO: 35.

[0198] In one embodiment, the mannanase variant of the enzyme composition is (i) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 39, and an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 24, 123, and 256 according to the numbering of SEQ ID NO: 39; or (ii) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 38, and an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 10, 19, 30, 38, 59, 60, 67, 97, 103, 129, 143, 167, 168, 184, 225, 228, 235, 244, 258, 261, and 298 according to the numbering of SEQ ID NO: 38; or (iii) an amino acid sequence that is at least 60% identical to SEQ ID NO: 40; or (iv) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 40, as well as an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 30, 81, 93, 95, 118, 138, 139, 200, 202, 210, 260, 283, 288, 294, and 295 according to the numbering of SEQ ID NO: 40. Includes:

[0199] In one embodiment, the mannanase variant has an amino acid sequence similar to SEQ ID NO:39 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%, at least 92%, 39, comprising an amino acid sequence that is 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 having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 24, 123 and 256 according to the numbering of SEQ ID NO:39.

[0200] In one embodiment, the mannanase variant has an amino acid sequence similar to SEQ ID NO:39 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%, at least 92%. , at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100%, identical to the amino acid sequence of SEQ ID NO: 39, and comprising one or more amino acid substitutions selected from the group consisting of X24I, X123I and X256G, preferably a combination of X24I and X256G, according to the numbering of SEQ ID NO: 39.

[0201] In one embodiment, the mannanase variant has an amino acid sequence similar to SEQ ID NO:39 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%, at least 92%. , at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100%, identical to the amino acid sequence of SEQ ID NO: 39, and comprising one or more amino acid substitutions selected from the group consisting of V24I, V123I and S256G, preferably a combination of V24I and S256G, according to the numbering of SEQ ID NO: 39.

[0202] In one embodiment, the mannanase variant has an amino acid sequence similar to SEQ ID NO:38 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%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, or at least 94%. 4%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100% identical to the amino acid sequence of SEQ ID NO: 38, and comprising an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 10, 19, 30, 38, 59, 60, 67, 97, 103, 129, 143, 167, 168, 184, 225, 228, 235, 244, 258 and 261 according to the numbering of SEQ ID NO: 38, preferably in combination with a deletion at position 298*.

[0203] In one embodiment, the mannanase variant has an amino acid sequence similar to SEQ ID NO:38 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%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99. 4.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100%, identical to the amino acid sequence of SEQ ID NO: 38, and comprising one or more amino acid substitutions selected from the group consisting of X10T, X19P, X30S, X38T, X59S, X60L, X67N, X97N, X103V, X129Y, X143Q, X167F, X168S, X184Q, X225P / G, X228T, X235Y, X244K, X258S and X261N according to the numbering of SEQ ID NO: 38, preferably in combination with the deletion X298*.

[0204] In one embodiment, the mannanase variant has an amino acid sequence similar to SEQ ID NO:38 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%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99. 4.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100%, identical to said sequence and comprising one or more amino acid substitutions selected from the group consisting of N10T, E19P, T30S, E38T, V59S, Q60L, D67N, D97N, I103V, M129Y, K143Q, Y167F, P168S, L184Q, C225P / G, V228T, L235Y, L244K, D258S and R261N according to the numbering of SEQ ID NO: 38, preferably in combination with the deletion Q298*.

[0205] In one embodiment, the mannanase variant has an amino acid sequence identical to SEQ ID NO:40 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%, or at least and / or 100% sequence identity.

[0206] In one embodiment, the mannanase variant has an amino acid sequence similar to SEQ ID NO:40 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%, 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 comprising an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 30, 81, 93, 95, 118, 138, 139, 200, 202, 210, 260, 283, 288, 294 and 295 according to the numbering of SEQ ID NO:40, preferably an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 260, 288, 294 and 295.

[0207] In one embodiment, the mannanase variant has an amino acid sequence similar to SEQ ID NO:40 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%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%. 5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100%, identical to the amino acid sequence of SEQ ID NO: 40, and which contains one or more amino acid substitutions selected from the group consisting of X30T, X81P, X93Q, X95D, X118K, X138P, X139R, X200T, X202R, X210G, X260F, X283H, X288I, X294P and X295V, preferably one or more amino acid substitutions selected from the group consisting of X260F, X288I, X294P and X295V according to the numbering of SEQ ID NO: 40.

[0208] In one embodiment, the mannanase variant has an amino acid sequence similar to SEQ ID NO:40 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%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%. 5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100% identical to the amino acid sequence of SEQ ID NO: 40, and comprising one or more amino acid substitutions selected from the group consisting of A30T, L81P, Y93Q, S95D, D118K, A138P, D139R, N200T, S202R, R210G, W260F, N283H, L288I, L294P and S295V, preferably one or more amino acid substitutions selected from the group consisting of W260F, L288I, L294P and S295V according to the numbering of SEQ ID NO: 40.

[0209] A mannanase having the above substitutions is set forth, for example, in SEQ ID NO: 41. Thus, in one embodiment, the mannanase variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 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 1195%, at least 1196%, at least 1197%, at least 1198%, at least 1199%, at least 1200%, at least 1211%, at least 1212%, at least 1213%, at least 1214%, at least 1215%, at least 1216%, at least 1218%, at least 1219%, at least 1221%, at least 1222, at least 1223, at least 1224, at least 1225, at least 1226, at least 1228, at least 1229, at least 1231, at least 1232, at least 1233, at least 1234 The amino acid sequence may be 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%, at least 99.5% or 100% identical.

[0210] cellulase In one embodiment, the enzyme composition of the present invention comprises a cellulase. The cellulases and cellulase variants described herein have cellulase activity. A "cellulase" is an enzyme capable of hydrolyzing cellulose. The cellulase may 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 the EC class 3.2.1.4 may be referred to as endoglucanases, endo-1,4-ss-D-glucan 4-glucanohydrolases, endo-1,4-beta-glucanases, carboxymethylcellulases, and beta-1,4-glucanases.

[0211] Endoglucanases can be classified by amino acid sequence similarity (Henrissat, B. UniProt accessed October 26, 2011) into family 5, which includes more than 20 EC 3.2.1.4 endoglucanases. T.-M Enveri, “Microbial Cellulases” in WM Fogarty, Microbial Enzymes and Biotechnology, Applied Science Publishers, p. 183-224 (1983), Methods in Enzymology, (1988) 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.

[0212] The cellulase of the enzyme composition is - a cellulase variant comprising an amino acid sequence that is at least 60% identical to SEQ ID NO: 42, and - a cellulase variant comprising an amino acid sequence that is at least 60% identical to SEQ ID NO: 46, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 44 or SEQ ID NO: 47 is selected from.

[0213] Preferably, the cellulase variant of the enzyme composition is a cellulase comprising an amino acid sequence that is at least 60% identical to SEQ ID NO:42.

[0214] Preferably, the cellulase variant has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 42.

[0215] Preferably, the cellulase variant has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to the amino acid sequence of SEQ ID NO:42.

[0216] Preferably, the cellulase variant has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 42.

[0217] Most preferably, the cellulase variant comprises or consists of SEQ ID NO:42.

[0218] In one embodiment, the cellulase of the enzyme composition has an amino acid sequence that is at least 60% identical to SEQ ID NO:46, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:44, or SEQ ID NO:47.

[0219] Preferably, the cellulase variant has a sequence similar to any of the amino acid sequences set forth in SEQ ID NOs: 43 to 47, 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%, or 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%, at least 99.5% or 100% sequence identity.

[0220] Preferably, the cellulase variant has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 43 to 47.

[0221] Preferably, the cellulase variant has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 43 to 47.

[0222] In one embodiment, the cellulase according to the invention preferably has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO:46.

[0223] In one embodiment, the cellulase variant has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO:43.

[0224] In one embodiment, the cellulase variant has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO:45.

[0225] In one embodiment, the cellulase variant has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to SEQ ID NO:44.

[0226] In one embodiment, the cellulase variant has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity.

[0227] Preferred herein is a composition comprising two or more enzymes selected from the group consisting of proteases, mannanases and cellulases, a. Proteases are I. Protease variants of a parent protease, (i) an amino acid sequence comprising at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16; and (ii) an amino acid sequence that is at least 80% but less than 100% identical to any of SEQ ID NO: 16 or a protease variant comprising II. Protease variants of a parent protease, (i) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 3, 4, 9, 19, 22, 43, 62, 63, 76, 87, 99, 101, 103, 104, 116, 118, 128, 129, 130, 156, 160, 167, 170, 182, 185, 188, 194, 205, 213, 217, 218, 232, 245, 256, 259, 262, and 271 according to the numbering of SEQ ID NO: 17, and an amino acid sequence having at least 60% but less than 100% sequence identity to SEQ ID NO: 22; or (ii) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 24, 53, 76, 78, 101, 128, and 217 according to the numbering of SEQ ID NO: 17, and having at least 60% but less than 100% sequence identity to SEQ ID NO: 17; or (iii) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 9, 89, 130, 133, 144, 159, 183, 189, 217, 224, 238, 243, 252, 259, 270, and 271 according to the numbering of SEQ ID NO: 19, and having at least 60% but less than 100% sequence identity to SEQ ID NO: 19; or (iv) an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 22, SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 31 Protease variants including is selected from B. Mannanase I. Mannanase variants of a parent mannanase, (i) one or more amino acid substitutions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379 according to the numbering of SEQ ID NO: 35, and (ii) an amino acid sequence that is at least 80% but less than 100% identical to the sequence of positions 31 to 490 of SEQ ID NO: 35 or a mannanase variant comprising II. Mannanase variants of a parent mannanase, (i) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 39, and an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 24, 123, and 256 according to the numbering of SEQ ID NO: 39; or (ii) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 38, and an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 10, 19, 30, 38, 59, 60, 67, 97, 103, 129, 143, 167, 168, 184, 225, 228, 235, 244, 258, 261, and 298 according to the numbering of SEQ ID NO: 38; or (iii) an amino acid sequence that is at least 60% identical to SEQ ID NO: 40; or (iv) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 40, as well as an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 30, 81, 93, 95, 118, 138, 139, 200, 202, 210, 260, 283, 288, 294, and 295 according to the numbering of SEQ ID NO: 40. Mannanase variants containing is selected from C. Cellulase I. A cellulase variant comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 42, or II. Cellulase variants comprising an amino acid sequence at least 60% identical to SEQ ID NO: 46, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 44, or SEQ ID NO: 47 is selected from At least one enzyme aa. A protease variant of a parent protease, (i) an amino acid sequence comprising at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16; and (ii) an amino acid sequence that is at least 80% but less than 100% identical to any of SEQ ID NO: 16 a protease variant comprising bb. A mannanase variant of a parent mannanase, (i) one or more amino acid substitutions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379 according to the numbering of SEQ ID NO: 35, and (ii) an amino acid sequence that is at least 80% but less than 100% identical to the sequence of positions 31 to 490 of SEQ ID NO: 35 mannanase variants comprising: cc. A cellulase variant comprising an amino acid sequence at least 80% identical to SEQ ID NO: 42 The composition is selected from the group consisting of:

[0228] Preferably, the protease variant has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5% but less than 100% sequence identity with SEQ ID NO: 16 and comprises the amino acid substitution X101D or X101E, preferably X101E, preferably R101E, according to the numbering of SEQ ID NO: 17 compared to SEQ ID NO: 16.

[0229] In certain embodiments, the protease variant comprises the amino acid substitution X101E according to the numbering of SEQ ID NO: 17, the amino acid substitutions X3T, X4I and X205I or the amino acid substitutions X156D and X262E, and an amino acid sequence that is at least 90% but less than 100% identical to SEQ ID NO: 16.

[0230] Alternatively, the protease variant may have a sequence similar to SEQ ID NO:22 by 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 the sequences X3T, X4I, X9D / E / R, X19L, X22R / A, X43R, X62D, X63D / E, X76D, X87N, X99A / D, X101M / R / E, X103A, X104I, X116L, X118V, X128L, X129Q, X130A, X156D / E, X160D / S, X167A, X170S, X182E / D, X185E / D, X188D, X189A, X190B, X191C, X192D, X193D, X194D, X195D, X196D, X197D, X198D, X199A, X200B, X201B, X202B, X203B, X204B, X205B, X206B, X207B, X210B, X211C, X212D, X213D, X214D, X215D, X216E, X217F, X218F, X219G, X220G, X221H, X222H, X223H, X224H, X225H, X226H, X227H, X228H, X230G, X231H, X232H, X233H, X234H, X235H, X236H, X237H, X238H, X240H, X241H, X242H, X243H, X244H, X245H, X24 and X271F, and preferably the protease variant further comprises an insertion of D and E adjacent to the C-terminus of position 99 according to the numbering of SEQ ID NO: 17, and preferably the protease variant has at least 90% similarity to any of SEQ ID NO: 24, at least 100% similarity to any of SEQ ID NO: 25. The amino acid sequence may be 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%, at least 99.5% or 100% identical.

[0231] Preferably, the mannanase variant comprises the amino acid substitutions X89V, X96D, X119H, X264Q, X289M, X312Y, X348N, X349G and X352G according to the numbering of SEQ ID NO: 35 and an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to the sequence of positions 31 to 490 of SEQ ID NO: 35.

[0232] Alternatively, the mannanase variants are at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, 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%, at least 99.5%, but less than 100% identical to SEQ ID NO:40 and include X30T, X118K, X93Q, X95D, X81P, X138P, X139R, X200T, X202R, X210G, X260F, X283H, X288I, X294P, and X295V according to the numbering of SEQ ID NO:40. and preferably the mannanase variant comprises an amino acid sequence that comprises one or more amino acid substitutions selected from the group consisting of X260F, X288I, X294P and X295V, and that is 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%, at least 99.5% or 100% identical to SEQ ID NO: 41.

[0233] Preferably, the cellulase variant has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 42.

[0234] Preferred herein is a composition comprising two or three enzymes selected from the group consisting of proteases, mannanases and cellulases, a. Proteases are (i) the amino acid substitution X101E according to the numbering of SEQ ID NO: 17, and (ii) an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to SEQ ID NO: 16 or a protease variant comprising the protease is a protease variant comprising the amino acid substitution X101E according to the numbering of SEQ ID NO: 17, and the amino acid substitutions X3T, X4I and X205I or the amino acid substitutions X156D and X262E, and an amino acid sequence which is at least 90% but less than 100% identical to SEQ ID NO: 16; or The protease may have a sequence similar to SEQ ID NO:22 by 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 less. X3T, X4I, X9D / E / R, X19L, X22R / A, X43R, X62D, X63D / E, X76D, X87N, X99A / D, X101M / R / E, X103A, X104I, X116L, X118V, X128L, X129Q, X130A, X156D / E, X160D / S, X167A, X170S, X182E / D, X185E / D, X188D, X194P, and X205I, X213A, X217E / D / Q, X218D, X232V, X245R, X256D / E, X259D / E, X262D / E and X271F, preferably the protease variant further comprises an insertion of D and E immediately adjacent to the C-terminus of position 99 according to the numbering of SEQ ID NO: 17, and preferably the protease variant has at least 90% similarity to any of SEQ ID NO: 24, at least and comprising an amino acid sequence that is 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%, at least 99.5% or 100% identical to the sequence of the amino acid sequence of the present invention. B. Mannanase (i) at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions X89V, X96D, X119H, X264Q, X289M, X312Y, X348N, X349G and X352G according to the numbering of SEQ ID NO: 35, and (ii) an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to the sequence of positions 31 to 490 of SEQ ID NO: 35 or a mannanase variant comprising The mannanase is at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, 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%, at least 99.5%, but less than 100% identical to SEQ ID NO:40 and is selected from the group consisting of X30T, X118K, X93Q, X95D, X81P, X138P, X139R, X200T, X202R, X210G, X260F, X283H, X288I, X294P, and X295V according to the numbering of SEQ ID NO:40. a mannanase variant comprising an amino acid sequence comprising one or more amino acid substitutions, preferably one or more amino acid substitutions selected from the group consisting of X260F, X288I, X294P and X295V, preferably the mannanase variant comprises an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO: 41; c. The composition is a cellulase variant comprising an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO:42.

[0235] More preferred herein is a composition comprising two or three enzymes selected from the group consisting of proteases, mannanases and cellulases, a. Proteases are (i) the amino acid substitution X101E according to the numbering of SEQ ID NO: 17, and (ii) an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to SEQ ID NO: 16 is a protease variant comprising B. Mannanase (i) at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions X89V, X96D, X119H, X264Q, X289M, X312Y, X348N, X349G and X352G according to the numbering of SEQ ID NO: 35, and (ii) an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to the sequence of positions 31 to 490 of SEQ ID NO: 35 and c. The composition is a cellulase variant comprising an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO:42.

[0236] Therefore, more preferred herein is a composition comprising a protease and a mannanase, a. Proteases are (i) the amino acid substitution X101E according to the numbering of SEQ ID NO: 17, and (ii) an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to SEQ ID NO: 16 and B. Mannanase (i) at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions X89V, X96D, X119H, X264Q, X289M, X312Y, X348N, X349G and X352G according to the numbering of SEQ ID NO: 35, and (ii) an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to the sequence of positions 31 to 490 of SEQ ID NO: 35 The composition is a mannanase variant comprising:

[0237] Therefore, more preferred herein is a composition comprising a protease and a cellulase, a. Proteases are (i) the amino acid substitution X101E according to the numbering of SEQ ID NO: 17, and (ii) an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to SEQ ID NO: 16 and b. The composition is a cellulase variant comprising an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO:42.

[0238] Therefore, more preferred herein is a composition comprising a mannanase and a cellulase, a. Mannanase is (i) at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions X89V, X96D, X119H, X264Q, X289M, X312Y, X348N, X349G and X352G according to the numbering of SEQ ID NO: 35, and (ii) an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to the sequence of positions 31 to 490 of SEQ ID NO: 35 and b. The composition is a cellulase variant comprising an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO:42.

[0239] More preferred herein is a composition comprising two or three enzymes selected from the group consisting of proteases, mannanases and cellulases, a. the protease is a protease variant comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16, with the amino acid substitution R101E according to the numbering of SEQ ID NO: 17, i.e., the only amino acid exchange compared to SEQ ID NO: 16; b. the mannanase is a mannanase variant comprising or consisting of the amino acid sequence of positions 31 to 490 of SEQ ID NO: 35, with amino acid substitutions Q89V, N96D, A119H, E264Q, W289M, N312Y, T348N, E349G, and S352G according to the numbering of SEQ ID NO: 35; c. The cellulase is a composition that is a cellulase variant comprising or consisting of an amino acid sequence that is 100% identical to SEQ ID NO:42.

[0240] Therefore, more preferred herein is a composition comprising a protease and a mannanase, a. the protease is a protease variant comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16, with the amino acid substitution R101E according to the numbering of SEQ ID NO: 17, i.e., the only amino acid exchange compared to SEQ ID NO: 16; b. The mannanase is a composition that is a mannanase variant comprising or consisting of the amino acid sequence of positions 31 to 490 of SEQ ID NO: 35, having amino acid substitutions Q89V, N96D, A119H, E264Q, W289M, N312Y, T348N, E349G and S352G according to the numbering of SEQ ID NO: 35.

[0241] Therefore, more preferred herein is a composition comprising a protease and a cellulase, a. the protease is a protease variant comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16, with the amino acid substitution R101E according to the numbering of SEQ ID NO: 17, i.e., the only amino acid exchange compared to SEQ ID NO: 16; b. The cellulase is a composition that is a cellulase variant comprising or consisting of an amino acid sequence that is 100% identical to SEQ ID NO:42.

[0242] Therefore, more preferred herein is a composition comprising a mannanase and a cellulase, a. the mannanase is a mannanase variant comprising or consisting of the amino acid sequence of positions 31 to 490 of SEQ ID NO: 35, with amino acid substitutions Q89V, N96D, A119H, E264Q, W289M, N312Y, T348N, E349G, and S352G according to the numbering of SEQ ID NO: 35; b. The cellulase is a composition that is a cellulase variant comprising or consisting of an amino acid sequence that is 100% identical to SEQ ID NO:42.

[0243] Preferably, the composition comprises a protease and a mannanase as described herein, a protease and a cellulase as described herein, or a protease, a mannanase, and a cellulase as described herein.

[0244] Even more preferably, the composition comprises a protease, a mannanase and a cellulase; a. Proteases are (i) the amino acid substitution X101E according to the numbering of SEQ ID NO: 17, and (ii) an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to SEQ ID NO: 16 is a protease variant comprising B. Mannanase (i) at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions X89V, X96D, X119H, X264Q, X289M, X312Y, X348N, X349G and X352G according to the numbering of SEQ ID NO: 35, and (ii) an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to the sequence of positions 31 to 490 of SEQ ID NO: 35 and c. The cellulase is a cellulase variant comprising an amino acid sequence that is 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%, at least 99.5% or 100% identical to SEQ ID NO:42.

[0245] Most preferably, the composition comprises a protease, a mannanase and a cellulase; a. the protease is a protease variant comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 16, with the amino acid substitution R101E according to the numbering of SEQ ID NO: 17, i.e., the only amino acid exchange compared to SEQ ID NO: 16; b. the mannanase is a mannanase variant comprising or consisting of the amino acid sequence of positions 31 to 490 of SEQ ID NO: 35, with amino acid substitutions Q89V, N96D, A119H, E264Q, W289M, N312Y, T348N, E349G, and S352G according to the numbering of SEQ ID NO: 35; c. The cellulase is a cellulase variant comprising or consisting of an amino acid sequence that is 100% identical to SEQ ID NO:42.

[0246] More enzymes In another embodiment, the composition comprising the enzyme composition described herein further comprises one or more additional enzymes different from the enzymes of the enzyme composition described herein.Preferably, the additional enzymes are protease, amylase, lipase, cellulase, mannanase, hemicellulase, phospholipase, esterase, pectinase, lactase, peroxidase, xylanase, cutinase, pectate lyase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase, beta-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, nuclease, DNase, phosphodiesterase, phytase, The additional enzyme is selected from the group consisting of carbohydrase, galactanase, xanthanase, xyloglucanase, oxidoreductase, perhydrolase, aminopeptidase, asparaginase, carbohydrase, carboxypeptidase, catalase, chitinase, cyclodextrin glycosyltransferase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, ribonuclease, transglutaminase, and dispersin, and combinations of at least two of the aforementioned types. More preferably, the additional enzyme is selected from the group consisting of amylase, lipase, hemicellulase, xylanase, DNase, dispersin, pectinase, oxidoreductase, cutinase, phosphodiesterase, licheninase, xanthan lyase, and hexosaminidase, and combinations of at least two of the aforementioned types. Most preferably, the further enzyme is an amylase and / or a lipase, preferably an amylase.

[0247] amylase The enzyme composition of the present invention may comprise an amylase. In another embodiment, the enzyme composition does not comprise an amylase.

[0248] The amylases and amylase variants described herein have amylase activity (also referred to herein as "amylolytic activity"). "Amylolytic activity" or "amylase activity" describes the ability to hydrolyze the glucosidic bonds of polysaccharides. Amylase activity can be determined by assays that measure amylase activity known to those skilled in the art. Examples of assays that measure 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 yield a soluble blue fragment. The absorbance of the resulting blue solution, measured at 620 nm by a spectrophotometer, 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.

[0249] 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 a free PNP molecule. This molecule has a yellow color and can therefore 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 (catalog number 10880078103). The slope of the time-dependent absorption curve is directly proportional to the specific activity (activity per mg of enzyme) of the amylase under a given set of conditions.

[0250] Amylase is - an amylase variant of a parent amylase, (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO:2; (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 4, 116, 176, 181, 186, 195, 206, 225, 251, 320, 405, and 482 according to the numbering of SEQ ID NO:2; (iii) an amylase variant comprising at least 60%, preferably at least 91%, but less than 100% sequence identity with any of SEQ ID NOs: 1 to 15, preferably SEQ ID NO: 1, wherein the parent amylase of the amylase variant is the amylase of SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1; and - an amylase variant of a parent amylase, (i) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 183, 243, 266, and 475 according to the numbering of SEQ ID NO: 9, and an amino acid sequence that has at least 60% but less than 100% sequence identity to SEQ ID NO: 9; or (ii) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 38, 126, 129, 153, 180, 187, 199, 203, 241, 362, 377, 458, 459, 460, 476, and 477 according to the numbering of SEQ ID NO: 7, and an amino acid sequence that has at least 60% but less than 100% sequence identity to SEQ ID NO: 7; or (iii) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 1, 7, 51, 52, 54, 109, 140, 195, 206, 243, 260, 280, 284, 304, 391, 476, and 477 according to the numbering of SEQ ID NO: 3, and an amino acid sequence that has at least 60% but less than 100% sequence identity to SEQ ID NO: 3; or (iv) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 9, 118, 149, 182, 186, 195, 202, 246, 257, 295, 299, 320, 323, 339, 345, 458, and 471 according to the numbering of SEQ ID NO: 13, and an amino acid sequence which has at least 60% but less than 100% sequence identity to SEQ ID NO: 13; or (v) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 1, 54, 56, 72, 109, 113, 116, 149, 167, 172, 174, 182, 195, 202, 206, 246, 257, 295, 299, 320, 339, 345, 391, 458, 471, 473, and 476 according to the numbering of SEQ ID NO: 15, and an amino acid sequence which has at least 60% but less than 100% sequence identity to SEQ ID NO: 15; or (vi) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 9, 118, 149, 182, 186, 195, 202, 246, 257, 295, 299, 320, 323, 339, 345, 405, 421, 422, 428, 458, and 471 according to the numbering of SEQ ID NO:4, and an amino acid sequence which has at least 60% but less than 100% sequence identity to SEQ ID NO:4; or (vii) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 1, 54, 56, 72, 109, 113, 116, 120, 134, 140, 167, 169, 172, 173, 174, 182, 183, 184, 195, 202, 206, 246, 255, 257, 260, 265, 295, 299, 304, 320, 339, 345, 391, 395, 410, 422, 429, 444, 451, 458, 473, 474, and 476 according to the numbering of SEQ ID NO:5, and an amino acid sequence that has at least 60% but less than 100% sequence identity to SEQ ID NO:5. Amylase variants including may be selected from:

[0251] An amylase variant is an amylase that does not occur in nature. Preferably, the amylase variant is a purified, isolated, synthetic, and / or recombinant amylase variant. Preferably, the amylase variant is a purified and recombinant amylase variant.

[0252] Preferably, the parent amylase of the amylase variant is an amylase having at least 60% sequence identity with any of SEQ ID NOs: 1 to 15, and more preferably, the parent amylase of the amylase variant is any of SEQ ID NOs: 1 to 15. Most preferably, the parent amylase of the amylase variant is the amylase of SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1.

[0253] Preferably, the amylase variants further comprise 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 variants of the invention having one or more amino acid substitutions described herein comprise 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: 2). Preferably, the amylase variants of the invention having one or more amino acid substitutions described herein comprise 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: 2.

[0254] Preferably, the amylase variant comprises a deletion at two amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion 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: 2.

[0255] The present invention is directed to amylase variants of a parent amylase that have amylase activity, wherein the variants comprise amino acid modifications, preferably insertions, deletions, substitutions or a combination thereof, most preferably substitutions, compared to the parent amylase at two or more positions corresponding to positions selected from the group consisting of 4, 25, 116, 176, 181, 186, 195, 206, 225, 251, 320, 405 and 482 according to the numbering of the amino acid sequence set forth in SEQ ID NO:2.

[0256] Alternatively, the numbering of SEQ ID NO: 1 can be used as a reference, i.e., amino acid positions 4, 25, 116, 176, 181, 186, 195, 206, 225, 251, 320, 405, and 482 (according to the numbering of SEQ ID NO: 2) can be described according to the numbering of SEQ ID NO: 1. In this alternative method of describing the amino acid positions of the present invention, amino acid positions 4, 25, 176, 186, 251, 405, and 482 according to the numbering of SEQ ID NO: 2 correspond to amino acid positions 4, 25, 116, 176, 181, 184, 193, 204, 223, 249, 318, 403, and 480 according to the numbering of SEQ ID NO: 1.

[0257] Preferably, the parent amylase of the amylase variant is an amylase having at least 60% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, or any of SEQ ID NOs: 3 to 15, and more preferably, the parent amylase of the amylase variant is an amylase of SEQ ID NO: 1, SEQ ID NO: 2, or any of SEQ ID NOs: 3 to 15. Most preferably, the parent amylase of the amylase variant is the amylase of SEQ ID NO: 1.

[0258] Preferably, the present invention is directed to amylase variants comprising one or more amino acid substitutions selected from the group consisting of X4Q, X25H, X116K, X176K, X186E, X195F, X206Y, X225A, X251E, X320K, X405M and X482W according to the numbering of the amino acid sequence set forth in SEQ ID NO:2.

[0259] Preferably, the present invention is directed to amylase variants comprising one or more amino acid substitutions selected from the group consisting of X4Q, X25H, X116K, X176K, X186E, X195F, X206Y, X225A, X251E, X320K, X405M and X482W according to the numbering of the amino acid sequence set forth in SEQ ID NO:2 compared to the parent sequence, wherein said variants have amylase activity; preferably, the parent amylase of the amylase variant is the amylase of SEQ ID NO:1 or any amylase having at least 60% sequence identity to SEQ ID NO:1; most preferably, the parent amylase of the amylase variant is the amylase of SEQ ID NO:1.

[0260] Preferably, in this embodiment, the amino acid residues in the parent amylase at the above-mentioned positions (ie, X) correspond to the amino acid residues shown in SEQ ID NO: 1 at each position according to the numbering of SEQ ID NO: 2.

[0261] Thus, preferably, the present invention is directed to amylase variants comprising amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of G4, N25, W116, R176, R181, G186, N195, I206, T225, T251, R320, L405 and Y482 according to the numbering of the amino acid sequence set forth in SEQ ID NO:2.

[0262] In a preferred embodiment, the present invention is directed to amylase variants comprising amino acid substitutions at one or more positions corresponding to positions selected from the group consisting of G4Q, N25H, W116K, R176K, R181T, G186E, N195F, I206Y, T225A, T251E, R320K, L405M and Y482W according to the numbering of the amino acid sequence set forth in SEQ ID NO:2.

[0263] The present invention is preferably directed to amylase variants comprising an amino acid substitution at one or more positions corresponding to position 25 according to the numbering of SEQ ID NO:2 and positions selected from the group consisting of 4, 116, 176, 181, 186, 195, 206, 225, 251, 320, 405 and 482.

[0264] Preferably, the present invention is directed to amylase variants comprising the amino acid substitutions X25H according to the numbering of the amino acid sequence set forth in SEQ ID NO:2 and one or more amino acid substitutions selected from the group consisting of X4Q, X116K, X176K, X186E, X195F, X206Y, X225A, X251E, X320K, X405M and X482W.

[0265] Preferably, therefore, the present invention is directed to amylase variants comprising an amino acid substitution at one or more positions corresponding to amino acid position N25 according to the numbering of SEQ ID NO: 2 and a position selected from the group consisting of G4, W116, R176, R181, G186, N195, I206, T225, T251, R320, L405 and Y482.

[0266] In a preferred embodiment, the present invention is directed to amylase variants comprising amino acid substitutions at one or more positions corresponding to the amino acid substitutions N25H according to the numbering of SEQ ID NO: 2 and positions selected from the group consisting of G4Q, W116K, R176K, R181T, G186E, N195F, I206Y, T225A, T251E, R320K, L405M and Y482W.

[0267] Preferably, the amylase variant comprises an amino acid substitution at one or more of the following amino acid positions (according to the numbering of the amino acid sequence set forth in SEQ ID NO: 2) compared to the parent amylase: Preferably, the parent amylase of the amylase variant is an amylase set forth in any of SEQ ID NOs: 1-15, and most preferably, the parent amylase of the amylase variant is the amylase of SEQ ID NO: 1. Preferably, the amino acid residue (i.e., X) of the parent amylase at the listed position corresponds to the amino acid residue shown in SEQ ID NO: 2 at the respective position (according to the numbering of SEQ ID NO: 2).

[0268] The amylase variant preferably comprises an amino acid substitution at position 25 (according to the numbering of SEQ ID NO: 2), preferably the substitution X25H. Alternatively, the amino acid substitution at position 25 is X25Y or X25D.

[0269] Preferably, the amylase variant comprises an amino acid substitution at position 4 (according to the numbering of SEQ ID NO: 2), preferably the substitution X4Q.

[0270] Preferably, the amylase variant comprises an amino acid substitution at position 116 (according to the numbering of SEQ ID NO: 2), preferably the substitution X116K.

[0271] Preferably, the amylase variant comprises an amino acid substitution at position 176 (according to the numbering of SEQ ID NO: 2), preferably the substitution X176K.

[0272] Preferably, the amylase variant comprises an amino acid substitution at position 181 (according to the numbering of SEQ ID NO: 2), preferably the substitution X181T.

[0273] Preferably, the amylase variant comprises an amino acid substitution at position 186 (according to the numbering of SEQ ID NO: 2), preferably the substitution X186E.

[0274] Preferably, the amylase variant comprises an amino acid substitution at position 195 (according to the numbering of SEQ ID NO: 2), preferably the substitution X195F.

[0275] Preferably, the amylase variant comprises an amino acid substitution at position 206 (according to the numbering of SEQ ID NO: 2), preferably the substitution X206Y.

[0276] Preferably, the amylase variant comprises an amino acid substitution at position 225 (according to the numbering of SEQ ID NO: 2), preferably the substitution X225A.

[0277] Preferably, the amylase variant comprises an amino acid substitution at position 251 (according to the numbering of SEQ ID NO: 2), preferably the substitution X251E.

[0278] Preferably, the amylase variant comprises an amino acid substitution at position 320 (according to the numbering of SEQ ID NO: 2), preferably the substitution X320K.

[0279] Preferably, the amylase variant comprises an amino acid substitution at position 405 (according to the numbering of SEQ ID NO: 2), preferably the substitution X405M.

[0280] Preferably, the amylase variant comprises an amino acid substitution at position 482 (according to the numbering of SEQ ID NO: 2), preferably the substitution X482W.

[0281] In one embodiment, the amylase variant of a parent amylase is (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO:2; (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 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO:2; and (iii) SEQ ID NO: 1 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably comprising at least 60%, preferably at least 91%, but less than 100% sequence identity to SEQ ID NO: 1, and preferably the parent amylase of the amylase variant is the amylase of SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1.

[0282] Preferably, the amylase variant comprises an amino acid substitution at position 25 according to the numbering of SEQ ID NO:2 and an amino acid substitution at one or more positions selected from the group consisting of 116, 181, 225 and 320.

[0283] Preferably, the amylase variant comprises an amino acid substitution at position 25 according to the numbering of SEQ ID NO:2 and one or more amino acid substitutions at positions selected from the group consisting of 176 and 186.

[0284] Preferably, the amylase variant comprises an amino acid substitution at position 25 according to the numbering of SEQ ID NO:2 and an amino acid substitution at one or more positions selected from the group consisting of 195 or 206.

[0285] Preferably, the amylase variant comprises an amino acid substitution at position 25 and an amino acid substitution at position 482 according to the numbering of SEQ ID NO:2.

[0286] Preferably, said amino acid substitution at position 25 is X25H.

[0287] Preferably, said amino acid substitution at position 116 is X116K, preferably said amino acid substitution at position 176 is X176K, preferably said amino acid substitution at position 181 is X181T, preferably said amino acid substitution at position 186 is X186E, preferably said amino acid substitution at position 195 is X195F, preferably said amino acid substitution at position 206 is X206Y, preferably said amino acid substitution at position 225 is X225A, preferably said amino acid substitution at position 320 is X320K and said amino acid substitution at position 482 is X482W.

[0288] Preferably, the amylase variant comprises an amino acid substitution at position 25, preferably the substitution X25H (according to the numbering of SEQ ID NO: 2) a) a substitution at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, or b) substitutions at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K, or c) substitutions at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and substitutions at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K; Includes:

[0289] Preferably, the amylase variant comprises an amino acid substitution at position 25, preferably the substitution X25H (according to the numbering of SEQ ID NO: 2) a) a substitution at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, or b) substitutions at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and substitutions at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K; Includes:

[0290] Preferably, the amylase variant comprises an amino acid substitution at position 25, preferably the substitution X25H (according to the numbering of SEQ ID NO: 2) a) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and a substitution at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, or b) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and a substitution at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K; or c) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and a substitution at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and a substitution at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K. Includes:

[0291] Preferably, the amylase variant comprises an amino acid substitution at position 25, preferably the substitution X25H (according to the numbering of SEQ ID NO: 2) a) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and a substitution at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, or b) a substitution at amino acid position 195, preferably X195F, or a substitution at amino acid position 206, preferably X206Y, and a substitution at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and a substitution at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K. Includes:

[0292] Preferably, the amylase variant comprises an amino acid substitution at position 25, preferably the substitution X25H (according to the numbering of SEQ ID NO: 2) a) a substitution at amino acid position 195, preferably X195F, and one or more substitutions at positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, or b) a substitution at amino acid position 195, preferably X195F, and one or more substitutions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and a substitution at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K. Includes:

[0293] Preferably, the amylase variant is (i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 2; (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 2. Includes:

[0294] Preferably, the present invention is directed to an amylase variant of a parent amylase, said variant comprising: (i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 2; (ii) the amino acid substitution X195F or X206Y according to the numbering of SEQ ID NO: 2; (iii) one or more, two or more, three or more, four or more, five or more, six or more, or all amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 2. Includes:

[0295] Preferably, the amylase variant comprises one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 2):

[0296] [Table 1]

[0297] Most preferably, the amylase variant comprises one of the following amino acid substitution combinations: X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W (according to the numbering of SEQ ID NO: 2).

[0298] Preferably, the amylase variant comprises one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 2):

[0299] [Table 2]

[0300] Most preferably, the amylase variant comprises the following amino acid substitution combination: X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W (according to the numbering of SEQ ID NO: 2).

[0301] In one embodiment, the amylase variant of a parent amylase is (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO:2; (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:2; and (iii) SEQ ID NO: 1 or SEQ ID NO: 2 or any of SEQ ID NOs: 3 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably comprising at least 60%, preferably 91% but less than 100% sequence identity to SEQ ID NO: 1, and preferably the parent amylase of the amylase variant is the amylase of SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1.

[0302] Preferably, the amylase variant comprises an amino acid substitution at position 4 and an amino acid substitution at position 25 according to the numbering of SEQ ID NO:2.

[0303] Preferably, the amylase variant comprises an amino acid substitution at position 4 according to the numbering of SEQ ID NO:2 and an amino acid substitution at one or more positions selected from the group consisting of 176 and 186.

[0304] Preferably, the amylase variant comprises an amino acid substitution at position 4 according to the numbering of SEQ ID NO:2 and an amino acid substitution at one or more positions selected from the group consisting of 251 and 405.

[0305] Preferably, the amylase variant comprises an amino acid substitution at position 4 and an amino acid substitution at position 482 according to the numbering of SEQ ID NO:2.

[0306] In one embodiment, the amylase variant of a parent amylase is (i) amino acid substitutions at positions 25 and 4 according to the numbering of SEQ ID NO:2; (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 176, 186, 251, 405, and 482 according to the numbering of SEQ ID NO: 2; and (iii) SEQ ID NO: 1 or SEQ ID NO: 2 or any of SEQ ID NOs: 3 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably having at least 60%, preferably 91% but less than 100% sequence identity to SEQ ID NO: 1, and preferably the parent amylase of the amylase variant is the amylase set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1.

[0307] Preferably, said amino acid substitution at position 4 is X4Q. Preferably, said amino acid substitution at position 25 is X25H.

[0308] Preferably, said amino acid substitution at position 176 is X176K, preferably said amino acid substitution at position 186 is X186E, preferably said amino acid substitution at position 251 is X251E, preferably said amino acid substitution at position 405 is X405M, and preferably said amino acid substitution at position 482 is X482W.

[0309] Preferably, the amylase variant is (according to the numbering of SEQ ID NO: 2) a) substitutions at one or more positions selected from 25, 176 and 186, preferably one or more substitutions selected from X25H, X176K 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 Includes:

[0310] Preferably, the amylase variant is (i) the amino acid substitution X4G according to the numbering of SEQ ID NO: 2; (ii) one or more, two or more, three or more, four or more, five or more, or all amino acid substitutions selected from the group consisting of X25H, X176K, X186E, X251E, X405M, and X482W according to the numbering of SEQ ID NO: 2. Includes:

[0311] Preferably, the amylase variant comprises an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 2, preferably the substitution X4Q, and a) substitutions at one or more positions selected from 25, 176 and 186, preferably one or more substitutions selected from X25H, X176K 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 Includes:

[0312] Preferably, the amylase variant is (i) the amino acid substitution X4G according to the numbering of SEQ ID NO: 2; (ii) the amino acid substitution X25H according to the numbering of SEQ ID NO: 2, and (iii) the amino acid substitutions X176K and X186E according to the numbering of SEQ ID NO: 2 Includes:

[0313] Preferably, the amylase variant is (i) the amino acid substitution X4G according to the numbering of SEQ ID NO: 2; (ii) the amino acid substitution X25H according to the numbering of SEQ ID NO:2; (iii) the amino acid substitutions X176K and X186E according to the numbering of SEQ ID NO: 2, and (iv) one or more, two or more, or all amino acid substitutions selected from the group consisting of X251E, X405M, and X482W according to the numbering of SEQ ID NO: 2. Includes:

[0314] Preferably, the amylase variant comprises one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 2):

[0315] [Table 3]

[0316] Most preferably, the amylase variant comprises the following amino acid substitution combination: X4Q+X25H+X176K+X186E+X251E+X405M+X482W (according to the numbering of SEQ ID NO: 2).

[0317] Amylase variants according to the invention having one or more amino acid substitutions as described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96.5%, at least 97%, at least 98.5%, at least 99%, at least 99.5%, at least 91.5%, at least 92.5%, at least 93.5%, at least 94.5%, at least 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 9 ... 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%, at least 99.5%, but less than 100% sequence identity.

[0318] Preferably, the amylase variants according to the present invention having one or more amino acid substitutions as described herein 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 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 115.5%, at least 116%, at least 117%, at least 118.5%, at least 119.5%, at least 120.5%, at least 121.5%, at least 122.5%, at least 123.5%, at least 124.5%, at least 125.5%, at least 126.5%, at least 127.5%, at least 128.5%, at least 129.5%, at least 130.5%, at least 131.5%, at least 132.5%, at least 133.5%, at least 134.5%, at least 135.5%, at least 136 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%, at least 99.5%, but less than 100% sequence identity.

[0319] Preferably, the amylase variant according to the invention having one or more amino acid substitutions as described herein preferably has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0320] Preferably, amylase variants according to the invention having one or more amino acid substitutions as described herein preferably have at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, but less than 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0321] 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 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, with one or more amino acid modifications as described herein.

[0322] 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 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, having the amino acid substitution X25H and preferably the amino acid substitution X195F or X206Y according to the numbering of SEQ ID NO: 2, and having 1 to 7, preferably 2 to 7 or 3 to 7, for example 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2.

[0323] 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 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, having the amino acid substitution X4Q according to the numbering of SEQ ID NO: 2 and having 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 X25H, X176K, X186E, X251E, X405M and X482W according to the numbering of SEQ ID NO: 2.

[0324] 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 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, having one or more of the above amino acid modifications, and further comprises 1 to 50, preferably 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10 or 1 to 5, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 8 or 2 to 5, preferably 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 8 or 3 to 5, preferably 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10 or 4 to 8 conservative amino acid exchanges, preferably one or more, preferably two or more amino acid deletions, for example corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 2 as described herein.

[0325] Preferably, the amylase variant according to the present invention having amylase activity comprises or has the amino acid sequence set forth in any of SEQ ID NOs: 1 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, having the amino acid substitution X25H and preferably the amino acid substitution X195F or X206Y according to the numbering of SEQ ID NO: 2, and having 1 to 7, preferably 2 to 7 or 3 to 7, for example 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2. and 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 a deletion of one or more, preferably two or more amino acids corresponding to positions selected from the group consisting of, for example, 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 2 described herein.

[0326] 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, having the amino acid substitution X25H and preferably the amino acid substitution X195F or X206Y according to the numbering of SEQ ID NO: 2, and having 1 to 7, preferably 2 to 7 or 3 to 7, for example 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2, and further comprising 1 to 10, preferably 1 to 5 conservative amino acid exchanges.

[0327] 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 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, having the amino acid substitution X4Q according to the numbering of SEQ ID NO: 2 and having 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 X25H, X176K, X186E, X251E, X405M and X482W according to the numbering of SEQ ID NO: 2, and further comprising 1 to 50, 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 X25H, X176K, X186E, X251E, X405M and X482W according to the numbering of SEQ ID NO: 2. or 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 15, 4 to 20, 4 to 10 or 4 to 8 conservative amino acid exchanges, preferably a deletion of one or more, preferably two or more amino acids corresponding to positions selected from the group consisting of, for example, 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 2 described herein.

[0328] 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, having the amino acid substitution X4Q according to the numbering of SEQ ID NO: 2, and having 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 X25H, X176K, X186E, X251E, X405M and X482W according to the numbering of SEQ ID NO: 2, and further comprising 1 to 10, preferably 1 to 5 conservative amino acid exchanges.

[0329] 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.

[0330] Preferably, the present invention is directed 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:2; (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: 2; (iii) SEQ ID NO: 1 or SEQ ID NO: 2 or any of SEQ ID NOs: 3 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably at least 60% identity, preferably 91% identity, but less than 100% identity to SEQ ID NO: 1 (preferably, where the parent amylase of the amylase variant is the amylase set forth in SEQ ID NO: 1 or SEQ ID NO: 2, 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: 2, 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: 2). Includes:

[0331] Preferably, the present invention is directed 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:2; (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: 2; (iii) SEQ ID NO: 1 or SEQ ID NO: 2 or any of SEQ ID NOs: 3 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably at least 80%, preferably 91%, but less than 100% sequence identity to SEQ ID NO: 1 (preferably, wherein the parent amylase of the amylase variant is the amylase set forth in SEQ ID NO: 1 or SEQ ID NO: 2, 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: 2, 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: 2). Includes:

[0332] Preferably, the amylase variant is (i) the amino acid substitution X4G according to the numbering of SEQ ID NO: 2; (ii) one or more, two or more, three or more, four or more, five or more or all amino acid substitutions selected from the group consisting of X25H, X176K, X186E, X251E, X405M and X482W according to the numbering of SEQ ID NO: 2; (iii) SEQ ID NO: 1 or SEQ ID NO: 2 or any of SEQ ID NOs: 3 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 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%, 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: 2, 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: 2). Includes:

[0333] Preferably, the amylase variant is (i) the amino acid substitution X4G according to the numbering of SEQ ID NO: 2; (ii) one or more, two or more, three or more, four or more, five or more, or all amino acid substitutions selected from the group consisting of X25H, X176K, X186E, X251E, X405M, and X482W according to the numbering of SEQ ID NO: 2. (iii) SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, or 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%, 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: 2, 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: 2). Includes:

[0334] Preferably, the amylase variant is (i) (following the numbering in SEQ ID NO: 2) 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 (ii) SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, or 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%, at least 99.5%, but less than 100% sequence identity; (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: 2, 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: 2). Includes:

[0335] Preferably, the amylase variant is (i) the amino acid substitution X4G according to the numbering of SEQ ID NO: 2; (ii) one or more, two or more, three or more, four or more, five or more or all amino acid substitutions selected from the group consisting of X25H, X176K, X186E, X251E, X405M and X482W according to the numbering of SEQ ID NO: 2; (iii) SEQ ID NO: 1 or SEQ ID NO: 2, preferably 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%, which have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, with SEQ ID NO: 1; 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%, 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: 2, 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: 2). Including, (v) The amylase variant preferably exhibits improved one or more properties compared to said parent amylase, preferably compared to the parent amylase as set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, preferably said amylase variant has improved 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 stability, preferably storage stability in a detergent composition, preferably the improved property is expressed as an Improvement Factor (IF) of >1.0, preferably the improvement factor is 1.1 or more, preferably 1.2 or more, more preferably 1.3 or more.

[0336] Particularly preferably, the amylase variant is (i) (following the numbering in SEQ ID NO: 2) 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 a combination of amino acid substitutions selected from (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%, but less than 100%, sequence identity to SEQ ID NO:1; (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: 2). Includes:

[0337] Particularly preferred are: (i) Amino acid substitution combinations (according to the numbering of SEQ ID NO: 2): 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; (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: 2). and amylase variants comprising:

[0338] 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: 2.

[0339] Preferably, the present invention is directed to an amylase variant of a parent amylase, said variant comprising: (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO:2; (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 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO: 2; (iii) SEQ ID NO: 1 or SEQ ID NO: 2 or any of SEQ ID NOs: 3 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably at least 60% identity, preferably 91% identity, but less than 100% identity to SEQ ID NO: 1 (preferably, where the parent amylase of the amylase variant is the amylase set forth in SEQ ID NO: 1 or SEQ ID NO: 2, 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: 2, 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: 2). Includes:

[0340] Preferably, the present invention is directed to an amylase variant of a parent amylase, said variant comprising: (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO:2; (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 116, 176, 181, 186, 195, 206, 225, 320, and 482 according to the numbering of SEQ ID NO: 2; (iii) SEQ ID NO: 1 or SEQ ID NO: 2 or any of SEQ ID NOs: 3 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably at least 80% identical, but preferably 91% identical, to SEQ ID NO: 1 (preferably, where the parent amylase of the amylase variant is the amylase set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1). and (iv) The invention of said amylase variant comprises one or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184 according to the numbering of SEQ ID NO: 2, preferably two or more amino acid deletions corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletions of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably deletions D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0341] Preferably, the amylase variant is (i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 2; (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 2; (iii) SEQ ID NO: 1 or SEQ ID NO: 2 or any of SEQ ID NOs: 3 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 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%, 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: 2, 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: 2). Includes:

[0342] Preferably, the amylase variant is (i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 2; (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 2; (iii) SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, or 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%, 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: 2, 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: 2). Includes:

[0343] Preferably, the present invention is directed to an amylase variant of a parent amylase, said variant comprising: (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO:2; (ii) an amino acid substitution at position 195 or 206 according to the numbering of SEQ ID NO:2; (iii) 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 116, 176, 181, 186, 225, 320, and 482 according to the numbering of SEQ ID NO: 2; (iv) SEQ ID NO: 1 or SEQ ID NO: 2 or any of SEQ ID NOs: 3 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably at least 60%, preferably at least 91%, but less than 100% sequence identity to SEQ ID NO: 1; and (v) 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: 2, preferably deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably deletions D183* and G184* (according to the numbering of SEQ ID NO: 2); Includes:

[0344] Preferably, the present invention is directed to an amylase variant of a parent amylase, said variant comprising: (i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 2; (ii) the amino acid substitution X195F or X206Y according to the numbering of SEQ ID NO: 2; (iii) one or more, two or more, three or more, four or more, five or more, six or more or all amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2; (iv) SEQ ID NO: 1 or SEQ ID NO: 2 or any of SEQ ID NOs: 3 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably at least 60%, preferably at least 91%, but less than 100% sequence identity to SEQ ID NO: 1; and (v) 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: 2, preferably deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably deletions D183* and G184* (according to the numbering of SEQ ID NO: 2); Includes:

[0345] Preferably, the present invention is directed to an amylase variant of a parent amylase, said variant comprising: (i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 2; (ii) the amino acid substitution X195F or X206Y according to the numbering of SEQ ID NO: 2; (iii) one or more, two or more, three or more, four or more, five or more, six or more or all amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2; (iv) at least 60%, preferably at least 91%, but less than 100% sequence identity to SEQ ID NO: 1; and (v) 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: 2, preferably deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably deletions D183* and G184* (according to the numbering of SEQ ID NO: 2); Includes:

[0346] Preferably, the amylase variant is (i) (following the numbering in SEQ ID NO: 2) X25H+X176K+X186E, X25H+X176K+X186E+X195F, X25H+X176K+X186E+X206Y, X25H+X116K+X181T+X206Y+X225A+X320K, X25H+X116K+X176K+X181T+X206Y+X225A+X320K, X25H+X116K+X181T+X186E+X206Y+X225A+X320K, X25H+X116K+X181T+X206Y+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K, X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W, X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W, X25H+X116K+X181T+X195F+X225A+X320K, X25H+X116K+X176K+X181T+X195F+X225A+X320K, X25H+X116K+X181T+X186E+X195F+X225A+X320K, X25H+X116K+X181T+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K, X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W, X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W, and X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W a combination of amino acid substitutions selected from the group consisting of: (ii) SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, or 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%, at least 99.5%, but less than 100% sequence identity; (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: 2, 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: 2). Includes:

[0347] Preferably, the amylase variant is (i) (following the numbering in SEQ ID NO: 2) X25H+X176K+X186E+X195F, X25H+X116K+X181T+X195F+X225A+X320K, X25H+X116K+X176K+X181T+X195F+X225A+X320K, X25H+X116K+X181T+X186E+X195F+X225A+X320K, X25H+X116K+X181T+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K, X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W, X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W, and X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W a combination of amino acid substitutions selected from the group consisting of: (ii) SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, or 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%, at least 99.5%, but less than 100% sequence identity; (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: 2, 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: 2). Includes:

[0348] Preferably, the amylase variant is (i) (following the numbering in SEQ ID NO: 2) X25H+X176K+X186E+X206Y, X25H+X116K+X181T+X206Y+X225A+X320K, X25H+X116K+X176K+X181T+X206Y+X225A+X320K, X25H+X116K+X181T+X186E+X206Y+X225A+X320K, X25H+X116K+X181T+X206Y+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K, X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W, X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W, and X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W a combination of amino acid substitutions selected from the group consisting of: (ii) SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, or 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%, at least 99.5%, but less than 100% sequence identity; (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: 2, 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: 2). Includes:

[0349] Preferably, the amylase variant is (i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 2; (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K, and X482W according to the numbering of SEQ ID NO: 2; (iii) SEQ ID NO: 1 or SEQ ID NO: 2, preferably 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%, which have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, with SEQ ID NO: 1; 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%, 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: 2, 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: 2). and (v) The amylase variant preferably exhibits improved one or more properties compared to said parent amylase, preferably compared to the parent amylase as set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, preferably said amylase variant has improved 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 stability, preferably storage stability in a detergent composition, preferably the improved property is expressed as an Improvement Factor (IF) of >1.0, preferably the improvement factor is 1.1 or more, preferably 1.2 or more, more preferably 1.3 or more.

[0350] Preferably, the amylase variant is (i) the amino acid substitution X25H according to the numbering of SEQ ID NO: 2; (ii) the amino acid substitution X195F or X206Y according to the numbering of SEQ ID NO: 2; (iii) one or more, two or more, three or more, four or more, five or more, six or more or all amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2; (iv) SEQ ID NO: 1 or SEQ ID NO: 2, preferably 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 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, or 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 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%, at least 99.5%, but less than 100% sequence identity; (v) 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: 2, preferably deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably deletions D183* and G184* (according to the numbering of SEQ ID NO: 2); and (vi) The amylase variant preferably exhibits improved one or more properties compared to said parent amylase, preferably compared to the parent amylase as set forth in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, and preferably said amylase variant has improved 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 stability, and preferably the improved property is expressed as an Improvement Factor (IF) of >1.0, preferably the improvement factor is 1.1 or more, preferably 1.2 or more, more preferably 1.3 or more.

[0351] Particularly preferably, the amylase variant is (i) (following the numbering in SEQ ID NO: 2) X25H+X176K+X186E, X25H+X176K+X186E+X195F, X25H+X176K+X186E+X206Y, X25H+X116K+X181T+X206Y+X225A+X320K, X25H+X116K+X176K+X181T+X206Y+X225A+X320K, X25H+X116K+X181T+X186E+X206Y+X225A+X320K, X25H+X116K+X181T+X206Y+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K, X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W, X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W, X25H+X116K+X181T+X195F+X225A+X320K, X25H+X116K+X176K+X181T+X195F+X225A+X320K, X25H+X116K+X181T+X186E+X195F+X225A+X320K, X25H+X116K+X181T+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K, X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W, X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W, and X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+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%, but less than 100%, sequence identity to SEQ ID NO:1; (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: 2). Includes:

[0352] Particularly preferred are: (i) Amino acid substitution combinations (according to the numbering of SEQ ID NO: 2): X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K, or X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W, preferably X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W, (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; (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: 2). and amylase variants comprising:

[0353] Most preferred are amino acid modifications according to the numbering of SEQ ID NO:2 X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K, or X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W The amylase variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, having the following structure:

[0354] In one embodiment, the amylase variant of the enzyme composition comprises (i) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 183, 243, 266, and 475 according to the numbering of SEQ ID NO: 9, and at least 60% but less than 100% sequence identity to SEQ ID NO: 9; or (ii) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 38, 126, 129, 153, 180, 187, 199, 203, 241, 362, 377, 458, 459, 460, 476, and 477 according to the numbering of SEQ ID NO: 7, and at least 60% but less than 100% sequence identity to SEQ ID NO: 7; or (iii) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 1, 7, 51, 52, 54, 109, 140, 195, 206, 243, 260, 280, 284, 304, 391, 476, and 477 according to the numbering of SEQ ID NO: 3, and at least 60% but less than 100% sequence identity to SEQ ID NO: 3; or (iv) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 9, 118, 149, 182, 186, 195, 202, 246, 257, 295, 299, 320, 323, 339, 345, 458, and 471 according to the numbering of SEQ ID NO: 13, and at least 60% but less than 100% sequence identity to SEQ ID NO: 13; or (v) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 1, 54, 56, 72, 109, 113, 116, 149, 167, 172, 174, 182, 195, 202, 206, 246, 257, 295, 299, 320, 339, 345, 391, 458, 471, 473, and 476 according to the numbering of SEQ ID NO: 15, and at least 60% but less than 100% sequence identity to SEQ ID NO: 15; or (vi) an amino acid sequence having an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 9, 118, 149, 182, 186, 195, 202, 246, 257, 295, 299, 320, 323, 339, 345, 405, 421, 422, 428, 458, and 471 according to the numbering of SEQ ID NO:4, and at least 60% but less than 100% sequence identity to SEQ ID NO:4; or (vii) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 1, 54, 56, 72, 109, 113, 116, 120, 134, 140, 167, 169, 172, 173, 174, 182, 183, 184, 195, 202, 206, 246, 255, 257, 260, 265, 295, 299, 304, 320, 339, 345, 391, 395, 410, 422, 429, 444, 451, 458, 473, 474, and 476 according to the numbering of SEQ ID NO:5, and an amino acid sequence having at least 60% but less than 100% sequence identity to SEQ ID NO:5. Includes:

[0355] In one embodiment, the amylase variant comprises an amino acid sequence having at least 60%, 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% of SEQ ID NO:9. , 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 an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 183, 243, 266 and 475 (according to the numbering of SEQ ID NO:9), more preferably an amino acid substitution selected from X183S, X243Q, X266Y and X475K.

[0356] In one embodiment, the amylase variant comprises an amino acid sequence having at least 60%, 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 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 110.5%, at least 111%, 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 129.5%, at least 129.5%, 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 and amino acid sequences having 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 an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 183, 243, 266 and 475 (according to the numbering of SEQ ID NO: 9), more preferably G183S, S243Q, F266Y, G475K.

[0357] In one embodiment, the amylase variant has an amino acid sequence identical to SEQ ID NO:9, 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 more identical to SEQ ID NO:9. has at least 99.5% but less than 100% sequence identity to SEQ ID NO:9, and comprises two amino acid deletions in the regions of R180, G181, T182, and G183 (according to the numbering of SEQ ID NO:9), optionally a truncated C-terminal portion (amino acids 485-583), and further comprises amino acid substitutions at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 183, 243, 266, and 475, more preferably all of X183S, X243Q, X266Y, and X475K.

[0358] In one embodiment, the amylase variant comprises an amino acid sequence having at least 60%, 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% SEQ ID NO:7. %, but less than 100% sequence identity and amino acid mutations at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 38, 126, 129, 153, 180, 187, 199, 203, 241, 362, 377, 458, 459, 460, 476 and 477 (according to the numbering of SEQ ID NO: 7), preferably X38N, X126Y, X129I, X153W, X180D / H, X187P, X199L, X203Y, X241Q, X362A, X The amino acid sequence includes an amino acid sequence having one or more, preferably all, mutations selected from X38N, X126Y, X129I, X153W, X178*, X179*, X180D, X187P, X203Y, X476K and X477E, more preferably all of X38N, X126Y, X129I, X153W, X178*, X179*, X187P, X203Y, X476K and X477E in combination or all of X178*, X179*, X187P, X203Y, X458N, X459S, X460T and X476K.

[0359] In one embodiment, the amylase variant has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100%, sequence identity to SEQ ID NO:7 and the amino acids 38, 126, 129, 153, 180, 187, 199, 203, 241, 362, 377, 458, 459, 460, 476 and (according to the numbering of SEQ ID NO:7) 477, preferably one or more selected from T38N, N126Y, T129I, F153W, T180D / H, E187P, M199L, I203Y, S241Q, S362A, R377Y, R458N, T459S, D460T, G476K and G477E , preferably all of the mutations, more preferably all of T38N, N126Y, T129I, F153W, R178*, G179*, T180D, E187P, I203Y, G476K and G477E or all of R178*, G179*, E187P, I203YR458N, T459S, D460T and G476K in combination.

[0360] In one embodiment, the amylase variant has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5% but less than 100% sequence identity to SEQ ID NO:3, and is selected from the group consisting of: R181, R201, R212, R214, R216, R218 ... The present invention further comprises two amino acid deletions in the regions of G182, D183, and G184, and further comprises amino acid mutations at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 1, 7, 51, 52, 54, 109, 140, 195, 206, 243, 260, 280, 284, 304, 391, 476, and 477, preferably X1*, X7A, X51I, X52Q, X54K, X109A, X140Y, X195F, X206Y, X243F, X260G, X270G, X280G, X290G, X300G, X310G, X320G, X330G, X340G, X350G, X360G, X370G, X380G, X390G, X400G, X410G, X420G, X430G, X440G, X450G, X460G, X470G, X480G, X490G, X511, X52Q, X54K, X109A, X140Y, X195F, X206Y, X243F, X260G, X470G, X540G, X550G, X560G, X570G, X580G, X590G, X600G, X610G, X620G, X630G, X640G, X650G, X660G, X670G, X680G, X690G, X700G, X711, X72Q, X750G, X760 The amino acid sequence includes one or more, preferably all, mutations selected from the group consisting of X1*, X7A, X51I, X52Q, X54K, X109A, X140Y, X195F, X206Y, X243F, X260G, X280S, X284F / D / R, X304R, X391A, X476E / K and X477R / K / E, preferably all of the mutations in combination.

[0361] In one embodiment, the amylase variant has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100%, sequence identity to SEQ ID NO:3 and comprises two amino acid deletions in the regions R181, G182, D183, G184 (according to the numbering of SEQ ID NO:2) and further comprises deletions of 1, 7, 51, 52, 54, 109, 140, 195, 206, 243, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318 Amino acid mutations at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 80, 284, 304, 391, 476 and 477, preferably H1*, G7A, T51I, S52Q, N54K, G109A, W140Y, N195F, V206Y, Y243F, E260G, N280S, W284F / D / R, G304R, E391A, G476E / K and G and 477R / K / E, preferably all of the mutations selected from the group consisting of H1*, G7A, T51I, S52Q, N54K, G109A, W140Y, N195F, V206Y, Y243F, E260G, N280S, W284F / D / R, G304R, E391A, G476E / K and G477R / K / E in combination.

[0362] In one embodiment, the amylase variant has an amino acid sequence similar to SEQ ID NO: 13 by 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 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 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%. 3.5%, 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 two amino acid deletions in the regions R181, G182, D183, G184 (according to the numbering of SEQ ID NO: 13), and further comprising 9, 118 , 149, 182, 186, 195, 202, 246, 257, 295, 299, 320, 323, 339, 345, 458 and 471, preferably amino acid mutations at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of X9I / L, X118K, X149A, X182T, X186A, X195F, X202L, X246V, X257I, X295F, X299Y, X320K, X323T, X339S, X345R, X458 X118K, X183*, X184*, X195F, X320K, X323T, X339S, X345R and X458K in combination.

[0363] In one embodiment, the amylase variant has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100%, sequence identity to SEQ ID NO: 13 and comprises two amino acid deletions in the regions R181, G182, D183, G184 (according to the numbering of SEQ ID NO: 13), and further comprises the deletion of 9, 118, 149, 182, 186, 195, 202, 246, 257, 295, 299, 320, 323, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, Amino acid mutations at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 345, 458 and 471, preferably one or more selected from M9I / L, R118K, G149A, G182T, G186A, N195F, M202L, T246V, T257I, Y295F, N299Y, R320K, M323T, A339S, E345R, R458K and N471E, preferably The amino acid sequence may comprise any or all of the mutations, more preferably the mutations R118K, D183*, G184*, N195F, R320K and R458K or all of the mutations M9L, R118K, G149A, G182T, D183*, G184*, G186A, N195F, M202L, T257I, Y295F, N299Y, R320K, M323T, A339S, E345R and R458K in combination.

[0364] An amylase having the above substitutions is shown, for example, in SEQ ID NO:14.

[0365] Thus, in one embodiment, the amylase variant comprises an amino acid sequence having 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%, at least 99.5% or 100% sequence identity to SEQ ID NO:14.

[0366] In one embodiment, the amylase variant has an amino acid sequence 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% identical to SEQ ID NO:15. and (f) a deletion of two amino acids in the regions R181, G182, D183, and G184 (according to the numbering of SEQ ID NO: 15), and further comprising: 1, 54, 56, 72, 109, 113, 116, 149, 167, 172, 174, 182, 195, 202, 206, 246, 252, 262, 272, 282, 295, 302, 306, 312, 314, 316, 322, 324, 326, 332, 336, 338, 340, 342, 344, 346, 352, 354, 356, 360, 362, 364, 366, 370, 372, 374, 376, 378, 378, 380, 382, 384, 386, 388, 389, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 452, 454, 456, 458, 460, 462, 464, 466, 470, 472, 474, 476, 478, Amino acid mutations at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 57, 295, 299, 320, 339, 345, 391, 458, 471, 473 and 476, preferably X1A, X54S, X56T, X72R, X109A, X113Q, X116Q, X149A, X167F, X172G, X174S, X182T, X195F, X202L, X206L / Y, X246V, X257I, X295F, X299Y, X320K, X339S, X345R, X391A, X45 The amino acid sequence includes one or more, preferably all, mutations selected from X1A, X54S, X56T, X72R, X109A, X113Q, X116Q, X149A, X167F, X172G, X174S, X182T, X195F, X202L, X206L / Y, X246V, X257I, X295F, X299Y, X320K, X339S, X345R, X391A, X458K, X471E, X473R and X476K, preferably all of the mutations in combination.

[0367] In one embodiment, the amylase variant has an identical sequence to SEQ ID NO:15 by 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%, of sequence identity, and comprising two amino acid deletions in the regions of R181, G182, D183, G184 (according to the numbering of SEQ ID NO: 15), and further comprising one of the following amino acids: 1, 54, 56, 72, 109, 113, 116, 149, 167, 172, 174, 182, 195, 202, 206, 246, 257, 295, 299, 320, 339, 345, 391, 458, 471, 473, and 476. amino acid mutations at one or more amino acid positions corresponding to amino acid positions selected from H1A, N54S, V56T, K72R, G109A, F113Q, R116Q, G149A, W167F, Q172G, A174S, G182T, N195F, M202L, V206L / Y, M246V, T257I, Y295F, Q299Y, R320K, A339S, Q345R, K391A, R458K, H471E, F473R and G476K and m is an amino acid sequence containing one or more, preferably all, of the mutations listed above, preferably all of the mutations listed below in combination: H1A, N54S, V56T, K72R, G109A, F113Q, R116Q, G149A, W167F, Q172G, A174S, G182T, N195F, M202L, V206L / Y, M246V, T257I, Y295F, Q299Y, R320K, A339S, Q345R, K391A, R458K, H471E, F473R, and G476K.

[0368] In one embodiment, the amylase variant has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5% but less than 100% sequence identity to SEQ ID NO:4 and includes (according to the numbering of SEQ ID NO:4) R181, G182, H183 , comprising two amino acid deletions in the region of G184, and further comprising amino acid mutations at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 9, 118, 149, 182, 186, 195, 202, 246, 257, 295, 299, 320, 323, 339, 345, 405, 421, 422, 428, 458 and 471, preferably X9I / L, X118K, X149A, X182T, X186A, The amino acid sequence may comprise one or more, preferably all, mutations selected from X195F, X202L, X246V, X257I, X295F, X299Y, X320K, X323T, X339S, X345R, X405L, X421H, X422P, X428T, X458K and X471E, more preferably all of the mutations X183*, X184*, X405L, X421H, X422P and X428T in combination.

[0369] In one embodiment, the amylase variant has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5% but less than 100% sequence identity to SEQ ID NO:4 and comprises two amino acid deletions in the regions R181, G182, H183, G184 (according to the numbering of SEQ ID NO:4) and further comprises the deletion of 9, 118, 149, 182, 186, 195, 20 Amino acid mutations at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 2, 246, 257, 295, 299, 320, 323, 339, 345, 405, 421, 422, 428, 458, and 471, preferably M9I / L, R118K, G149A, G182T, G186A, N195F, M202L, T246V, T257I, Y The present invention also includes an amino acid sequence comprising one or more, preferably all, mutations selected from 295F, N299Y, R320K, M323T, A339S, E345R, I405L, A421H, A422P, A428T, R458K and N471E, more preferably all of the mutations H183*, G184*, I405L, A421H, A422P and A428T in combination.

[0370] In one embodiment, the amylase variant comprises an amino acid sequence having at least 60%, 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 selected from the group consisting of 1, 54, 56, 72, 109, 113, 116, 120, 134, 140, 167, 169, 172, 173, 174, 182, 183, 184, 195, 202, 206, 246, 255, 257, 260, 265, 295, 299, 304, 320, 339, 345, 391, 395, 410, 422, 429, 444, 451, 458, 473, 474 and 476 (according to the numbering of SEQ ID NO: 5). amino acid substitutions at one or more amino acid positions corresponding to amino acid positions in the nucleotide sequence of the present invention, preferably X1*, X54S, X56T, X72R, X109A, X113Q, X116H, X116Q, X120L, X134E, X140Y, X167F, X169E, X172E, X172G, X172K, X172N, X172R, X173P, X173V, X174*, X174S, X174K, X182T, X195F, X202L, X206L, X206Y, X246V, X255A, One or more, preferably all, mutations selected from the group consisting of X257I, X260G, X265G, X295F, X299Y, X304R, X320A, X320K, X339S, X345R, X391A, X395P, X410I, X422P, X429I, X444Q, X451W, X458K, X473R, X474V and X476K, more preferably X1*, X54S, X56T, X72R, X109A, X113Q, X116Q, X167F, X172G, X The amino acid sequence includes one or more, preferably all, mutations selected from the group consisting of X1*, X54S, X56T, X72R, X109A, X113Q, X116Q, X167F, X172G, X174S, X182*, X183*, X184T, X195F, X206L, X391A, X473R, and X476K, preferably all of the mutations in combination.

[0371] In one embodiment, the amylase variant has an amino acid sequence 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% identical to SEQ ID NO:5.5%, but less than 100%, of sequence identity, and containing two amino acid deletions in the regions of R181, G182, D183, and G184 (according to the numbering of SEQ ID NO: 5), and further including 1, 54, 56, 72, 109, 113, 116, 120, 134, 140, 167, 169, 172, 173, 174, 182, 183, 184, 195, 202, 206, 246, 255, 257, 260, 265, 295, 299, 304, 320, 339, 345, 391, 395, 410, 422, 429, 444 , 451, 458, 473, 474 and 476, preferably H1*, N54S, V56T, K72R, G109A, F113Q, R116H, R116Q, V120L, T134E, W140Y, W167F, Q169E, Q172E, Q172G, Q172K, Q172N, Q172R, L173P, L173V, A174*, A174S, A174K, G182T, N195F, M202L, One or more, preferably all, selected from the group consisting of V206L, V206Y, M246V, G255A, T257I, N260G, A265G, Y295F, Q299Y, S304R, R320A, R320K, A339S, Q345R, K391A, Q395P, V410I, A422P, V429I, T444Q, F451W, R458K, P473R, C474V and G476K, more preferably H1*, N54S, V56T, K72R, G109A, F113Q, R116Q, W The amino acid sequence includes one or more, preferably all, selected from the group consisting of H1*, N54S, V56T, K72R, G109A, F113Q, R116Q, W167F, Q172G, A174S, G182*, D183*, G184T, N195F, V206L, K391A, P473R and G476K, preferably all of H1*, N54S, V56T, K72R, G109A, F113Q, R116Q, W167F, Q172G, A174S, G182*, D183*, G184T, N195F, V206L, K391A, P473R and G476K in combination.

[0372] An amylase having the above substitutions is shown, for example, in SEQ ID NO:6.

[0373] Thus, in one embodiment, the amylase variant comprises an amino acid sequence having 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%, at least 99.5% or 100% sequence identity to SEQ ID NO:6.

[0374] Preferably, the composition comprises: a) the amino acid substitution X25H according to the numbering of SEQ ID NO: 2 and one or more amino acid substitutions selected from the group consisting of X4Q, X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X251E, X320K, X405M and X482W; b) deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably deletion of two or more, most preferably two amino acids corresponding to positions selected from the group consisting of R181, G182, D183 and G184, preferably D183* and G184* (wherein the numbering is according to the amino acid sequence set out in SEQ ID NO: 2); and further comprising an amylase variant comprising: c) The amylase variant has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100%, sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1.

[0375] Alternatively, preferably, the composition further comprises an amylase variant that has a similar affinity to SEQ ID NO:5 by 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 from the group consisting of 1, 54, 56, 72, 109, 113, 116, 120, 134, 140, 167, 169, 172, 173, 174, 182, 183, 184, 195, 202, 206, 246, 255, 257, 260, 265, 295, 299, 304, 320, 339, 345, 391, 395, 410, 422, 429, 444, 451, 458, 473, 474 and 476 (according to the numbering of SEQ ID NO: 5). Amino acid substitutions at one or more amino acid positions corresponding to the selected amino acid positions, preferably mutations X1*, X54S, X56T, X72R, X109A, X113Q, X116H, X116Q, X120L, X134E, X140Y, X167F, X169E, X172E, X172G, X172K, X172N, X172R, X173P, X173V, X174*, X174S, X174K, X182T, X195F, X202L, X206L, X206Y, X206R, X207R, X208R, X210R, X211R, X212R, X213R, X214R, X215R, X216R, X217R, X218R, X219R, X220R, X221G, X222R, X223R, X224R, X225R, X226R, X227R, X228R, X230R, X231R, X232R, X233R, X234R, X235R, X236R, X237R, X238R, X240R, X241R, X242R, X243R, X244R, X245R, X246R, X247R, X248R, X250R, X251R, X252R, X253R, X254R, X255R, X256R, X257R, X258R, X260R, X261R, X262R, X263R, X264R, X 46V, X255A, X257I, X260G, X265G, X295F, X299Y, X304R, X320A, X320K, X339S, X345R, X391A, X395P, X410I, X422P, X429I, X444Q, X451W, X458K, X473R, X474V and X476K, more preferably X1*, X54S, X56T, X72R, X109A, X113Q, X116Q, X167F, X172G, X174S, X182*, X18 The amino acid sequence comprises an amino acid sequence having one or more alterations selected from the group consisting of X1*, X54S, X56T, X72R, X109A, X113Q, X116Q, X167F, X172G, X174S, X182*, X183*, X184T, X195F, X206L, X391A, X473R and X476K, preferably all of these alterations in combination, most preferably the alterations X1*, X54S, X56T, X72R, X109A, X113Q, X116Q, X167F, X172G, X174S, X182*, X183*, X184T, X195F, X206L, X391A, X473R and X476K in combination. Thus, in another preferred alternative embodiment, the composition further comprises an amylase variant, which has an affinity for SEQ ID NO:6 by 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%, or at least 89%.The amino acid sequence may include an amino acid sequence having 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%, at least 99.5%, or 100% sequence identity.

[0376] Preferably, the composition comprises a protease, a mannanase, a cellulase and an amylase; a. Proteases are (i) the amino acid substitution X101E according to the numbering of SEQ ID NO: 17, and (ii) an amino acid sequence that is 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%, at least 99.5%, but less than 100% identical to SEQ ID NO: 16 is a protease variant comprising B. Mannanase (i) at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or all of the amino acid substitutions X89V, X96D, X119H, X264Q, X289M, X312Y, X348N, X349G and X352G according to the numbering of SEQ ID NO: 35, and (ii) an amino acid sequence that is at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5%, but less than 100%, identical to the sequence of positions 31 to 490 of SEQ ID NO: 35 and c. the cellulase is a cellulase variant comprising an amino acid sequence 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%, at least 99.5% or 100% identical to SEQ ID NO:42; Amylase is a) the amino acid substitution X25H according to the numbering of SEQ ID NO: 2 and one or more amino acid substitutions selected from the group consisting of X4Q, X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X251E, X320K, X405M and X482W; b) deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletion of two or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably deletion of amino acids corresponding to positions 181 and 182, 182 and 183 or 183 and 184, preferably deletion of two or more, most preferably two amino acids corresponding to positions selected from the group consisting of R181, G182, D183 and G184, preferably D183* and G184* (wherein the numbering is according to the amino acid sequence set out in SEQ ID NO: 2); and wherein the amylase variant comprises c) The amylase variant has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100%, sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1.

[0377] 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.

[0378] In one aspect of the invention, suitable lipases are selected from: lipases from the genus Humicola (synonym Thermomyces) as described in EP 258068, EP 305216, WO 92 / 05249 and WO 2009 / 109500, such as lipases 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 Lipases from P. sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (WO 96 / 12012), Pseudomonas mendocina (WO 95 / 14783), P. glumae (WO 95 / 35381, WO 96 / 00292), Streptomyces griseus (WO 2011 / 150157) and S.Lipases from S. pristinaespiralis (WO 2012 / 137147), GDSL-type Streptomyces lipases (WO 2010 / 065455), lipases from Thermobifida fusca disclosed in WO 2011 / 084412, lipases from Geobacillus stearothermophilus disclosed in WO 2011 / 084417, Bacillus lipases, for example, as disclosed in WO 00 / 60063, Dartois et al. (1992), Biochemica et Biophysica Lipases derived from B. subtilis, B. stearothermophilus (JP 64-074992 A) or B. pumilus (WO 91 / 16422 A) disclosed in Acta, 1131,253-360 or WO 2011 / 084599, lipases derived from Candida antarctica (WO 94 / 01541 A), cutinase derived from Pseudomonas mendocina (U.S. Pat. No. 5,389,536 A, WO 88 / 09367 A), cutinase derived from Magnaporthe grisea (U.S. Pat. No. 5,389,536 B), and the like are also useful. grisea (WO 2010 / 107560), cutinase from Fusarium solani pisi disclosed in WO 90 / 09446, WO 00 / 34450 and WO 01 / 92502, and cutinase from Humicola lanuginosa disclosed in WO 00 / 34450 and WO 01 / 92502.

[0379] Such suitable lipase variants are, for example, those developed by the methods disclosed in WO 95 / 22615, WO 97 / 04079, WO 97 / 07202, WO 00 / 60063, WO 2007 / 087508, EP 407225 and EP 260105.

[0380] 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), Biotouch LL100 (AB Enzymes) and Lipomax (Gist-Brocades / now DSM).

[0381] 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.

[0382] 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.

[0383] 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.

[0384] 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.

[0385] 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.

[0386] 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.

[0387] The Thermomyces lanuginosa lipase may be a variant of amino acids 1 to 269 of SEQ ID NO: 2 of U.S. Patent No. 5,869,438 having lipolytic activity, wherein the variant of amino acids 1 to 269 of SEQ ID NO: 2 of U.S. Patent No. 5,869,438 is characterized by containing amino acid substitutions T231R and N233R.

[0388] The Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity that contain at least one, preferably two or more, and more preferably all of the following amino acid substitutions within the polypeptide sequence of amino acids 1-269 of SEQ ID NO: 1 of WO 2015 / 010009: N11K, A18K, G23K, K24A, V77I, D130A, V154I, V187T, T189Q, and that are at least 95%, at least 96%, or at least 97% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 1 of WO 2015 / 010009.

[0389] Methods for obtaining enzymes and producing enzyme compositions Another embodiment of the present invention is a method for producing an enzyme composition comprising the proteases, mannanases and / or cellulases described herein, preferably comprising: a) obtaining the sequence of a variant of a protease, mannanase and / or cellulase described herein; b) producing said variants of proteases, mannanases or cellulases using the sequences obtained under a) as described herein; c) in one or more steps, combining two or more enzymes selected from the group consisting of proteases, mannanases and cellulases as described herein; The method includes:

[0390] Methods for obtaining specific enzyme variants are known to those skilled in the art. Methods for introducing the amino acid modifications described in protein sequences, such as substitutions or deletions, are well known in the art. The enzyme variants described herein can be produced using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc. Site-directed mutagenesis is a technique for generating one or more (several) mutations at one or more defined sites in a polynucleotide. Site-directed mutagenesis can be achieved in vitro by PCR, which involves the use of oligonucleotide primers containing the desired mutations. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis, which involves cleaving a site in a plasmid containing a polynucleotide encoding the nucleotide to be mutated with a restriction enzyme, followed by ligation of an oligonucleotide containing the mutation into the polynucleotide. Typically, the restriction enzymes used to digest the plasmid and the oligonucleotide are the same, allowing the plasmid and insert to be ligated together. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949-4955 and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966. Site-directed mutagenesis can also be achieved in vivo by methods known in the art. See, for example, U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16. Any site-directed mutagenesis procedure can be used in the present invention. Many commercially available kits are available that can be used to prepare variants.

[0391] Synthetic gene construction involves the in vitro synthesis of designed polynucleotide molecules that encode a polypeptide of interest. Gene synthesis can be performed using a number of techniques, including the multiplexed microchip-based technology described by Tian et al. (2004, Nature 432:1050-1054) and similar techniques for synthesizing and assembling oligonucleotides on optically programmable microfluidic chips.

[0392] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested by using known methods of mutagenesis, recombination and / or shuffling followed by associated screening procedures such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86, 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30; 10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204) and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46; 145; Ner et al., 1988, DNA 7; 127).

[0393] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17;893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0394] Semisynthetic gene construction is achieved by combining aspects of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semisynthetic construction typically involves a process that utilizes synthesized polynucleotide fragments in combination with PCR techniques. Thus, defined regions of a gene can be synthesized de novo, while other regions can be amplified using site-directed mutagenesis primers, and still other regions can be subjected to error-prone or non-error-prone PCR amplification. The polynucleotide subsequences can then be shuffled.

[0395] The resulting enzymes can be produced on an industrial scale and then purified. Industrial production of enzymes is usually achieved by culturing host cells that express the enzymes (also known as fermentation). Suitable host cells are described herein. A nucleic acid sequence encoding each of the enzymes described herein can be transformed into a host cell, which is then cultured under conditions suitable for producing the enzyme. In a preferred embodiment, the enzyme is purified from the host cell.

[0396] The production of the enzyme is preferably (a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding an enzyme described herein by introducing into the host cell a nucleic acid construct comprising a polynucleotide encoding an enzyme described herein; (b) culturing the recombinant host cell of step (a) under conditions conducive to expression of the polynucleotide; (c) optionally recovering the enzyme encoded by the polynucleotide; Includes:

[0397] 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 the fermentation, the fermentation broth is collected and may be further processed, and includes a liquid fraction and a solid fraction. The enzyme of interest may be further purified from the fermentation broth.

[0398] The amylases, proteases, mannanases, and cellulases described herein may or may not be secreted from the microbial cells (into the liquid fraction of the fermentation broth) (and thus contained within the cells in the fermentation broth). Accordingly, the enzymes can be recovered from the liquid fraction of the fermentation broth or from cell lysates. Preferably, the enzymes are secreted from the cells into the fermentation broth via a secretory signal peptide attached to the end of the amino acid sequence of each enzyme. Recovery of the enzymes 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, collection, 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.

[0399] WO 00 / 43502 A1, WO 2008 / 110498 A1, and WO 2017 / 097869 A1 describe methods for recovering proteins of interest from a fermentation broth that precipitate and / or crystallize 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.

[0400] The proteases, mannanases, and cellulases described herein can be purified from the fermentation broth by methods known in the art. For example, each enzyme 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 polypeptides can then be purified by various procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoresis (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 polypeptides can then be concentrated by procedures known in the art, including, but not limited to, ultrafiltration and evaporation.

[0401] Preparations containing enzyme compositions The enzyme compositions described herein may be further processed to form enzyme composition-containing preparations. Accordingly, preparations comprising the enzyme compositions described herein and at least one additional component are also claimed herein.

[0402] The present invention therefore provides a method for producing a preparation comprising the steps of: a) an enzyme composition as described herein; and b) one or more components described herein; The present invention also relates to a method comprising the step of mixing

[0403] The preparation may be a non-complex formulation, such as an enzyme composition-containing formulation, or a complex formulation, such as a detergent composition.

[0404] In one embodiment of the present invention, the enzyme composition is formulated as an enzyme composition-containing formulation, preferably a concentrated enzyme composition-containing formulation. The enzyme composition-containing formulation may be solid or liquid. The protein formulation may be obtained using techniques known in the art. For example, but not limited to, a solid enzyme formulation may 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.

[0405] The liquid enzyme formulation may contain an amount of enzyme in the range of 0.012% to 40%, 0.02% to 20%, 0.04% to 12%, 0.1% to 8% or preferably 0.2 to 6% by weight based on the total weight of the enzyme formulation.

[0406] Preferably, the enzyme preparation comprises the enzyme of the invention in an amount of 2 to 120 mg of active enzyme per gram of enzyme preparation, preferably 6 to 80 mg / g, 6 to 60 mg / g or 10 to 40 mg / g.

[0407] In one embodiment, the enzyme composition-containing formulation, particularly a liquid enzyme formulation, further comprises one or more additional compounds selected from the group consisting of solvents, salts, pH adjusters, preservatives, enzyme stabilizers, and thickeners. Preferably, the enzyme composition-containing formulation does not contain a surfactant. The solvent can be water and / or an organic solvent. The aqueous enzyme composition-containing formulation of the present invention can comprise water in an amount greater than about 50 wt%, greater than about 60 wt%, greater than about 70 wt%, or greater than about 80 wt%, based on the total weight of the enzyme formulation. The enzyme composition-containing formulation of the present invention can comprise an organic solvent in an amount greater than about 30 wt%, greater than 40 wt%, greater than about 50 wt%, greater than about 60 wt%, greater than about 70 wt%, or greater than about 80 wt%, 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.

[0408] In one embodiment, the enzyme composition-containing composition contains at least one preservative. Preferably, the term "preservative" refers to a substance added to a liquid composition for preservative purposes, and more preferably, the term "preservative" excludes compounds known to have a preservative function that are present in th...

Claims

1. A composition comprising two or more enzymes selected from the group consisting of proteases, mannanases, and cellulases, a. The aforementioned protease is I. A protease variant of the parent protease, (i) an amino acid sequence that, compared to SEQ ID NO: 16, includes at least two additional negative charges in the loop region of residues 98-104 according to the numbering of SEQ ID NO: 17, and (ii) Any of SEQ ID NOs: 16 to 34, preferably an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 16 Protease variants containing, or II. A protease variant of the parent protease, (i) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 3, 4, 9, 19, 22, 43, 62, 63, 76, 87, 99, 101, 103, 104, 116, 118, 128, 129, 130, 156, 160, 167, 170, 182, 185, 188, 194, 205, 213, 217, 218, 232, 245, 256, 259, 262 and 271 according to the numbering of SEQ ID NO: 17, and an amino acid sequence having at least 60% but less than 100% sequence identity with SEQ ID NO: 22, or (ii) Amino acid substitutions at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 24, 53, 76, 78, 101, 128 and 217 according to the numbering of Sequence ID No. 17, and an amino acid sequence having at least 60% but less than 100% sequence identity with Sequence ID No. 17, or (iii) Amino acid substitutions at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 9, 89, 130, 133, 144, 159, 183, 189, 217, 224, 238, 243, 252, 259, 270, and 271 according to the numbering of Sequence ID No. 19, and an amino acid sequence having at least 60% but less than 100% sequence identity with Sequence ID No. 19, or (iv) Amino acid sequences having at least 60% sequence identity with SEQ ID NO: 22, SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 31 Protease variants containing Selected from, b. The aforementioned mannanase is I. A mannanase variant of the parent mannanase, (i) One or more amino acid substitutions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379, according to the numbering of Sequence ID No. 35, and (ii) An amino acid sequence that is at least 60% but less than 100% identical to the sequence of positions 31-490 of Sequence ID No.

35. Mannanase variants containing, or II. A mannanase variant of the parent mannanase, (i) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 24, 123, and 256 according to the numbering of SEQ ID NO: 39, and an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NO: 39, or (ii) Amino acid substitutions at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 10, 19, 30, 38, 59, 60, 67, 97, 103, 129, 143, 167, 168, 184, 225, 228, 235, 244, 258, 261 and 298 according to the numbering of Sequence ID No. 38, and an amino acid sequence that is at least 60% but less than 100% identical to Sequence ID No. 38, or (iii) An amino acid sequence that is at least 60% identical to SEQ ID NO: 40, or (iv) Amino acid substitutions at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 30, 81, 93, 95, 118, 138, 139, 200, 202, 210, 260, 283, 288, 294, and 295 according to the numbering of Sequence ID No. 40, and amino acid sequences that are at least 60% but less than 100% identical to Sequence ID No.

40. Mannanase variants containing Selected from, c. The cellulase is, I. A cellulase variant containing an amino acid sequence that is at least 60% identical to SEQ ID NO: 42, or II. Cellulase variants containing an amino acid sequence that is at least 60% identical to SEQ ID NO: 46, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 44, or SEQ ID NO: 47 Selected from, At least one type of enzyme, aa. A protease variant of the parent protease, (i) an amino acid sequence that, compared to SEQ ID NO: 16, includes at least two additional negative charges in the loop region of residues 98-104 according to the numbering of SEQ ID NO: 17, and (ii) Amino acid sequences of SEQ ID NOs: 16-34, preferably SEQ ID NO: 16, which are at least 60% but less than 100% identical. Protease variants including bb. A mannanase variant of the parent mannanase, (i) One or more amino acid substitutions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, 122, 124, 126, 127, 129, 231, 233, 235, 244, 254, 255, 264, 270, 272, 273, 274, 281, 286, 289, 290, 296, 301, 309, 312, 314, 317, 319, 341, 346, 348, 349, 352, 356 and 379, according to the numbering of Sequence ID No. 35, and (ii) An amino acid sequence that is at least 60% but less than 100% identical to the sequence of positions 31-490 of Sequence ID No.

35. Mannanase variants including, cc. Cellulase variant containing an amino acid sequence that is at least 60% identical to SEQ ID NO: 42 A composition selected from the group consisting of the following.

2. The composition according to claim 1, wherein the protease variant has at least 80%, but less than 100%, sequence identity with SEQ ID NO: 16, and comprises an amino acid substitution at position 101, preferably X101E, according to the numbering of SEQ ID NO:

17.

3. The aforementioned mannanase variants have at least 80% but less than 100% sequence identity with SEQ ID NO: 35, and follow the numbering of SEQ ID NO: 35, and are X86N, X89V / L, X96D, X101T / V, X103Y / E / A, X107N, X108G, X109Q / A, X112N, X119Y / H / T, X122S, X1 24E / C / D, X126E, X127A, X129M / L / F, X231Q / K / L / P / Y, X233V, X235H / R / L / Q / N / Y, X244I / V / N, X254W, X255Y / H / R, X264Q / V, X270T, X272I, X273T, X274E / C / Q, X281L, Q / A, X289F / M / H, X290A, X296H / F / Y, X301E / C / T, X309L, X312F / Y, X314P, X317T, X319D / E, X341F, X346T, X348S / R / N / M / G, X349T / S / G / D, X352N / G, X356Y / V / T / Q / H / C and X379V The composition according to claim 1, comprising one or more amino acid substitutions selected from the group consisting of, preferably from X31V, X89V, X96D, X119Y / H / T, X264Q / V, X289F / M / H, X312F / Y, X348S / R / N / M / G, X349T / S / D / G, X352N / G, and X379V.

4. The composition according to claim 1, wherein the cellulase variant comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to SEQ ID NO:

42.

5. Preferably, a) An amylase variant of the parent amylase, (i) Consists of an amino acid substitution at position 25 in accordance with the numbering of Sequence ID No. 2, (ii) comprising amino acid substitutions at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 4, 116, 176, 181, 186, 195, 206, 225, 251, 320, 405 and 482 according to the numbering of Sequence ID No. 2, and (iii) Any of sequence numbers 1 to 15, preferably an amylase variant having at least 60%, preferably at least 91%, identity with sequence number 1, but less than 100% sequence identity, and b) An amylase variant of the parent amylase, (i) an amino acid substitution at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 183, 243, 266, and 475 according to the numbering of Sequence ID No. 9, and an amino acid sequence having at least 60% but less than 100% sequence identity with Sequence ID No. 9, or (ii) Amino acid substitutions at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 38, 126, 129, 153, 180, 187, 199, 203, 241, 362, 377, 458, 459, 460, 476 and 477 according to the numbering of Sequence ID No. 7, and an amino acid sequence having at least 60% but less than 100% sequence identity with Sequence ID No. 7, or (iii) Amino acid substitutions at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 1, 7, 51, 52, 54, 109, 140, 195, 206, 243, 260, 280, 284, 304, 391, 476 and 477 according to the numbering of Sequence ID No. 3, and an amino acid sequence having at least 60% but less than 100% sequence identity with Sequence ID No. 3, or (iv) Amino acid substitutions at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 9, 118, 149, 182, 186, 195, 202, 246, 257, 295, 299, 320, 323, 339, 345, 458 and 471 according to the numbering of Sequence ID No. 13, and an amino acid sequence having at least 60% but less than 100% sequence identity with Sequence ID No. 13, or (v) Amino acid substitutions at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 1, 54, 56, 72, 109, 113, 116, 149, 167, 172, 174, 182, 195, 202, 206, 246, 257, 295, 299, 320, 339, 345, 391, 458, 471, 473 and 476 according to the numbering of Sequence ID No. 15, and an amino acid sequence having at least 60% but less than 100% sequence identity with Sequence ID No. 15, or (vi) Amino acid substitutions at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 9, 118, 149, 182, 186, 195, 202, 246, 257, 295, 299, 320, 323, 339, 345, 405, 421, 422, 428, 458 and 471 according to the numbering of Sequence ID No. 4, and an amino acid sequence having at least 60% but less than 100% sequence identity with Sequence ID No. 4, or (vii) Amino acid substitutions at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of 1, 54, 56, 72, 109, 113, 116, 120, 134, 140, 167, 169, 172, 173, 174, 182, 183, 184, 195, 202, 206, 246, 255, 257, 260, 265, 295, 299, 304, 320, 339, 345, 391, 395, 410, 422, 429, 444, 451, 458, 473, 474, and 476 according to the numbering of Sequence ID No. 5, and amino acid sequences having at least 60% but less than 100% sequence identity with Sequence ID No.

5. amylase variants containing The composition according to claim 1, further comprising an amylase selected from.

6. The composition according to claim 5, wherein the amylase variant has at least 80%, but less than 100%, sequence identity with SEQ ID NO: 1, and one or more amino acid substitutions selected from the group consisting of X25H, X4Q, X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X251E, X320K, X405M, and X482W, according to the numbering of SEQ ID NO:

2.

7. The composition according to claim 1, comprising one or more additional enzymes selected from the group consisting of lipase, hemicellulase, xylanase, DNase, dispersin, pectinase, oxidoreductase, cutinase, phosphodiesterase, licheninase, xanthan lyase, and hexosaminidase, preferably lipase.

8. The composition according to claim 1, which is a detergent composition, preferably a laundry detergent composition or a hard surface cleaning detergent composition.

9. The composition according to claim 8, wherein the detergent composition comprises detergent components selected from the group consisting of enzyme stabilizers, surfactants, defoamers, builders, polymers, bleaching agents (bleachers), rheological modifiers, hydrotropes, softeners, desiccants, whitening agents, buffer solutions, antifoaming agents, preservatives, corrosion inhibitors, dyes, and fragrances.

10. The composition according to claim 1, wherein a combination of at least two enzymes selected from protease, mannanase, and cellulase in the enzyme composition provides a synergistic cleaning effect.

11. A method for producing the composition described in claim 1, comprising the step of mixing at least two enzymes selected from protease, mannanase, and cellulase.

12. A cleaning method comprising the step of bringing a textile product or a hard surface into contact with the composition described in claim 1, preferably at a low temperature.

13. The method according to claim 12, for cleaning composite stains from textile products or hard surfaces.

14. Preferably, use of the composition according to claim 1 to provide a synergistic cleaning effect at low temperatures.

15. Use of the composition according to claim 1 for removing complex stains, preferably at a low temperature.