Enzyme composition comprising protease, mannanase and / or cellulase

Abstract

Novel enzyme compositions are provided in the present invention. More particularly, the present invention provides enzyme compositions comprising at least two enzymes selected from the group consisting of proteases, mannanases and cellulases and methods of making and using these enzyme compositions.

Description

Technical Field

[0001] In the present invention, novel enzyme compositions are provided. More particularly, the present invention provides enzyme compositions comprising at least two enzymes selected from the group consisting of proteases, mannanases and cellulases, and methods of making and using these enzyme compositions. Background Art

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

[0003] The most typically used enzymes in detergents are proteases and amylases, while mannanases and cellulases are used less frequently. Proteases, amylases, mannanases, and cellulases are all hydrolases that degrade different substrates. Proteases degrade proteins, amylases degrade starch, mannanases degrade galactomannan, and cellulases degrade cellulose.

[0004] However, successful cleaning often depends on the effectiveness of removing stains composed of different types of soils. Therefore, successful cleaning depends on the effective removal of stains that contain a complex mixture of proteins, starches, galactomannans, cellulose and other types of partially undefined soils. The inventors of the present application have identified a combination of specific enzymes that are particularly suitable for removing such complex stains. Summary of the invention

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

[0006] Compositions using the specific enzymes of the present invention provide beneficial cleaning effects when cleaning objects. Therefore, the present invention also relates to the use of the enzyme composition for cleaning objects and detergent compositions comprising the enzyme composition. DETAILED DESCRIPTION

[0007] The present invention may be understood more readily by reference to the following detailed description of embodiments of the invention and the Examples included therein.

[0008] Although the invention will be described with respect to specific embodiments, this description should not be construed in a limiting sense.

[0009] definition

[0010] Unless otherwise noted, terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0011] Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given. Unless otherwise stated or obvious from the nature of the definitions, the definitions apply to all compounds, methods and uses described herein.

[0012] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include the corresponding plural forms as well, unless the context clearly dictates otherwise.

[0013] In the context of the present invention, the terms "about" and "approximately" represent an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the discussed feature. The term typically indicates a deviation from the indicated numerical value of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5%.

[0014] In addition, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" and the like in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequential order or a chronological order. It should be understood that the terms so used are interchangeable where appropriate, and that the embodiments of the invention described herein are capable of operating in other sequences than those described or illustrated herein. Where the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", "i", "ii" and the like refer to steps of a method or use or an assay, there is no temporal or temporal time interval coherence between these steps, i.e., these steps may be performed simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in this application as described above or below.

[0015] Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0016] It should be understood that the term "comprising" is not limiting. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising at least a certain number of members, this means that a group consisting only of these members is also encompassed.

[0017] A "variant" enzyme differs from a "parent" enzyme by certain amino acid alternations, preferably by amino acid substitutions at one or more amino acid positions.

[0018] In describing the variants of the invention, abbreviations for the individual amino acids are used according to the generally accepted IUPAC single-letter or three-letter amino acid abbreviations.

[0019] As used herein, "amino acid change" refers to an amino acid substitution, deletion or insertion.

[0020] "Substitutions" are described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the amino acid that replaces the original amino acid. For example, a histidine at position 120 replaced by an alanine is represented as "His120Ala" or "H120A". Substitutions can also be described by naming only the resulting amino acid in the variant, e.g., "X120A" or "120A" or "Xaa120Ala" or "120Ala", without specifying the parent amino acid at that position.

[0021] "Deletions" are described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by *. Accordingly, the deletion of glycine at position 150 is indicated as "Gly150*" or "G150*". Alternatively, a deletion is indicated by, for example, "deletion of D183 and G184".

[0022] "Insertion" is described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the original amino acid and the newly added amino acid. For example, the insertion of lysine at position 180 near glycine is represented as "Gly180GlyLys" or "G180GK". When more than one amino acid residue is inserted, such as, for example, Lys and Ala are inserted after Gly180, such insertion may be represented as: "Gly180GlyLysAla" or "G195GKA".

[0023] In the case where the substitution and insertion occur at the same position, this can be expressed as "S99SD+S99A" or simply "S99AD". Variants containing multiple changes are separated by a plus sign "+", for example, "Arg170Tyr+Gly195Glu", "R170Y+G195E" or "X170Y+X195E" represent that the arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively. Alternatively, multiple changes can be separated by spaces or commas, for example, "R170Y G195E" or "R170Y, G195E". When different substitution changes can be introduced at a certain position, the different changes are separated by commas, for example, "Arg170Tyr, Glu" and "R170T, E" represent that the arginine at position 170 is replaced by tyrosine or glutamic acid, respectively. The substitution of a specific position can also be represented as "X120A, G, H", "120A, G, H", "X120A / G / H" or "120A / G / H". Alternatively, different changes or optional substitutions can be represented by brackets, such as "Arg170[Tyr, Gly]" or "Arg170{Tyr, Gly}" or abbreviated as "R170[Y, G]" or "R170{Y, G}".

[0024] The term "native" (or naturally or wild-type or endogenous) cell or organism or polynucleotide or polypeptide refers to a cell or organism or polynucleotide or polypeptide as found in nature (ie, without any human intervention).

[0025] The term "introducing at least two negative charges into a specific amino acid sequence" refers to an increase in the net charge of a specific amino acid sequence of at least two negative charges. Such an increase in the net charge of a specific amino acid sequence of at least two negative charges is achieved by changing the amino acid sequence, and can be achieved by one or more amino acid sequence changes selected from the group consisting of substitution, deletion and insertion, preferably by one or more amino acid substitutions. The net charge of a specific amino acid sequence can be increased by at least two negative charges by removing positive charges or by introducing negative charges or a combination thereof. The four amino acids aspartic acid (Asp, D), glutamic acid (Glu, E), lysine (Lys, K) and arginine (Arg, R) have side chains that can be charged at neutral pH. At pH 7.0, two are negatively charged: aspartic acid (Asp, D) and glutamic acid (Glu, E) (acidic side chains), and two are positively charged: lysine (Lys, K) and arginine (Arg, R) (basic side chains). Thus, the introduction of at least two negative charges in the amino acid sequence can be achieved, for example, by replacing arginine with glutamic acid, replacing two uncharged leucine residues with two glutamic acid residues, by inserting two aspartic acid residues or by deleting two lysine residues. The introduction of at least two negative charges by amino acid sequence modification is preferably evaluated under conditions that typically occur in a washing step, preferably at pH 6-11, preferably at pH 7-9, more preferably at pH 7.5-8.5, further preferably at pH 7.0-8.0, and most preferably at pH 7.0 or pH 8.0.

[0026] The term "heterologous" (or exogenous or foreign or recombinant or non-original or non-naturally) polypeptide is defined herein as a polypeptide that is not native to a host cell; a polypeptide that is native to a host cell, wherein structural modifications (e.g., deletions, substitutions, and / or insertions) have been made by recombinant DNA techniques to alter the native polypeptide; or a polypeptide that is native to a host cell, the expression of which is quantitatively altered or the expression of which is from a genomic location different from that of the native host cell due to manipulation of the host cell DNA by recombinant DNA techniques (e.g., a stronger promoter). Similarly, the term "heterologous" (or exogenous or foreign or recombinant or non-original or non-naturally) polynucleotide refers to a polynucleotide that is not native to a host cell; a polynucleotide that is native to a host cell, wherein structural modifications (e.g., deletions, substitutions, and / or insertions) have been made by recombinant DNA techniques to alter the native polynucleotide; or a polynucleotide that is native to a host cell, whose expression is quantitatively altered due to manipulation of the regulatory elements of the polynucleotide by recombinant DNA techniques (e.g., a stronger promoter); or a polynucleotide that is native to a host cell, but has not been integrated into its natural genetic environment due to genetic manipulation by recombinant DNA techniques. With respect to the relationship between two or more polynucleotide sequences or the relationship between two or more amino acid sequences, the term "heterologous" is used to characterize that the two or more polynucleotide sequences or the two or more amino acid sequences do not naturally occur in a specific combination with each other.

[0027] For the purposes of this invention, "recombinant" (or transgenic) with respect to a cell or organism means that the cell or organism contains a heterologous polynucleotide, which has been introduced by a human using genetic techniques. With respect to polynucleotides, "recombinant" includes all constructs produced using genetic techniques / recombinant DNA technology, wherein

[0028] (a) a polynucleotide sequence or a portion thereof, or

[0029] (b) one or more genetic control sequences, including but not limited to a promoter, operably linked to the polynucleotide, or

[0030] (c) Both a) and b)

[0031] Not in its wild-type genetic context or has been artificially modified.

[0032] "Synthetic" compounds are obtained by in vitro chemical and / or enzymatic synthesis.

[0033] Variant polynucleotides and variant polypeptide sequences can be defined by their sequence identity when compared to another sequence. Sequence identity is usually provided in the form of "% sequence identity" or "% identity". In order to calculate sequence identity, the first step is to generate a sequence alignment. According to the present invention, a paired global alignment is generated, which means that the two sequences are aligned over their entire length, which is usually generated by using a mathematical method known as an alignment algorithm.

[0034] According to the present invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J.Mol.Biol. [Journal of Molecular Biology] (1979) 48, pp. 443-453). Preferably, the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the present invention, wherein the program default parameters are used (polynucleotides: gap opening = 10.0, gap extension = 0.5 and matrix = EDNAFULL; polypeptides: gap opening = 10.0, gap extension = 0.5, matrix = EBLOSUM62). After the two sequences are aligned, in a second step, the identity value is determined based on the alignment generated. For this purpose, % identity is calculated by dividing the number of identical residues by the length of the alignment region showing the corresponding sequence of the present invention in its full length multiplied by 100: % identity = (identical residues / length of the alignment region showing the corresponding sequence of the present invention in its full length) * 100.

[0035] For calculating the percentage identity of two nucleic acid sequences, it is also applicable to calculate the percentage identity of two amino acid sequences with certain specifications. For the nucleic acid sequence of coded protein, pairwise comparison should be carried out on the complete length (from start codon to stop codon, excluding intron) of the coding region of the sequence of the present invention. Introns present in other sequences compared with the sequence of the present invention should also be removed for pairwise comparison. After comparing the two sequences, in the second step, the identity value is determined according to the comparison produced. Percent identity is calculated by: %-identity=(same residue / show the length of the comparison region of the sequence of the present invention from start codon to stop codon and excluding intron on its complete length)*100.

[0036] In addition, a preferred alignment program for nucleic acid sequences implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, pp. 443-453) is "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)), using the program default parameters (gap opening = 10.0, gap extension = 0.5, and matrix = EDNAFULL).

[0037] Variant polypeptides can also be defined by their sequence similarity when compared to another sequence. Sequence similarity is usually provided as "% sequence similarity" or "%-similarity". The % sequence similarity takes into account that the defined amino acid sets have similar properties, for example by their size, by their hydrophobicity, by their charge or by other features. Here, the exchange of one amino acid with another can be referred to as a "conservative mutation". The definition of similar amino acids according to the present invention is as follows, which should also apply to the determination of %-similarity according to the present invention, which is also consistent with the BLOSUM62 matrix used by the program "NEEDLE", which is one of the most commonly used amino acid similarity matrices for database searches and sequence alignments:

[0038] Amino acid A is similar to amino acid S

[0039] Amino acid D is similar to amino acid E; N

[0040] Amino acid E is similar to amino acid D; K; Q

[0041] Amino acid F is similar to amino acid W; Y

[0042] Amino acid H is similar to amino acid N; Y

[0043] Amino acid I is similar to amino acid L; M; V

[0044] Amino acid K is similar to amino acid E; Q; R

[0045] Amino acid L is similar to amino acid I; M; V

[0046] Amino acid M is similar to amino acid I; L; V

[0047] Amino acid N is similar to amino acid D; H; S

[0048] Amino acid Q is similar to amino acid E; K; R

[0049] Amino acid R is similar to amino acid K; Q

[0050] Amino acid S is similar to amino acid A; N; T

[0051] Amino acid T is similar to amino acid S

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

[0053] Amino acid W is similar to amino acid F; Y

[0054] Amino acid Y is similar to amino acid F; H; W

[0055] To calculate sequence similarity, in a first step, a sequence alignment is generated as described above. After the two sequences have been aligned, in a second step, a similarity value is determined based on the alignment generated. For this purpose, %-similarity is calculated by adding the number of identical residues to the number of similar residues divided by the length of the alignment region showing the sequence of the invention over its full length multiplied by 100: %-similarity=[(identical residues+similar residues) / length of the alignment region showing the sequence of the invention over its full length]*100.

[0056] "Fragment" or "subsequence" used herein is a part of a polynucleotide or amino acid sequence. The term "functional fragment" refers to any nucleic acid or amino acid sequence that only comprises a part of a full-length amino acid sequence, but still has the same or similar activity and / or function. Preferably, the functional fragment is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99% or at least 99.5% identical to the original full-length amino acid sequence. The functional fragment comprises consecutive nucleotides or amino acids compared to the original nucleic acid or original amino acid sequence, respectively.

[0057] As used herein, a "gene construct" or "expression cassette" is a nucleic acid molecule consisting of at least one sequence of interest to be expressed, which is operably linked to one or more control sequences as described herein (at least to a promoter).

[0058] As used herein, the term "vector" includes any kind of construct suitable for carrying an exogenous polynucleotide sequence to transfer to another cell or to stably or transiently express in a given cell. As used herein, the term "vector" encompasses any kind of cloning vehicle, such as, but not limited to, plasmids, phagemids, viral vectors (e.g., phage), phages, baculoviruses, cosmids, forsmids, artificial chromosomes, and any other vectors specific to a particular host of interest. Exogenous polynucleotide sequences typically include a coding sequence that may be referred to herein as a "target gene". The target gene may include introns and exons, depending on the source or destination of the host cell.

[0059] The term "introduction of polynucleotides" or "transformation of polynucleotides" referred to herein encompasses the transfer of exogenous polynucleotides into host cells, regardless of the method used for transfer. That is, as used herein, the term "transformation of polynucleotides" is independent of vectors, shuttle systems or host cells, and it not only relates to polynucleotide transfer methods of transformation known in the art (see, e.g., Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), but it also encompasses any other kind of polynucleotide transfer method, such as, but not limited to, transduction or transfection.

[0060] A polynucleotide encoding a polypeptide can be "expressed". The term "expression" or "gene expression" means that one or more genes or gene constructs are transcribed into structural RNA (e.g., rRNA, tRNA) or mRNA, which is then translated into protein or not. The process includes DNA transcription and processing of the resulting mRNA product.

[0061] The term "purification" or "purifying" refers to a process in which at least one component (e.g., a protein of interest) is separated from at least another component (e.g., particulate matter of a fermentation broth) and transferred to different compartments or phases, wherein the different compartments or phases do not necessarily need to be separated by a physical barrier. Examples of such different compartments are two compartments separated by a filter membrane or cloth, namely a filtrate and a retentate; examples of such different phases are precipitate and supernatant or filter cake and filtrate, respectively. The solution obtained after purifying the enzyme of interest from the fermentation broth is referred to herein as a "purified enzyme solution."

[0062] By "protein formulation" (or "enzyme preparation"), e.g., "protein variant formulation", it is meant any non-complex formulation comprising minor ingredients, wherein these ingredients are used for the purpose of stabilizing the protein contained in the protein formulation and / or the stability of the protein formulation itself. Preferably, the non-complex protein formulation comprises a higher concentration of protein than a complex formulation (e.g., than a detergent composition). Thus, preferably, the non-complex protein formulation is a concentrated protein variant formulation. Preferably, the non-complex protein formulation comprises 2 mg / g to 120 mg / g active enzyme, wherein a complex formulation such as a detergent composition comprises 0.002 mg / g to 6 mg / g active enzyme.

[0063] "Enzyme properties" include, but are not limited to, catalytic activity, substrate / cofactor specificity, product specificity, stability over time, thermal stability, pH stability, and chemical stability. "Enzyme activity" or "catalytic activity" means the catalytic effect exerted by an enzyme, expressed in units / mg of enzyme (specific activity) or substrate molecules converted / minute / enzyme molecule (molecular activity). Enzyme activity can be specified by the actual function of the enzyme, for example, proteases exert proteolytic activity by catalyzing the hydrolytic cleavage of peptide bonds, lipases exert lipolytic activity by hydrolytic cleavage of ester bonds, amylase activity involves the hydrolysis of glycosidic linkages in polysaccharides, etc.

[0064] 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.

[0065] In order to determine and quantify the catalytic activity of an enzyme stored or used under certain conditions over time, the "initial enzyme activity" is measured at time zero (100%) and at a later time point (x%) under the specified conditions. By comparing the measured values, the potential loss of enzyme activity can be determined. The extent of enzyme activity loss determines the stability or instability of the enzyme itself or in the formulation.

[0066] As used herein, "enzyme inhibitors" are compounds that slow down or stop the activity of an enzyme. Enzyme inhibitors can also typically stabilize the three-dimensional structure of an enzyme. Therefore, enzyme inhibitors also typically act as "enzyme stabilizers."

[0067] "pH stability" refers to the ability of an enzyme to exert enzymatic activity after exposure to a certain pH value.

[0068] The terms "thermal stability", "thermostability" or "temperature-dependent activity" refer to the ability of an enzyme to exert catalytic activity or washing performance after exposure to elevated temperatures, preferably at a temperature of 40°C for 28 days, more preferably 56 days, preferably in a detergent composition (preferably, in a model ES1-C detergent), or at 92°C for at least 10 min.

[0069] The term "detergent stability" or "stability under storage in a detergent composition" refers to the ability of an enzyme to exert catalytic activity or washing performance after storage in a detergent composition, preferably in a detergent composition (preferably, in a model ES1-C detergent) at a temperature of 40°C or 50°C for 28 days, more preferably 56 days.

[0070] As used herein, the "washing performance" (also referred to herein as "cleaning performance") of an enzyme refers to the contribution of the enzyme to the cleaning performance of a detergent composition, i.e., the cleaning performance added to the detergent composition by the performance of the enzyme. The term "washing performance" is similarly used herein for laundry and hard surface cleaning. Washing performance is compared under relevant washing conditions. The term "relevant washing conditions" is used herein to indicate the conditions actually used by households in the detergent market segment, particularly washing temperature, time, washing machinery, foam concentration, detergent type, and water hardness. The term "improved washing performance" is used to indicate that a better final result is obtained in stain removal under relevant washing conditions, or that less enzyme is required to obtain the same final result on a weight basis relative to corresponding control conditions.

[0071] In this article, the term "specific performance" refers to the ability of each unit of active enzyme to clean and remove specific stains or dirt. In some embodiments, specific performance is determined using stains or dirt (e.g., eggs, egg yolks, milk, grass, minced meat, blood, chocolate sauce, baby food, sebum, etc.).

[0072] "Detergent composition", "detergent formulation" or "detergent" refers to a composition designated for cleaning soiled materials. Detergent compositions according to the present invention include detergent compositions for different applications such as laundry and hard surface cleaning. The term "detergent component" is defined herein to mean a class of chemicals that can be used in detergent compositions. Typical detergent components are surfactants. "Surfactant" (herein used synonymously with "surfactant (surfaceactive agent)") means an organic chemical that changes the properties of a liquid at an interface when added to a liquid. Depending on its ionic charge, surfactants are referred to as nonionic surfactants, anionic surfactants, cationic surfactants or amphoteric surfactants. The term "effective amount of a detergent component" includes the amount of certain components that provide effective stain removal and / or effective cleaning conditions (e.g., pH, temperature, water hardness), the amount that effectively provides optical benefits (e.g., optical brightening, dye transfer inhibition, color care), and the amount of certain components that effectively assist processing (maintaining physical properties during processing, storage and use; e.g., rheology modifiers, hydrotropes, desiccants).

[0073] The term "laundry" or "laundering" refers to both domestic and industrial laundry and means the process of treating textiles and / or fabrics with a solution containing the detergent composition of the present invention. The laundering process can be carried out using technical equipment such as domestic or industrial washing machines. Alternatively, the laundering process can be done manually.

[0074] The term "textile" means any textile material, including yarn (thread made of natural or synthetic fibers for knitting or weaving), yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and fabrics such as clothing, cloth and other articles made from these materials. As used herein, the term "fabric" (a textile made from woven, knitted or felted fibers) or "garment" (any clothing made from textiles) is also meant to include the broader term textiles.

[0075] The term "fiber" includes natural fibers, synthetic fibers and mixtures thereof. Examples of natural fibers are plant fibers (e.g. flax, jute and cotton) or fibers of animal origin, containing proteins such as collagen, keratin and fibroin (e.g. silk, sheep wool, angora wool, mohair, cashmere). Examples of fibers of synthetic origin are polyurethane fibers such as or Polyester fibers, polyolefins such as elastofin, or polyamide fibers such as nylon. The fibers may be single fibers or part of a textile, such as a knitted, woven or nonwoven fabric.

[0076] The term "hard surface cleaning" refers to both domestic hard surface cleaning and industrial hard surface cleaning and means the process of treating hard surfaces with a solution containing the detergent composition of the present invention. Hard surfaces can include any hard surface in the home or industry, such as floors, furniture, walls, sanitary ceramics, glass, metal surfaces (including medical equipment), knives and tableware. A special form of hard surface cleaning is dishwashing, manual dishwashing (MDW) or automatic dishwashing (ADW).

[0077] The term "dishwashing" refers to all forms of dishwashing, such as manual dishwashing or automatic dishwashing. Dishwashing includes, but is not limited to, cleaning all forms of crockery, such as plates, cups, glasses, bowls, all forms of cutlery, such as spoons, knives, forks and serving utensils, as well as ceramics, plastics, such as melamine, metal, porcelain, glass and acrylic.

[0078] The cleaning performance is evaluated under relevant cleaning conditions. In the present context, the term "relevant cleaning conditions" refers to the conditions actually used in washing machines, automatic dishwashers or in manual cleaning processes, in particular cleaning temperature, time, cleaning regime, foam concentration, detergent type and water hardness.

[0079] The term "medical device cleaning" refers to the cleaning steps involved in reprocessing reusable medical devices. Medical device cleaning methods can be divided into two categories: manual cleaning methods and mechanical / automatic cleaning methods. Manual cleaning is used when no mechanical device is available or the medical device to be cleaned is too fragile or difficult to clean with mechanical devices. Mechanical / automatic cleaning methods remove dirt and microorganisms through an automated cleaning and rinsing process that includes ultrasonic cleaning and washing.

[0080] In the field of soil removal, the term "stain" is generally used to refer to laundry, such as the cleaning of textiles, fabrics or fibers, while the term "soil" is generally used to refer to hard surface cleaning, such as the cleaning of dishes and cutlery. However, the terms "stain" and "soil" are used interchangeably herein.

[0081] As used herein, "chelating builders" are distinguished from precipitating builders because no substantial precipitate is formed when the builder is used in an amount sufficient to combine with all calcium ions in an aqueous solution initially having a hardness of 7° dH (German hardness) at neutral pH. "Strong builders" are classified as highly efficient chelating agents that are capable of strongly binding divalent cations (e.g., Ca2+), with a logarithmic stability constant (Log K of the cation / chelating agent complex) of 1.17kcal / cm2. Ca ) is greater than 4, especially greater than 5, greater than 6 or greater than 7. The stability constant is determined at an ionic strength of 0.1 M and a temperature of 25° C. "Strong chelating builders" combine the above two properties.

[0082] "Complex stains" (also referred to herein as "mixed stains") are stains comprising more than one soil component selected from fat, protein, starch, mannan, cellulose, preferably wherein the stain comprises a thickener such as galactomannan or locust bean gum. An example of a complex stain is a salad dressing stain. Other food stains such as chocolate pudding or baby food are considered complex stains.

[0083] As used herein, the term "synergistic action" or "synergistic effect" refers to the interaction of two or more enzymes to produce a combined effect (eg, wash performance) that is greater than the sum of their individual effects.

[0084] As used herein, the term "low temperature" refers to a temperature range equal to or lower than 40°C, preferably 10-40°C, more preferably 20-40°C, more preferably 20-35°C.

[0085] Detailed Description

[0086] In the present invention, novel enzyme compositions are provided. More particularly, the present invention provides enzyme compositions comprising two or more enzymes selected from the group consisting of proteases, mannanases and cellulases, and methods of making and using these enzyme compositions.

[0087] Specifically, the inventors have found that at least one enzyme selected from the following is used in such an enzyme composition:

[0088] aa. A protease variant of a parent protease, wherein the protease variant comprises

[0089] (i) compared to SEQ ID NO: 16, comprises at least two additional negatively charged amino acid sequences in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, and

[0090] (ii) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NOs: 16-34, preferably SEQ ID NO: 16,

[0091] bb. a mannanase variant of a parent mannanase, wherein the mannanase variant comprises

[0092] (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

[0093] (ii) 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

[0094] cc. A cellulase comprising an amino acid sequence at least 60% identical to SEQ ID NO: 42,

[0095] Provides a beneficial cleaning effect.

[0096] Composition

[0097] In one embodiment, the present invention is directed to an enzyme composition.

[0098] In particular, the present invention relates to a composition comprising two or more enzymes selected from the group consisting of proteases, mannanases and cellulases, wherein

[0099] a. The protease is selected from

[0100] I. A protease variant of a parent protease, wherein the protease variant comprises

[0101] (i) compared to SEQ ID NO: 16, comprises at least two additional negatively charged amino acid sequences in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, and

[0102] (ii) an amino acid sequence that is at least 60% but less than 100% identical to any one of SEQ ID NOs: 16-34, preferably SEQ ID NO: 16, or

[0103] II. A protease variant of a parent protease, wherein the protease variant comprises

[0104] (i) an amino acid substitution at one or more amino acid positions corresponding to the 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

[0105] (ii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO: 17, or

[0106] (iii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO: 19, or

[0107] (iv) an amino acid sequence that has 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,

[0108] b. The mannanase is selected from

[0109] I. A mannanase variant of a parent mannanase, wherein the mannanase variant comprises

[0110] (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

[0111] (ii) 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, or

[0112] II. A mannanase variant of a parent mannanase, wherein the mannanase variant comprises

[0113] (i) an amino acid substitution at one or more amino acid positions corresponding to the 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 at least 60% but less than 100% identical to SEQ ID NO:39, or

[0114] (ii) an amino acid substitution at one or more amino acid positions corresponding to the 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, and amino acid sequences that are at least 60% but less than 100% identical to SEQ ID NO:38, or

[0115] (iii) an amino acid sequence that is at least 60% identical to SEQ ID NO: 40, or

[0116] (iv) an amino acid substitution at one or more amino acid positions corresponding to the 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, and an amino acid sequence at least 60% but less than 100% identical to SEQ ID NO:40,

[0117] c. The cellulase is selected from

[0118] I. A cellulase variant comprising an amino acid sequence at least 60% identical to SEQ ID NO: 42, or

[0119] II. A cellulase variant 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, and

[0120] Wherein at least one enzyme is selected from the group consisting of

[0121] aa. A protease variant of a parent protease, wherein the protease variant comprises

[0122] (i) compared to SEQ ID NO: 16, comprises at least two additional negatively charged amino acid sequences in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, and

[0123] (ii) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NOs: 16-34, preferably SEQ ID NO: 16,

[0124] bb. a mannanase variant of a parent mannanase, wherein the mannanase variant comprises

[0125] (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

[0126] (ii) an amino acid sequence that is at least 60% but less than 100% identical to the sequence according to positions 31-490 of SEQ ID NO:35, and

[0127] cc. A cellulase comprising an amino acid sequence at least 60% identical to SEQ ID NO:42.

[0128] In a preferred embodiment, the present invention relates to an enzyme composition comprising two or more enzymes selected from the group consisting of proteases, mannanases and cellulases, wherein

[0129] a. The protease is selected from

[0130] I. A protease variant of a parent protease, wherein the protease variant comprises

[0131] (i) compared to SEQ ID NO: 16, comprises at least two additional negatively charged amino acid sequences in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, and

[0132] (ii) an amino acid sequence that is at least 60% but less than 100% identical to any one of SEQ ID NOs: 16 to 34, preferably SEQ ID NO: 16,

[0133] b. The mannanase is selected from

[0134] I. A mannanase variant of a parent mannanase, wherein the mannanase variant comprises

[0135] (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

[0136] (ii) an amino acid sequence that is at least 60% but less than 100% identical to the sequence according to positions 31-490 of SEQ ID NO:35, and

[0137] c. The cellulase is selected from

[0138] I. A cellulase variant comprising an amino acid sequence at least 60% identical to SEQ ID NO:42.

[0139] 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 relates to a composition comprising a protease, a mannanase and a cellulase as described herein. In one embodiment, the composition further comprises an amylase. Therefore, in another embodiment, the composition comprises a protease, a mannanase, and a cellulase and an amylase as described herein.

[0140] Preferably, the composition comprises protease and mannanase, protease and cellulase, mannanase and cellulase, or protease, mannanase and cellulase, wherein the protease, cellulase and mannanase are selected from

[0141] aa. A protease variant of a parent protease, wherein the protease variant comprises

[0142] (i) compared to SEQ ID NO: 16, comprises at least two additional negatively charged amino acid sequences in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, and

[0143] (ii) an amino acid sequence that is at least 60% but less than 100% identical to SEQ ID NOs: 16-34, preferably SEQ ID NO: 16,

[0144] bb. a mannanase variant of a parent mannanase, wherein the mannanase variant comprises

[0145] (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

[0146] (ii) 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

[0147] cc. A cellulase comprising an amino acid sequence at least 60% identical to SEQ ID NO:42.

[0148] In one embodiment the invention relates 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 as described herein, e.g., anti-ashing and anti-pilling cellulases.

[0149] Protease

[0150] In one embodiment, the enzyme composition of the present invention comprises a protease.

[0151] The proteases and protease variants described herein have protease activity (also referred to herein as "proteolytic activity"). This property is related to the hydrolytic activity (proteolysis, which means the hydrolysis of the peptide bonds that link the amino acids in a polypeptide chain together) of the protease on a substrate containing protein (e.g., casein, hemoglobin, and BSA). Quantitatively, proteolytic activity is related to the rate at which a protease or proteolytic enzyme degrades a protein within a specified time. Methods for analyzing proteolytic activity are well known in the literature (see, e.g., Gupta et al. (2002), Appl. Microbiol. Biotechnol. 60: pp. 381-395).

[0152] For example, proteolytic activity can be determined by 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 free pNA, which can be quantified by measuring the optical density at 405 nm (OD405).

[0153] The protease enzyme of the enzyme composition is selected from

[0154] - a protease variant of a parent protease, wherein the protease variant comprises

[0155] 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 an amino acid sequence at least 60% but less than 100% identical to SEQ ID NO: 16-34, preferably SEQ ID NO: 16, or

[0156] - a protease variant of a parent protease, wherein the protease variant comprises

[0157] (i) an amino acid substitution at one or more amino acid positions corresponding to the 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

[0158] (ii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO: 17, or

[0159] (iii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO: 19, or

[0160] (iv) an amino acid sequence that has 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.

[0161] Preferably, the protease of the enzyme composition is a protease variant of a parent protease, wherein the protease variant comprises 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 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.

[0162] Preferably, the protease is a variant protease of a parent protease, which is preferably a subtilisin.

[0163] Preferably, the parent protease of the protease variant is a protease having at least 60% sequence identity with any one of SEQ ID NOs: 16-34, preferably the parent protease of the protease variant is a protease according to any one of SEQ ID NOs: 16-34. Most preferably, the parent protease of the protease variant is a protease according to SEQ ID NO: 16.

[0164] Preferably, in this embodiment, the amino acid residue in the parent protease at the above cited position (ie, X) corresponds to the amino acid residue shown in SEQ ID NO: 16 at the corresponding position (according to the numbering of SEQ ID NO: 17).

[0165] The variant protease 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 (BPN' numbering, i.e., wherein positions are numbered as they correspond to the amino acid sequence of subtilisin BPN' of Bacillus amyloliquefaciens (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 catalytic triad, preferably the protease is a subtilisin.

[0166] 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 changes selected from the group consisting of substitution, deletion and insertion, preferably by substitution or insertion, preferably substitution.

[0167] Preferably, in the protease, at least two additional negative charges in the loop region of residues 98 to 104 compared to SEQ ID NO: 16 are caused by one or more amino acid substitutions at amino acid positions numbered according to SEQ ID NO: 17, the amino acid positions being selected from the group consisting of: 98, 99, 100, 101, 102, 103 and 104, preferably at position 101.

[0168] 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 changes selected from the group consisting of X99E, X101D and X101E (preferably D99E, R101D and R101E).

[0169] In a preferred embodiment, the protease comprises an amino acid sequence comprising the amino acid substitution R101E or R101D (according to the numbering of SEQ ID NO: 17) compared to SEQ ID NO: 16. In another embodiment, at least two additional negative charges in the loop region of residues 98 to 104 are not caused by the amino acid substitutions X101E or X101D compared to SEQ ID NO: 16. Most preferably, the protease comprises an amino acid sequence comprising the amino acid substitution R101E (according to the numbering of SEQ ID NO: 17) compared to SEQ ID NO: 16.

[0170] In a preferred embodiment, loop sequence 98-104 has two additional negative charges compared to SEQ ID NO: 16, having the following sequence ADGEGAI, ADGDGAI, ADGDGSV, ADGEGSV, AADGSGSV, AADGEGSV or ASEGEGSV, having a longer sequence with an insertion in the loop sequence.

[0171] In a further preferred embodiment, the amino acid sequence of the protease comprises alanine at position 103 (103A) and isoleucine at position 104 (104I) compared to SEQ ID NO: 16 (according to the numbering of SEQ ID NO: 17), more preferably, 101R, 104I and 103A.

[0172] In a further preferred embodiment, the amino acid sequence of the protease does not comprise 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 comprise additional amino acid residues between positions 42-43, 51-55, 155-165, 187-189, 217-218 or 218-219 (according to the numbering of SEQ ID NO: 17) compared to SEQ ID NO: 16.

[0173] In a preferred embodiment, the protease comprises one or more further substitutions compared to SEQ ID NO: 16 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] Preferably, the protease is identical to SEQ ID Any of NOs: 16-34 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least to 99.5% but less than 100% sequence identity.

[0179] 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.

[0180] Preferably, the protease is identical to SEQ ID NO:16 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5% but less than 100% sequence identity and has a sequence identity of at least 100% to SEQ. Compared to SEQ ID NO: 16, at least two additional negative charges are contained in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17.

[0181] 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% or 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.

[0182] Preferably, the protease has 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 comprises a polypeptide according to SEQ ID NO: 16. The amino acid substitution numbered in NO: 17 is X101D or X101E, preferably X101E, preferably R101E.

[0183] Preferably, the protease has 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 to 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.

[0184] 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 33, 36, 45, 55, 57, 58, 59, 61, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 117, 118, 120, 123, 124, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 240, 241, 242, 243, 244, 245 131, 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.

[0185] In another embodiment of the present invention, the protease as described herein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and wherein the amino acid sequence of the protease comprises at least two additional negative charges in the loop region of residues 98 to 104 compared to SEQ ID NO: 16, comprises the amino acid substitution X101E or X101D, preferably X101E (according to the numbering of SEQ ID NO: 17), and further comprises, according to the numbering of SEQ ID NO: 17, at least one amino acid residue selected from the group consisting of:

[0186] a. Threonine or serine at position 3 (X3T or X3S),

[0187] b. Isoleucine or valine at position 4 (X4I or X4V),

[0188] c. serine, alanine, threonine or arginine at position 63 (X63S, X63A, X63T or X63R),

[0189] d. Threonine, aspartic acid or glutamic acid at position 156 (X156T, X156D or X156E),

[0190] e. serine or proline at position 194 (X194S or X194P),

[0191] f. Serine, valine or methionine at position 199 (X199S, X199V or X199M)

[0192] g. Isoleucine or valine at position 205 (X205I or X205V); and

[0193] h. Aspartic acid, glutamic acid, glutamine, glycine or leucine at position 217 (X217D, X217E, X217Q, X217G or X217L).

[0194] Preferably, the protease has 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 has a sequence identity to SEQ ID NO: 17. Compared with NO:16, it comprises amino acid substitution X101D or X101E, preferably X101E, and one or more of the following: amino acid substitutions X3T, X4I and X205I, preferably S3T, V4I and V205I (according to the numbering of SEQ ID NO:17). Further preferred, 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 to SEQ ID NO: 16 and comprises the amino acid substitutions R101D or R101E, preferably R101E, and the amino acid substitutions S3T, V4I and V205I, according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16.

[0195] In another embodiment, the protease comprises an amino acid sequence at least 80% identical to SEQ ID NO: 16, and compared to SEQ ID NO: 16, the protease comprises the amino acid substitution X101E or X101D, preferably X101E, and one or more substitutions selected from the group consisting of X156D, X262E, X137H, X3T, X45E,D,Q, X55N, X58W,Y,L, X59D,M,N,T, X61D,R, X87E, X97S, X98D,E,R, X106A,W, X117E, X120V,D,K,N, X125M, X129D, X136Q, X144W, X161T, X163A,G, X171 L, X172S, X185Q, X199M, X209W, X222Q, X238H, X244T, X261T,D and

[0196] In one embodiment, the protease comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and compared to SEQ ID NO: 16, the protease comprises the amino acid substitution X101E or X101D, preferably X101E, and one, preferably both, of the substitutions selected from the group consisting of X156D and X262E (according to the numbering of SEQ ID NO: 17).

[0197] In another embodiment, the protease comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and compared to SEQ ID NO: 16, the protease comprises the amino acid substitution R101E or R101D, preferably R101E, and one or more substitutions selected from the group consisting of S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61 D,R, S87E, G97S, A98D,E,R, S106A,W, N117E, H120V,D,K,N, S125M, P129D, E136Q, S144W, S161T, S163A,G, Y171 L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D, and L262N,Q,D.

[0198] In one embodiment, the protease comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and compared to SEQ ID NO: 16, the protease comprises the amino acid substitution R101E or R101D, preferably R101E, and one, preferably both, of the substitutions selected from the group consisting of S156D and L262E (according to the numbering of SEQ ID NO: 17).

[0199] Further preferred, 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 to SEQ ID NO: 16 and comprises the amino acid substitutions R101D or R101E, preferably R101E, and the amino acid substitutions S156D and L262E, according to the numbering of SEQ ID NO: 17, compared to SEQ ID NO: 16.

[0200] A protease having substitutions as described above is shown, for example, in SEQ ID NO:28.

[0201] Thus, in one embodiment, the protease variant comprises NO:28 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 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 an amino acid sequence.

[0202] In another embodiment, the protease comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and compared to SEQ ID NO: 16, the protease comprises the amino acid substitutions R101E, and one or more of S156D and L262E, preferably both, and optionally at least one further mutation selected from I104T, H120D, Q137H, S141H, R145H and S163G (according to the numbering of SEQ ID NO: 17).

[0203] Preferably, the protease has at least 80% 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, wherein the protease comprises one or more conservative amino acid exchanges as described herein compared to SEQ ID NO: 16. Preferably, the protease comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, 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.

[0204] Preferably, the protease described herein, in addition to comprising a modification producing at least two additional negative charges in the loop region of residues 98 to 104, preferably in addition to comprising 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 (according to the numbering of SEQ ID NO: 17), may also comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid exchanges compared to SEQ ID NO: 16.

[0205] 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 to 104 according to the numbering of SEQ ID NO: 17 compared to SEQ ID NO: 16, wherein the remaining differences in the amino acid sequence compared to SEQ ID NO: 16 are due to conservative amino acid exchanges as described herein.

[0206] Preferably, the protease is a protease variant comprising amino acid substitution X101E and optionally amino acid substitutions X3T, X4I and X205I or amino acid substitutions X156D and X262E (according to the numbering of SEQ ID NO: 17), and an amino acid sequence at least 80% but less than 100% identical to SEQ ID NO: 16.

[0207] 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 at least 80% but less than 100% identical to SEQ ID NO:16.

[0208] Most preferred is a protease variant comprising or consisting of the amino acid sequence shown in SEQ ID NO:16, having the amino acid substitution R101E according to the numbering of SEQ ID NO:17, i.e. only one amino acid is exchanged compared to SEQ ID NO:16.

[0209] Alternatively preferred is a protease variant comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16, having the amino acid substitutions R101E and S3T, V4I and V205I (according to the numbering of SEQ ID NO: 17).

[0210] Alternatively preferred is a protease variant comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16, having the amino acid substitutions R101E and S156D and L262E (according to the numbering of SEQ ID NO: 17).

[0211] In one embodiment, the protease variant of the enzyme composition comprises

[0212] (i) an amino acid substitution at one or more amino acid positions corresponding to the 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

[0213] (ii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO: 17, or

[0214] (iii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO: 19, or

[0215] (iv) an amino acid sequence that has 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.

[0216] In one embodiment, the protease variant comprises a protease 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 93.5%, at least 94.5%, at least 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 99.6%, at least 100. 5%, at least 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 amino acid substitutions at one or more amino acid positions corresponding to the amino acid positions selected from the group consisting of: according to SEQ ID NO: 1. 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 of ID NO:17.

[0217] Preferably, the protease variant comprises 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 92.5%, at least 93.5%, at least 94.5%, at least 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 99.6%, at least 100. , 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 one or more amino acid substitutions selected from the group consisting of 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, X131 156D / E, X160D / S, X167A, X170S, X182E / D, X185E / D, X188D, X194P, 213A, X217E / D / Q, X218D, X232V, X245R, X256D / E, X259D / E, X262D / E and

[0218] Preferably, the protease variant comprises 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 92.5%, at least 93.5%, at least 94.5%, at least 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 99.6%, at least 100. , 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 one or more amino acid substitutions selected from the group consisting of S3T, V4I, S9D / E / R, R19L, T22R / A, N43R, N62D, G63D / E, N76D, S87N, S99A / D, S101M / R / E, S103A, V104I, N116L, G118V, S128L, P129Q, S130A, S131 156D / E, G160D / S, Y167A, R170S, Q182E / D, N185E / D, S188D, A194P, V205I, T 213A, L217E / D / Q, N218D, A232V, N245R, S256D / E, S259D / E, L262D / E and E271F.

[0219] In a preferred embodiment, the above-mentioned protease variant further comprises an insertion of D and E directly adjacent to the C-terminus of position 99 according to the numbering of SEQ ID NO:17.

[0220] Proteases having substitutions as described above 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.

[0221] Thus, in one embodiment, the protease variant comprises a residue corresponding to SEQ ID NO: 24, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: %, 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%, 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 one of NO:29.

[0222] 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, or SEQ ID NO:29.

[0223] 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.

[0224] 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:23.

[0225] 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.

[0226] 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:27.

[0227] 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:29.

[0228] In another embodiment, the protease variant comprises a protease variant 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 93.5%, at least 94.5%, at least 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 99.6%, at least 100. 5%, at least 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 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 SEQ ID NO: 17.

[0229] Preferably, the protease variant comprises 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 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 120%, at least 121%, , 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 one or more 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).

[0230] Preferably, the protease variant comprises 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 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 120%, at least 121%, , 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 one or more 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).

[0231] Proteases having substitutions as described above are shown, for example, in SEQ ID NOs: 32, 33 and 34.

[0232] Thus, in one embodiment, the protease variant comprises 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 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%, 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.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] In one embodiment, the protease variant comprises a protease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, 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 %, 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, and amino acid substitutions at one or more amino acid positions corresponding to the amino acid positions selected from the group consisting of: according to SEQ ID 9, 89, 130, 133, 144, 159, 183, 189, 217, 224, 238, 243, 252, 259, 270 and 271 of NO: 19, preferably one or more, preferably all selected from the group consisting of X159T, X183S, X238N, X243N, X259N and X270A, more preferably one or more, preferably all selected from the group consisting of X9T, X130D, X133A, X144K, X159T, X183S, X189T, X217M, X224A, X238N, X243N, X252T, X259N, X270A and X271E, even more preferably one or more, preferably all, selected from the group consisting of X9T, X89A, X130D, X133A, X144K, X159T, X183S, X189T, X217M, X224A, X238N, X243N, X252T, X259N, X270A and X271E.

[0237] In one embodiment, the protease variant comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, but less than 100% sequence identity to SEQ ID NO: 19, and one or more, preferably all, selected from Substitutions from the group consisting of X159T, X183S, X238N, X243N, X259N and X270A, more preferably one or more, preferably all selected from the group consisting of X9T, X130D, X133A, X144K, X159T, X183S, X189T, X217M, X224A, X238N, X243N, X252T, X259N, X270A and X271E, even more preferably one or more, preferably all selected from the group consisting of X9T, X89A, X130D, X133A, X144K, X159T, X183S, X189T, X217M, X224A, X238N, X243N, X252T, X259N, X270A and X271E, according to SEQ ID NO: 1 ID NO:19.

[0238] In one embodiment, the protease variant comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, but less than 100% sequence identity to SEQ ID NO: 19, and one or more, preferably all 9T, N183S, Y238N, T243N, D259N, and V270A, more preferably one or more, preferably all selected from the group consisting of P9T, N130D, T133A, N144K, F159T, N183S, S189T, Y217M, S224A, Y238N, T243N, N252T, D259N, V270A, and Q271E, even more preferably one or more, preferably all selected from the group consisting of P9T, S89A, N130D, T133A, N144K, F159T, N183S, S189T, Y217M, S224A, Y238N, T243N, N252T, D259N, V270A, and Q271E (according to SEQ ID NO: 11). ID NO:19).

[0239] In one embodiment, the protease variant is identical 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 Any of the amino acid sequences shown in NO:31 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.

[0240] Preferably, the protease variant is identical 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 Any of the amino acid sequences shown in NO:31 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.

[0241] 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 shown 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.

[0242] In one embodiment, the protease according to the present 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] In one embodiment, the protease according to the present 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.

[0249] 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.

[0250] Mannanase

[0251] In one embodiment, the enzyme composition of the invention comprises a mannanase.

[0252] Mannanases and mannanase variants described herein have mannanase activity. "Mannanase" described herein is an enzyme selected from the group of (galacto)mannan degrading enzymes. The (galacto)mannan degrading enzyme can be selected from β-mannosidase (EC 3.2.1.25), endo-1,4-β-mannosidase (EC 3.2.1.78) and 1,4-β-mannobiosidase (EC 3.2.1.100). Preferably, the mannan degrading enzyme is selected from the group of endo-1,4-mannanase, preferably endo-1,4-β-mannosidase (EC 3.2.1.78), which may be referred to herein as a group of enzymes of endo-β-1,4-D-mannanase, β-mannanase or mannanase. Endo-1,4-mannanase randomly hydrolyzes 1,4-linkages within the mannan backbone to release manno-oligosaccharides (MOS).

[0253] The mannanase may be selected from alkaline mannanases of family 5 or 26 (ie GH5 or GH26). The term "alkaline mannanase" is intended to encompass a mannanase having an enzyme activity of at least 40% of its maximum activity at a given pH of 7 to 12, preferably 7.5 to 10.5.

[0254] The mannanase of the enzyme composition is selected from

[0255] - 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 amino acid substitutions with the amino acid sequence of SEQ ID NO: 36. An amino acid sequence that is at least 60% but less than 100% identical to the sequence at positions 31-490 of NO:35, and

[0256] - a mannanase variant of a parent mannanase, wherein the mannanase variant comprises

[0257] (i) an amino acid substitution at one or more amino acid positions corresponding to the 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 at least 60% but less than 100% identical to SEQ ID NO:39, or

[0258] (ii) an amino acid substitution at one or more amino acid positions corresponding to the 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, and amino acid sequences that are at least 60% but less than 100% identical to SEQ ID NO:38, or

[0259] (iii) an amino acid sequence that is at least 60% identical to SEQ ID NO: 40, or

[0260] (iv) 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, and an amino acid sequence at least 60% but less than 100% identical to SEQ ID NO:40.

[0261] Preferably, the mannanase of the enzyme composition is a mannanase variant of a parent mannanase, wherein 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.

[0262] Preferably, the mannanase of the enzyme composition is a mannanase variant of a parent mannanase, wherein 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, and amino acid substitutions at one or more amino acid positions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, Any of NOs: 35-37 has an amino acid sequence with at least 60% but less than 100% sequence identity.

[0263] Preferably, the parent mannanase of the mannanase variant is a mannanase having at least 60% sequence identity with any one of SEQ ID NOs: 35-37, preferably the parent mannanase of the mannanase variant is a mannanase according to any one of SEQ ID NOs: 35-37. Most preferably, the parent mannanase of the mannanase variant is a mannanase according to SEQ ID NO: 35.

[0264] Preferably, in this embodiment, the amino acid residue in the parent protease at the above-cited position (ie, X) corresponds to the amino acid residue shown in SEQ ID NO:35 at the corresponding position (according to the numbering of SEQ ID NO:35).

[0265] Preferably, the mannanase variant comprises one or more amino acid substitutions 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, X264Q / V, X270T, L / Q / A, X289F / M / H, X290A, X296H / F / Y, X301E / C / T, X309L,

[0266] Preferably, the mannanase variant comprises one or more amino acid substitutions 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, 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, A319D / E.

[0267] 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.

[0268] 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.

[0269] 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 as numbered according to SEQ ID NO:35.

[0270] 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.

[0271] Preferably, the mannanase variant comprises amino acid substitutions at positions 89 and 96 and amino acid substitutions at one or more amino acid positions selected from the group consisting of 31, 119, 264, 289 and 312 according to the numbering of SEQ ID NO:35.

[0272] 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.

[0273] 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 as numbered according to SEQ ID NO:35.

[0274] Preferably, the mannanase variant comprises at least one of the amino acid substitutions X89V and X96D and one or more amino acid substitutions selected from the group consisting of X31V, X119Y / H / T, X264Q / V, X289F / M / H and X312F / Y (according to the numbering of SEQ ID NO: 35).

[0275] 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 as numbered according to SEQ ID NO: 35.

[0276] Preferably, the mannanase comprises at least one of the amino acid substitutions Q89V and N96D and one or more amino acid substitutions selected from the group consisting of A31V, A119Y / H / T, E264Q / V, W289F / M / H and N312F / Y (according to the numbering of SEQ ID NO: 35).

[0277] 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).

[0278] 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).

[0279] 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).

[0280] 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).

[0281] 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.

[0282] 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).

[0283] 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.

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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.

[0288] 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 as numbered according to SEQ ID NO: 35.

[0289] 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 as numbered according to SEQ ID NO: 35.

[0290] Preferably, the mannanase variant comprises any one of the amino acid sequences shown in SEQ ID NOs: 35-37 or the sequence according to positions 31-490 of SEQ ID NO: 35, preferably the sequence according to positions 31-490 of SEQ ID NO: %, 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%, 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 amino acid sequence of positions 31-490 of NO:35.

[0291] More preferably, the mannanase variant comprises %, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, but less than 100% identical to the amino acid sequence of positions 31-490 of NO:35.

[0292] 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 31-490 of SEQ ID NO:35.

[0293] Preferably, the mannanase of the enzyme composition is a mannanase variant of a parent mannanase, wherein 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, and amino acid substitutions at one or more amino acid positions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, Any of the amino acid sequences shown in NO:35-37 or the sequence according to positions 31-490 of SEQ ID NO:35, preferably the sequence according to positions 31-490 of SEQ ID NO:35. %, 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%, 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 amino acid sequence of positions 31-490 of NO:35.

[0294] Preferably, the mannanase of the enzyme composition is a mannanase variant of a parent mannanase, wherein 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, and amino acid substitutions at one or more amino acid positions selected from the group consisting of 86, 89, 96, 101, 103, 107, 108, 109, 112, 119, %, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, but less than 100% identical to the amino acid sequence of positions 31-490 of NO:35.

[0295] Preferably, the mannanase variant comprises one or more amino acid substitutions 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, 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, with any one of the amino acid sequences shown in SEQ ID NOs: 35-37 or with the sequence according to positions 31-490 of SEQ ID NO: 35, preferably with the sequence according to positions 31-490 of SEQ ID NO: 35. %, 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%, 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 amino acid sequence of positions 31-490 of NO:35.

[0296] Preferably, the mannanase variant comprises one or more amino acid substitutions 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, X236V / L / Q / N / Y, X237N / L / Q / N / Y, X238 44I / 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, 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 the amino acid sequence at positions 31-490 of ID NO:35.

[0297] Preferably, the mannanase variant comprises one or more amino acid substitutions 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, X236V / L / Q / N / Y, X237N / L / Q / N / Y, X238 44I / 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, 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 the amino acid sequence at positions 31-490 of ID NO:35.

[0298] 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 numbered according to SEQ ID NO: 35, and amino acid substitutions corresponding to the amino acid sequence of SEQ ID NO: 36. The amino acid sequence of positions 31-490 of NO:35 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 amino acid sequence of positions 31-490 of NO:35.

[0299] 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), and the amino acid substitutions according to SEQ ID NO: 36. The amino acid sequence of positions 31-490 of NO:35 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 amino acid sequence of positions 31-490 of NO:35.

[0300] 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, and the amino acid substitutions X89V, X96D, X119H, X264Q, X289M, X312Y, X348N, X349G and X352G according to the numbering of SEQ ID NO: The amino acid sequence of positions 31-490 of NO:35 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 amino acid sequence of positions 31-490 of NO:35.

[0301] More preferably, the mannanase variant comprises amino acid substitutions X89V, X96D, X119H, X264Q, X289M and X312Y according to the numbering of SEQ ID NO: 35, and amino acid substitutions X89V, X96D, X119H, X264Q, X289M and X312Y according to the numbering of SEQ ID NO: The amino acid sequence of positions 31-490 of NO:35 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 amino acid sequence of positions 31-490 of NO:35.

[0302] Most preferably, the mannanase variant comprises 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 31-490 of NO:35 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 amino acid sequence of positions 31-490 of NO:35.

[0303] Most preferred is a mannanase variant comprising or consisting of the amino acid sequence according to positions 31-490 of SEQ ID NO:35, having the amino acid substitutions Q89V, N96D, A119H, E264Q, W289M, N312Y, T348N, E349G and S352G according to the numbering of SEQ ID NO:35.

[0304] In one embodiment, the mannanase variant of the enzyme composition comprises

[0305] (i) an amino acid substitution at one or more amino acid positions corresponding to the 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 at least 60% but less than 100% identical to SEQ ID NO:39, or

[0306] (ii) an amino acid substitution at one or more amino acid positions corresponding to the 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, and amino acid sequences that are at least 60% but less than 100% identical to SEQ ID NO:38, or

[0307] (iii) an amino acid sequence that is at least 60% identical to SEQ ID NO: 40, or

[0308] (iv) 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, and an amino acid sequence at least 60% but less than 100% identical to SEQ ID NO:40.

[0309] In one embodiment, the mannanase variant comprises 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 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 120%, : The invention relates to an amino acid sequence that is 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 amino acid sequence of FIG. 39, and amino acid substitutions at one or more amino acid positions corresponding to the amino acid positions selected from the group consisting of: 24, 123 and 256 according to the numbering of SEQ ID NO: 39.

[0310] In one embodiment, the mannanase variant comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92.5%, at least 93.5%, at least 94.5%, at least 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 99.6%, at least 100.6%, at least 101.5%, at least 102.5%, at least 103.5%, at least 104.5%, at least 105.5%, at least 106.5%, at least 107.5%, at least 108.5%, at least 109.5%, at least 110.5%, at least 111.5%, at least 112.5%, at least 113.5%, at least 114.5%, at least 115.5%, at least 116.5%, at least 117.5%, at least 118.5%, at least 119.5%, at least 119.5%, at least 119.5%, at least An amino acid sequence that is 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, and comprising one or more amino acid substitutions selected from the group consisting of X24I, X123I and X256G according to the numbering of SEQ ID NO: 39, preferably a combination of X24I and X256G.

[0311] In one embodiment, the mannanase variant comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92.5%, at least 93.5%, at least 94.5%, at least 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 99.6%, at least 100.6%, at least 101.5%, at least 102.5%, at least 103.5%, at least 104.5%, at least 105.5%, at least 106.5%, at least 107.5%, at least 108.5%, at least 109.5%, at least 110.5%, at least 111.5%, at least 112.5%, at least 113.5%, at least 114.5%, at least 115.5%, at least 116.5%, at least 117.5%, at least 118.5%, at least 119.5%, at least 119.5%, at least 119.5%, at least An amino acid sequence that is 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, and comprising one or more amino acid substitutions selected from the group consisting of V24I, V123I and S256G according to the numbering of SEQ ID NO:39, preferably a combination of V24I and S256G.

[0312] In one embodiment, the mannanase variant comprises 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 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 120%, at least 121%, at least 122%, : The invention relates to an amino acid sequence which is 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 amino acid sequence of FIG. 38, and comprises an amino acid substitution at one or more amino acid positions corresponding to the amino acid position 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 the deletion at position 298*.

[0313] In one embodiment, the mannanase variant comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92.5%, at least 93.5%, at least 94.5%, at least 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 99.6%, at least 100.5%, at least 101.5%, at least 102.5%, at least 103.5%, at least 104.5%, at least 105.5%, at least 106.5%, at least 107.5%, at least 108.5%, at least 109.5%, at least 110.5%, at least 111.5%, at least 112.5%, at least 113.5%, at least 114.5%, at least 115.5%, at least 116.5%, at least 117.5%, at least 118.5%, at least 119 ... , 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 amino acid sequence and comprises one or more amino acid substitutions selected from the group consisting of: according to SEQ X10T, X19P, X30S, X38T, X59S, X60L, X67N, X97N, X103V, X129Y, X143Q, X167F, X168S, X184Q, X225P / G, X228T, X235Y, X244K, X258S and X261N of ID NO: 38 are preferably combined with the deletion X298*.

[0314] In one embodiment, the mannanase variant comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92.5%, at least 93.5%, at least 94.5%, at least 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 99.6%, at least 100.5%, at least 101.5%, at least 102.5%, at least 103.5%, at least 104.5%, at least 105.5%, at least 106.5%, at least 107.5%, at least 108.5%, at least 109.5%, at least 110.5%, at least 111.5%, at least 112.5%, at least 113.5%, at least 114.5%, at least 115.5%, at least 116.5%, at least 117.5%, at least 118.5%, at least 119 ... , 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 amino acid sequence and comprises one or more amino acid substitutions selected from the group consisting of: according to SEQ N10T, E19P, T30S, E38T, V59S, Q60L, D67N, D97N, I103V, M129Y, K143Q, Y167F, P168S, L184Q, C225P / G, V228T, L235Y, L244K, D258S and R261N of ID NO: 38 are preferably combined with deletion Q298*.

[0315] In one embodiment, the mannanase variant is identical to SEQ ID NO:40 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% sequence identity.

[0316] In one embodiment, the mannanase variant comprises 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% identical to SEQ ID NO:40. , 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 amino acid sequence 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, 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 (according to the numbering of SEQ ID NO: 40).

[0317] In one embodiment, the mannanase variant comprises 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% identical amino acid sequence and comprises 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).

[0318] In one embodiment, the mannanase variant comprises 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% identical amino acid sequence and comprises 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).

[0319] A mannanase having substitutions as described above is shown, for example, in SEQ ID NO:41.

[0320] Thus, in one embodiment, the mannanase variant comprises NO:41 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 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 an amino acid sequence.

[0321] Cellulase

[0322] In one embodiment, the enzyme composition of the present invention comprises cellulase.

[0323] Cellulase and cellulase variants described herein have cellulase activity. "Cellulase" is an enzyme capable of hydrolyzing cellulose. Cellulase can be selected from cellobiohydrolase (1,4-PD-glucan cellobiohydrolase, EC3.2.1.91), endo-ss-1,4-glucanase (EC 3.2.1.4) and ss-glucosidase (EC 3.2.1.21). The endoglucanase of EC category 3.2.1.4 can be named endoglucanase, endo-1,4-ss-D-glucan 4-glucanohydrolase, endo-1,4-β-glucanase, carboxymethyl cellulase and β-1,4-glucanase.

[0324] Endoglucanases can be classified into Family 5 based on amino acid sequence similarity (Henrissat, B. Available in UniProt 10 / 26 / 2011), which contains more than 20 endoglucanases of EC 3.2.1.4. Reference is also made to T.-M. Enveri, "Microbial Cellulases," in WM Fogarty, Microbial Enzymes and Biotechnology, Applied Science Publishers, pp. 183-224 (1983); Methods in Enzymology, (1988) Vol. 160, pp. 200-391 (edited by Wood, WA and Kellogg, ST); Béguin, P., "Molecular Biology of Cellulose Degradation," Annu. Rev. Microbiol. (1990), Vol. 44, pp. 219-248; Begun, P. and Aubert, JP., "The biological degradation of cellulose," FEMS Microbiology. Reviews [FEMS Microbiology] 13 (1994) pp. 25-58; Henrissat, B., "Cellulases and their interaction with cellulose", Cellulose [Cellulose] (1994), Vol. 1, pp. 169-196.

[0325] The cellulase of the enzyme composition is selected from

[0326] - a cellulase variant comprising an amino acid sequence at least 60% identical to SEQ ID NO: 42, and

[0327] - 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.

[0328] Preferably, the cellulase variant of the enzyme composition is a cellulase comprising an amino acid sequence at least 60% identical to SEQ ID NO:42.

[0329] 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.

[0330] 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.

[0331] 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.

[0332] Most preferably, the cellulase variant comprises or consists of SEQ ID NO:42.

[0333] 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.

[0334] Preferably, the cellulase variant is identical to SEQ ID Any of the amino acid sequences set forth in NOs:43-47 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.

[0335] 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 one of the amino acid sequences shown in SEQ ID NOs: 43-47.

[0336] 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 one of the amino acid sequences shown in SEQ ID NOs:43-47.

[0337] In one embodiment, the cellulase according to the present 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 with SEQ ID NO:46.

[0338] 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.

[0339] 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.

[0340] 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.

[0341] In one embodiment, the cellulase variant is identical to SEQ ID Any of the amino acid sequences shown in NO:47 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.

[0342] Preferred herein is a composition comprising two or more enzymes selected from the group consisting of proteases, mannanases and cellulases, wherein

[0343] a. The protease is selected from

[0344] I. A protease variant of a parent protease, wherein the protease variant comprises

[0345] (i) compared to SEQ ID NO: 16, comprises at least two additional negatively charged amino acid sequences in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, and

[0346] (ii) an amino acid sequence that is at least 80% but less than 100% identical to any one of SEQ ID NO: 16, or

[0347] II. A protease variant of a parent protease, wherein the protease variant comprises

[0348] (i) an amino acid substitution at one or more amino acid positions corresponding to the 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

[0349] (ii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO: 17, or

[0350] (iii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO: 19, or

[0351] (iv) an amino acid sequence that has 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,

[0352] b. The mannanase is selected from

[0353] I. A mannanase variant of a parent mannanase, wherein the mannanase variant comprises

[0354] (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

[0355] (ii) an amino acid sequence that is at least 80% but less than 100% identical to the sequence according to positions 31 to 490 of SEQ ID NO: 35, or

[0356] II. A mannanase variant of a parent mannanase, wherein the mannanase variant comprises

[0357] (i) an amino acid substitution at one or more amino acid positions corresponding to the 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 at least 60% but less than 100% identical to SEQ ID NO:39, or

[0358] (ii) an amino acid substitution at one or more amino acid positions corresponding to the 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, and amino acid sequences that are at least 60% but less than 100% identical to SEQ ID NO:38, or

[0359] (iii) an amino acid sequence that is at least 60% identical to SEQ ID NO: 40, or

[0360] (iv) an amino acid substitution at one or more amino acid positions corresponding to the 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, and an amino acid sequence at least 60% but less than 100% identical to SEQ ID NO:40,

[0361] c. The cellulase is selected from

[0362] I. A cellulase variant comprising an amino acid sequence at least 80% identical to SEQ ID NO: 42, or

[0363] II. A cellulase variant 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, and

[0364] Wherein at least one enzyme is selected from the group consisting of

[0365] aa. A protease variant of a parent protease, wherein the protease variant comprises

[0366] (i) compared to SEQ ID NO: 16, comprises at least two additional negatively charged amino acid sequences in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, and

[0367] (ii) an amino acid sequence that is at least 80% but less than 100% identical to SEQ ID NO: 16,

[0368] bb. a mannanase variant of a parent mannanase, wherein the mannanase variant comprises

[0369] (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

[0370] (ii) an amino acid sequence that is at least 80% but less than 100% identical to the sequence according to positions 31-490 of SEQ ID NO:35, and

[0371] cc. A cellulase variant comprising an amino acid sequence at least 80% identical to SEQ ID NO:42.

[0372] 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 to SEQ ID NO: 16 and comprises an 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.

[0373] In particular embodiments, the protease variant comprises amino acid substitution X101E and amino acid substitutions X3T, X4I and X205I or amino acid substitutions X156D and X262E according to the numbering of SEQ ID NO: 17, and an amino acid sequence that is at least 90% but less than 100% identical to SEQ ID NO: 16.

[0374] Alternatively, the protease variant comprises an amino acid sequence having 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: 22, and one or more amino acid substitutions selected from the group consisting of: NO:17 numbered 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, X16 OD / 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, preferably the protease variant further comprises the protease according to SEQ 99, at least 99.5%, at least 99.5%, at least 99.5%, at least 99.5%, or 100% identical to any one of SEQ ID NO:24.

[0375] 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 amino acid sequence according to positions 31-490 of SEQ ID NO:35.

[0376] Alternatively, the mannanase variant comprises 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:40. The amino acid sequence of the present invention comprises 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 SEQ ID NO: 1). %, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO:41.

[0377] Preferably, the cellulase 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:42.

[0378] Preferred herein is a composition comprising two or three enzymes selected from the group consisting of proteases, mannanases and cellulases, wherein

[0379] a. The protease is a protease variant comprising

[0380] (i) amino acid substitution X101E according to the numbering of SEQ ID NO: 17, and

[0381] (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,

[0382] or the protease is a protease variant comprising amino acid substitution X101E and amino acid substitutions X3T, X4I and X205I or amino acid substitutions X156D and X262E according to the numbering of SEQ ID NO: 17, and an amino acid sequence that is at least 90% but less than 100% identical to SEQ ID NO: 16,

[0383] or the protease is a protease variant comprising an amino acid sequence having 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: 22, and one or more amino acid substitutions selected from the group consisting of: 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, X205I, X213A, X217E / D / Q, X218D, X232V, X245R, X256D / E, X259D / E, X262D / E, and X271F (according to SEQ %, 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 one of SEQ ID NO: 24,

[0384] b. The mannanase is a mannanase variant comprising

[0385] (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

[0386] (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 according to positions 31-490 of SEQ ID NO:35,

[0387] or the mannanase is a mannanase variant comprising 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:40. The amino acid sequence of the present invention comprises 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 SEQ ID NO: 1). %, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% identical to SEQ ID NO:41,

[0388] c. the cellulase comprises 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.

[0389] More preferred herein is a composition comprising two or three enzymes selected from the group consisting of proteases, mannanases and cellulases, wherein

[0390] a. The protease is a protease variant comprising

[0391] (i) amino acid substitution X101E according to the numbering of SEQ ID NO: 17, and

[0392] (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 SEQ ID NO:16,

[0393] b. The mannanase is a mannanase variant comprising

[0394] (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

[0395] (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 according to positions 31-490 of SEQ ID NO:35, and

[0396] 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.

[0397] Therefore, more preferred herein is a composition comprising a protease and a mannanase, wherein

[0398] a. The protease is a protease variant comprising

[0399] (i) amino acid substitution X101E according to the numbering of SEQ ID NO: 17, and

[0400] (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 SEQ ID NO:16, and

[0401] b. The mannanase is a mannanase variant comprising

[0402] (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

[0403] (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 according to positions 31-490 of SEQ ID NO:35.

[0404] Therefore, more preferred herein is a composition comprising a protease and a cellulase, wherein

[0405] a. The protease is a protease variant comprising

[0406] (i) amino acid substitution X101E according to the numbering of SEQ ID NO: 17, and

[0407] (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 SEQ ID NO:16, and

[0408] b. 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.

[0409] Therefore, more preferred herein is a composition comprising mannanase and cellulase, wherein

[0410] a. The mannanase is a mannanase variant comprising

[0411] (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

[0412] (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 according to positions 31-490 of SEQ ID NO:35, and

[0413] b. 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.

[0414] More preferred herein is a composition comprising two or three enzymes selected from the group consisting of proteases, mannanases and cellulases, wherein

[0415] a. the protease is a protease variant comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16, having an amino acid substitution R101E according to the numbering of SEQ ID NO: 17, i.e. only one amino acid is exchanged compared to SEQ ID NO: 16,

[0416] b. the mannanase is a mannanase variant comprising or consisting of the amino acid sequence according to positions 31-490 of SEQ ID NO: 35, having amino acid substitutions Q89V, N96D, A119H, E264Q, W289M, N312Y, T348N, E349G and S352G numbered according to SEQ ID NO: 35,

[0417] c. The cellulase is a cellulase variant, which comprises or consists of an amino acid sequence that is 100% identical to SEQ ID NO: 42.

[0418] Therefore, more preferred herein is a composition comprising a protease and a mannanase, wherein

[0419] a. the protease is a protease variant comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16, having an amino acid substitution R101E according to the numbering of SEQ ID NO: 17, i.e. only one amino acid is exchanged compared to SEQ ID NO: 16, and

[0420] b. The mannanase is a mannanase variant comprising or consisting of the amino acid sequence according to positions 31-490 of SEQ ID NO: 35, having amino acid substitutions Q89V, N96D, A119H, E264Q, W289M, N312Y, T348N, E349G and S352G numbered according to SEQ ID NO: 35.

[0421] Therefore, more preferred herein is a composition comprising a protease and a cellulase, wherein

[0422] a. the protease is a protease variant comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16, having an amino acid substitution R101E according to the numbering of SEQ ID NO: 17, i.e. only one amino acid is exchanged compared to SEQ ID NO: 16, and

[0423] b. The cellulase is a cellulase variant, which comprises or consists of an amino acid sequence that is 100% identical to SEQ ID NO: 42.

[0424] Therefore, more preferred herein is a composition comprising mannanase and cellulase, wherein

[0425] a. the mannanase is a mannanase variant comprising or consisting of the amino acid sequence according to positions 31-490 of SEQ ID NO: 35, having amino acid substitutions Q89V, N96D, A119H, E264Q, W289M, N312Y, T348N, E349G and S352G numbered according to SEQ ID NO: 35, and

[0426] b. The cellulase is a cellulase variant, which comprises or consists of an amino acid sequence that is 100% identical to SEQ ID NO: 42.

[0427] Preferably, the composition comprises a protease as described herein and a mannanase, a protease as described herein and a cellulase, or a protease, a mannanase and a cellulase as described herein.

[0428] Even more preferably, the composition comprises protease, mannanase and cellulase, wherein

[0429] a. The protease is a protease variant comprising

[0430] (i) amino acid substitution X101E according to the numbering of SEQ ID NO: 17, and

[0431] (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 SEQ ID NO:16,

[0432] b. The mannanase is a mannanase variant comprising

[0433] (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

[0434] (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 according to positions 31-490 of SEQ ID NO:35, and

[0435] 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.

[0436] Most preferably, the composition comprises protease, mannanase and cellulase, wherein

[0437] a. the protease is a protease variant comprising or consisting of the amino acid sequence shown in SEQ ID NO: 16, having an amino acid substitution R101E according to the numbering of SEQ ID NO: 17, i.e. only one amino acid is exchanged compared to SEQ ID NO: 16,

[0438] b. the mannanase is a mannanase variant comprising or consisting of the amino acid sequence according to positions 31-490 of SEQ ID NO: 35, having amino acid substitutions Q89V, N96D, A119H, E264Q, W289M, N312Y, T348N, E349G and S352G numbered according to SEQ ID NO: 35, and

[0439] c. The cellulase is a cellulase variant, which comprises or consists of an amino acid sequence that is 100% identical to SEQ ID NO: 42.

[0440] Other enzymes

[0441] In another embodiment, the composition comprising the enzyme composition described herein further comprises one or more other enzymes different from the enzymes of the enzyme composition described herein. Preferably, the other enzyme is selected from the group consisting of proteases, amylases, lipases, cellulases, mannanases, hemicellulases, phospholipases, esterases, pectinases, lactases, peroxidases, xylanases, cutinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases, nucleases, , DNA enzymes, phosphodiesterases, phytases, carbohydrases, galactanases, xanthanases, xyloglucanases, oxidoreductases, perhydrolases, aminopeptidases, asparaginases, carbohydrases, carboxypeptidases, catalases, chitinases, cyclodextrin glycosyltransferases, α-galactosidases, β-galactosidases, glucoamylases, α-glucosidases, β-glucosidases, invertases, ribonucleases, transglutaminases and dispersins, and combinations of at least two of the foregoing types. More preferably, the other enzymes are selected from the group consisting of amylases, lipases, hemicellulases, xylanases, DNA enzymes, dispersins, pectinases, oxidoreductases, cutinases, phosphodiesterases, licheninases, xanthan lyases and hexosaminidase, and combinations of at least two of the foregoing types. Most preferably, the additional enzyme is an amylase and / or a lipase, preferably an amylase.

[0442] Amylase

[0443] The enzyme composition of the present invention may comprise an amylase. In another embodiment, the enzyme composition does not comprise an amylase.

[0444] 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 glycosidic linkages in polysaccharides. Amylase activity can be determined by assays known to those skilled in the art for measuring amylase activity. Examples of assays for measuring amylase activity are the Phadebas assay or the EPS assay ("Infinity reagent"). In the Phadebas assay, amylase activity is determined by using Phadebas tablets as substrates (Phadebas amylase test, provided by Magle Life Science). Starch is hydrolyzed by amylase, producing soluble blue fragments. The absorbance of the resulting blue solution (measured spectrophotometrically at 620 nm) is a function of amylase activity. The measured absorbance is directly proportional to the specific activity (activity / mg pure amylase protein) of the amylase in question under given conditions.

[0445] Alternatively, amylase activity can also be determined by the method using ethylidene-4-nitrophenyl-α-D-maltoheptaglycoside (EPS). D-maltoheptaglycoside is a closed oligosaccharide that can be cut by endoamylase. After cutting, the α-glucosidase digestion substrate included in the test kit releases yellow free PNP molecules, and therefore can be measured at 405nm by visible spectrophotometry. The test kit containing EPS substrate and α-glucosidase is manufactured by, for example, Roche Costum Biotech (Cat. No. 10880078103). The slope of the time-dependent absorption curve is directly proportional to the specific activity (activity / mg enzyme) of the amylase discussed under given conditions.

[0446] The amylase may be selected from

[0447] - an amylase variant of a parent amylase, wherein the amylase variant comprises

[0448] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 2, and

[0449] (ii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of 4, 116, 176, 181, 186, 195, 206, 225, 251, 320, 405 and 482 according to the numbering of SEQ ID NO: 2,

[0450] (iii) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to any one of SEQ ID NOs: 1-15, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, and

[0451] - an amylase variant of a parent amylase, wherein the amylase variant comprises

[0452] (i) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO:9, or

[0453] (ii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO:7, or

[0454] (iii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO:3, or

[0455] (iv) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO: 13, or

[0456] (v) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity to SEQ ID NO: 15, or

[0457] (vi) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO:4, or

[0458] (vii) an amino acid substitution at one or more amino acid positions corresponding to the 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.

[0459] An amylase variant is a non-naturally occurring amylase. 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.

[0460] Preferably, the parent amylase of the amylase variant is an amylase having at least 60% sequence identity with any one of SEQ ID NOs: 1 to 15, preferably the parent amylase of the amylase variant is an amylase according to any one of SEQ ID NOs: 1 to 15. Most preferably, the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1.

[0461] Preferably, the amylase variant further comprises a deletion at one or more, preferably two or more amino acids corresponding to positions selected from the group consisting of: 181, 182, 183 and 184. Preferably, the amylase variant of the invention having one or more amino acid substitutions as described herein comprises a deletion of one or more amino acids corresponding to positions 183 and 184, preferably a deletion of two amino acids corresponding to positions 183 and 184 (according to the numbering of SEQ ID NO: 2). Preferably, the amylase variant of the invention having one or more amino acid substitutions as described herein comprises a deletion of one or more, preferably two or more, most preferably two amino acids corresponding to positions selected from the group consisting of: R181, G182, D183 and G184, preferably D183* and G184*, wherein the numbering is according to the amino acid sequence shown in SEQ ID NO: 2.

[0462] Preferably, the amylase variant comprises a deletion at two amino acids corresponding to positions selected from the group consisting of: 181, 182, 183 and 184, preferably a deletion corresponding to amino acids at positions 181 and 182, 182 and 183, or 183 and 184, preferably deletion D183* and G184*, wherein numbering is according to SEQ ID NO:2.

[0463] The present invention relates to an amylase variant of a parent amylase having amylase activity, wherein the variant comprises an amino acid change, preferably an insertion, a deletion, a substitution or a combination thereof, most preferably a substitution, at two or more positions corresponding to the 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 shown in SEQ ID NO: 2, compared to the parent amylase.

[0464] In an alternative manner, 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) may be described with reference to the numbering of SEQ ID NO: 1 (i.e., according to the numbering of SEQ ID NO: 1). In this alternative manner of describing the amino acid positions of the invention, amino acid positions 4, 25, 176, 186, 251, 405 and 482 according to the numbering of SEQ ID NO: 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.

[0465] Preferably, the parent amylase of the amylase variant is an amylase having at least 60% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2, or any one of SEQ ID NO: 3-15, preferably the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 2, or any one of SEQ ID NO: 3-15. Most preferably, the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1.

[0466] Preferably, the present invention relates to an amylase variant 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, numbered according to the amino acid sequence shown in SEQ ID NO:2.

[0467] Preferably, the present invention relates to an amylase variant 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, compared to the parent sequence, and wherein the variant has amylase activity, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or any amylase having at least 60% sequence identity with SEQ ID NO: 1, most preferably the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1.

[0468] Preferably, in this embodiment, the amino acid residue in the parent amylase at the above cited position (ie, X) corresponds to the amino acid residue shown in SEQ ID NO: 1 at the corresponding position (according to the numbering of SEQ ID NO: 2).

[0469] Thus, preferably, the present invention relates to an amylase variant comprising an amino acid substitution at one or more positions corresponding to a position selected from the group consisting of: G4, N25, W116, R176, R181, G186, N195, 1206, T225, T251, R320, L405 and Y482 according to the numbering of the amino acid sequence shown in SEQ ID NO: 2.

[0470] In a preferred embodiment, the present invention relates to an amylase variant comprising an amino acid substitution at one or more positions corresponding to a position 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 shown in SEQ ID NO:2.

[0471] The present invention preferably relates to amylase variants comprising an amino acid substitution at position 25 and at one or more positions corresponding to 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.

[0472] Preferably, the present invention relates to an amylase variant comprising the amino acid substitution X25H and one or more amino acid substitutions selected from the group consisting of X4Q, X116K, X176K, X186E, X195F, X206Y, X225A, X251E, X320K, X405M and X482W, numbered according to the amino acid sequence shown in SEQ ID NO: 2.

[0473] Thus, preferably, the present invention relates to an amylase variant comprising an amino acid substitution at amino acid position N25 and at one or more positions corresponding to positions selected from the group consisting of: G4, W116, R176, R181, G186, N195, 1206, T225, T251, R320, L405 and Y482 according to the numbering of SEQ ID NO:2.

[0474] In a preferred embodiment, the present invention relates to an amylase variant comprising the amino acid substitution N25H and an amino acid substitution at one or more positions corresponding to the positions selected from the group consisting of: G4Q, W116K, R176K, R181T, G186E, N195F, I206Y, T225A, T251E, R320K, L405M and Y482W according to the numbering of SEQ ID NO:2.

[0475] Preferably, the amylase variant comprises an amino acid substitution at one or more of the amino acid positions (according to the numbering of the amino acid sequence shown in SEQ ID NO: 2) as compared to the parent amylase. Preferably, the parent amylase of the amylase variant is an amylase according to any one of SEQ ID NOs: 1-15, most preferably the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1. Preferably, the amino acid residue at the referenced position (i.e., X) in the parent amylase corresponds to the amino acid residue shown in SEQ ID NO: 2 at the corresponding position (according to the numbering of SEQ ID NO: 2).

[0476] 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 acid substitution at position 25 is X25Y or X25D.

[0477] 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.

[0478] 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.

[0479] 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.

[0480] 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.

[0481] 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.

[0482] 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.

[0483] 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.

[0484] 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.

[0485] 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.

[0486] 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.

[0487] 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.

[0488] 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.

[0489] In one embodiment, the amylase variant of a parent amylase comprises

[0490] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 2,

[0491] (ii) amino acid substitutions at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of 116, 176, 181, 186, 195, 206, 225, 320 and 482 according to the numbering of SEQ ID NO: 2, and

[0492] (iii) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1-15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1.

[0493] Preferably, the amylase variant comprises an amino acid substitution at position 25 and an amino acid substitution at one or more positions selected from the group consisting of 116, 181, 225 and 320 according to the numbering of SEQ ID NO:2.

[0494] Preferably, the amylase variant comprises an amino acid substitution at position 25 and an amino acid substitution at one or more positions selected from the group consisting of 176 and 186 according to the numbering of SEQ ID NO:2.

[0495] Preferably, the amylase variant comprises an amino acid substitution at position 25 and an amino acid substitution at one or more positions selected from the group consisting of: 195 or 206 according to the numbering of SEQ ID NO:2.

[0496] 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.

[0497] Preferably, the amino acid substitution at position 25 is X25H.

[0498] Preferably, the amino acid substitution at position 116 is X116K, preferably the amino acid substitution at position 176 is X176K, preferably the amino acid substitution at position 181 is X181T, preferably the amino acid substitution at position 186 is X186E, preferably the amino acid substitution at position 195 is X195F, preferably the amino acid substitution at position 206 is X206Y, preferably the amino acid substitution at position 225 is X225A, preferably the amino acid substitution at position 320 is X320K, and preferably the amino acid substitution at position 482 is X482W.

[0499] Preferably, the amylase variant comprises an amino acid substitution at position 25, preferably X25H, and (according to the numbering of SEQ ID NO: 2)

[0500] a) substitution at one or more positions selected from 176 and 186; preferably, one or more substitutions selected from X176K and X186E, or

[0501] 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

[0502] 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.

[0503] Preferably, the amylase variant comprises an amino acid substitution at position 25, preferably X25H, and (according to the numbering of SEQ ID NO: 2)

[0504] a) substitution at one or more positions selected from 176 and 186; preferably, one or more substitutions selected from X176K and X186E, or

[0505] 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.

[0506] Preferably, the amylase variant comprises an amino acid substitution at position 25, preferably X25H, and (according to the numbering of SEQ ID NO: 2)

[0507] 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

[0508] 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

[0509] 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.

[0510] Preferably, the amylase variant comprises an amino acid substitution at position 25, preferably X25H, and (according to the numbering of SEQ ID NO: 2)

[0511] 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

[0512] 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.

[0513] Preferably, the amylase variant comprises an amino acid substitution at position 25, preferably X25H, and (according to the numbering of SEQ ID NO: 2)

[0514] a) a substitution at amino acid position 195, preferably X195F, and a substitution at one or more positions selected from 176 and 186; preferably, one or more substitutions selected from X176K and X186E, or

[0515] b) a substitution at amino acid position 195 (preferably X195F), and a substitution at one or more positions selected from 176 and 186, preferably one or more substitutions selected from X176K and X186E, and a substitution at one or more positions selected from 116, 181, 225 and 320, preferably one or more substitutions selected from X116K, X225A and X320K.

[0516] Preferably, the amylase variant comprises

[0517] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 2, and

[0518] (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, numbered according to SEQ ID NO: 2.

[0519] Preferably, the present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0520] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 2,

[0521] (ii) any one of the amino acid substitutions X195F or X206Y according to the numbering of SEQ ID NO: 2,

[0522] (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.

[0523] Preferably, the amylase variant comprises one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 2):

[0524] X25H+X176K+X186E X25H+X176K+X186E+X195F X25H+X116K+X181T+X195F+X225A+X320K X25H+X116K+X176K+X181T+X195F+X225A+X320K X25H+X116K+X181T+X186E+X195F+X225A+X320K X25H+X116K+X181T+X195F+X225A+X320K+X482W X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W

[0525] Most preferably, the amylase variant comprises one of the following combinations of amino acid substitutions: X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W (according to the numbering of SEQ ID NO: 2).

[0526] Preferably, the amylase variant comprises one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 2):

[0527] X25H+X176K+X186E 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

[0528] 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).

[0529] In one embodiment, the amylase variant of a parent amylase comprises

[0530] (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 2,

[0531] (ii) amino acid substitutions at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of 25, 176, 186, 251, 405 and 482 according to the numbering of SEQ ID NO: 2, and

[0532] (iii) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or any one of SEQ ID NO: 3-15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1.

[0533] 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.

[0534] Preferably, the amylase variant comprises an amino acid substitution at position 4 and an amino acid substitution at one or more positions selected from the group consisting of 176 and 186 according to the numbering of SEQ ID NO:2.

[0535] Preferably, the amylase variant comprises an amino acid substitution at position 4 and an amino acid substitution at one or more positions selected from the group consisting of: 251 and 405 according to the numbering of SEQ ID NO:2.

[0536] 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.

[0537] In one embodiment, the amylase variant of a parent amylase comprises

[0538] (i) amino acid substitutions at positions 25 and 4 according to the numbering of SEQ ID NO: 2,

[0539] (ii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of 176, 186, 251, 405 and 482 according to the numbering of SEQ ID NO: 2, and

[0540] (iii) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or any one of SEQ ID NO: 3-15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1.

[0541] Preferably, the amino acid substitution at position 4 is X4Q. Preferably, the amino acid substitution at position 25 is X25H.

[0542] Preferably, the amino acid substitution at position 176 is X176K, preferably the amino acid substitution at position 186 is X186E, preferably the amino acid substitution at position 251 is X251E, preferably the amino acid substitution at position 405 is X405M, and preferably the amino acid substitution at position 482 is X482W.

[0543] Preferably, the amylase variant comprises (according to the numbering of SEQ ID NO: 2)

[0544] a) substitution at one or more positions selected from 25, 176 and 186; preferably, one or more substitutions selected from X25H, X176K and X186E, or

[0545] b) substitutions at one or more positions selected from 251, 405 and 482, preferably one or more substitutions selected from X251E, X405M and X482W, or

[0546] 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.

[0547] Preferably, the amylase variant comprises

[0548] (i) amino acid substitution X4G according to the numbering of SEQ ID NO: 2,

[0549] (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.

[0550] Preferably, the amylase variant comprises an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 2, preferably X4Q, and

[0551] a) substitution at one or more positions selected from 25, 176 and 186; preferably, one or more substitutions selected from X25H, X176K and X186E, or

[0552] b) substitution at one or more positions selected from 251, 405 and 482, preferably one or more substitutions selected from X251E, X405M and X482W, or

[0553] 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.

[0554] Preferably, the amylase variant comprises

[0555] (i) amino acid substitution X4G according to the numbering of SEQ ID NO: 2,

[0556] (ii) amino acid substitution X25H according to the numbering of SEQ ID NO: 2, and

[0557] (iii) amino acid substitutions X176K and X186E according to the numbering of SEQ ID NO:2.

[0558] Preferably, the amylase variant comprises

[0559] (i) amino acid substitution X4G according to the numbering of SEQ ID NO: 2,

[0560] (ii) amino acid substitution X25H according to the numbering of SEQ ID NO: 2,

[0561] (iii) amino acid substitutions X176K and X186E according to the numbering of SEQ ID NO: 2, and

[0562] (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.

[0563] Preferably, the amylase variant comprises one of the following combinations of amino acid substitutions (according to the numbering of SEQ ID NO: 2):

[0564] 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 X4Q+X25H+X176K+X186E+X251E+X405M+X482W

[0565] Most preferably, the amylase variant comprises the following combination of amino acid substitutions: X4Q+X25H+X176K+X186E+X251E+X405M+X482W (according to the numbering of SEQ ID NO: 2).

[0566] The amylase variants according to the invention having one or more amino acid substitutions as described herein preferably have at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, %, at least 98%, at least 98.5%, at least 99% or at least 99.5% but less than 100% sequence identity.

[0567] Preferably, the amylase variants of the present invention having one or more amino acid substitutions as described herein are preferably SEQ ID NOs: 1-15, preferably SEQ ID NOs: 1 or 2, preferably SEQ ID The amino acid sequence shown in NO:1 has at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100% sequence identity.

[0568] Preferably, the amylase variants of the invention having one or more amino acid substitutions as described herein preferably have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5%, but less than 100% sequence identity to the amino acid sequence shown in SEQ ID NO:1.

[0569] Preferably, the amylase variants according to the invention having one or more amino acid substitutions as described herein preferably have at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5% but less than 100% sequence identity to the amino acid sequence shown in SEQ ID NO:1.

[0570] In another embodiment, the amylase variant according to the present invention having amylase activity comprises or consists of: any one of SEQ ID NO: 1-15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably the amino acid sequence shown in SEQ ID NO: 1, having one or more amino acid changes described herein.

[0571] In another embodiment, the amylase variant according to the invention having amylase activity comprises or consists of: any one of SEQ ID NO: 1-15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably the amino acid sequence shown in SEQ ID NO: 1, having an amino acid substitution X25H according to the numbering of SEQ ID NO: 2, and, preferably any of amino acid substitutions X195F or X206Y, and having 1 to 7, preferably 2 to 7 or 3 to 7, such as 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions selected from the group consisting of: X116K, X176K, X181T, X186E, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2.

[0572] In another embodiment, the amylase variant according to the invention having amylase activity comprises or consists of: any one of SEQ ID NO: 1-15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably the amino acid sequence shown 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, such as 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.

[0573] Preferably, the amylase variant according to the present invention having amylase activity comprises or consists of any one of SEQ ID NOs: 1-15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 3. The amino acid sequence shown in NO:1 has one or more amino acid changes as described above and further comprises 1 to 50, preferably 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 to 5, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 8, or 2 to 5, preferably 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 8, or 3 to 5, preferably 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10 or 4 to 8 conservative amino acid exchanges, preferably including a deletion at one or more, preferably two or more amino acids corresponding to the positions selected from the group consisting of: 181, 182, 183 and 184 according to the numbering of SEQ ID NO:2, as described herein.

[0574] Preferably, the amylase variant according to the invention having amylase activity comprises or consists of: any one of SEQ ID NO: 1-15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably the amino acid sequence shown in SEQ ID NO: 1, having the amino acid substitution X25H and, preferably any one of the amino acid substitutions 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, such as 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions selected from the group consisting of: according to SEQ ID NO: 2 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 including one or more, preferably two or more amino acid deletions at positions corresponding to the group consisting of: according to SEQ ID Numbers 181, 182, 183 and 184 of NO:2 are as described herein.

[0575] Preferably, the amylase variant according to the present invention having amylase activity comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, having an amino acid substitution X25H and, preferably any of amino acid substitutions 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, such as 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions selected from the group consisting of: X116K, X176K, X181T, X186E, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2, and further comprising 1 to 10, preferably 1 to 5 conservative amino acid exchanges.

[0576] Preferably, the amylase variant according to the invention having amylase activity comprises or consists of: any one of SEQ ID NOs: 1-15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably the amino acid sequence shown in SEQ ID NO: 1, having an 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, such as 1, 2, 3, 4, 5 or 6 amino acid substitutions selected from the group consisting of: according to SEQ ID NO: 1 to 15, preferably SEQ ID NO: 1 or SEQ ID NO: 2, preferably the amino acid sequence shown in SEQ ID NO: 1, having an 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, such as 1, 2, 3, 4, 5 or 6 amino acid substitutions selected from the group consisting of: according to 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 including one or more, preferably two or more amino acid deletions at positions corresponding to the group consisting of: according to SEQ ID Numbers 181, 182, 183 and 184 of NO:2 are as described herein.

[0577] Preferably, the amylase variant according to the invention having amylase activity comprises or consists of the amino acid sequence shown 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, such as 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.

[0578] Conservative amino acid substitutions may occur over the entire length of the amylase variant sequence. In one embodiment, such mutations do not belong to a functional domain of the amylase variant. In one embodiment, conservative mutations do not belong to the catalytic center of the amylase variant.

[0579] Preferably, the present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0580] (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 2,

[0581] (ii) amino acid substitutions at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of 25, 176, 186, 251, 405 and 482 according to the numbering of SEQ ID NO: 2, and

[0582] (iii) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or any one of SEQ ID NO: 3-15, preferably to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, and

[0583] (iv) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0584] Preferably, the present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0585] (i) an amino acid substitution at position 4 according to the numbering of SEQ ID NO: 2,

[0586] (ii) amino acid substitutions at one or more, preferably two or more, more preferably three or more amino acid positions corresponding to the 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

[0587] (iii) having at least 80% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or any one of SEQ ID NO: 3-15, preferably to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, and

[0588] (iv) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0589] Preferably, the amylase variant comprises

[0590] (i) amino acid substitution X4G according to the numbering of SEQ ID NO: 2,

[0591] (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,

[0592] (iii) with SEQ ID NO: 1 or SEQ ID NO: 2, or any one of SEQ ID NO: 3-15, preferably with SEQ ID NO: 1 or SEQ ID NO: 2, preferably with SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% or at least 99.5% but less than 100% sequence identity,

[0593] (iv) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0594] Preferably, the amylase variant comprises

[0595] (i) amino acid substitution X4G according to the numbering of SEQ ID NO: 2,

[0596] (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,

[0597] (iii) with SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity,

[0598] (iv) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0599] Preferably, the amylase variant comprises

[0600] (i) a combination of amino acid substitutions (according to the numbering of SEQ ID NO: 2) selected from the group consisting of:

[0601] X4Q+X25H+X176K+X186E,

[0602] X4Q+X25H+X176K+X186E+X251E,

[0603] X4Q+X25H+X176K+X186E+X405M,

[0604] X4Q+X25H+X176K+X186E+X482W,

[0605] X4Q+X25H+X176K+X186E+X251E+X405M,

[0606] X4Q+X25H+X176K+X186E+X251E+X482W,

[0607] X4Q+X25H+X176K+X186E+X405M+X482W, and

[0608] X4Q+X25H+X176K+X186E+X251E+X405M+X482W,

[0609] (ii) with SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity, and

[0610] (iii) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0611] Preferably, the amylase variant comprises

[0612] (i) amino acid substitution X4G according to the numbering of SEQ ID NO: 2,

[0613] (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,

[0614] (iii) with SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity,

[0615] (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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2), and

[0616] (v) wherein the amylase variant exhibits one or more improved properties, preferably relative to the parent amylase, preferably relative to the parent amylase shown in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, preferably the amylase variant has an improvement in stability, thermostability, storage stability, storage stability in detergent compositions, wash performance, wash performance in laundry detergents and / or wash performance in dishwashing detergents, preferably the improved property is improved stability, preferably wherein the improved property is expressed as an improvement factor (IF) > 1.0, and wherein preferably the improvement factor is equal to or greater than 1.1, preferably, equal to or greater than 1.2, more preferably, equal to or greater than 1.3.

[0617] Particularly preferably, the amylase variant comprises

[0618] (i) a combination of amino acid substitutions (according to the numbering of SEQ ID NO: 2) selected from the group consisting of:

[0619] X4Q+X176K+X186E,

[0620] X4Q+X25H+X176K+X186E,

[0621] X4Q+X25H+X176K+X186E+X251E,

[0622] X4Q+X25H+X176K+X186E+X405M,

[0623] X4Q+X25H+X176K+X186E+X482W,

[0624] X4Q+X25H+X176K+X186E+X251E+X405M,

[0625] X4Q+X25H+X176K+X186E+X251E+X482W,

[0626] X4Q+X25H+X176K+X186E+X405M+X482W, and

[0627] X4Q+X25H+X176K+X186E+X251E+X405M+X482W,

[0628] (ii) has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity to SEQ ID NO: 1, and

[0629] (iii) a deletion of two amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of the amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0630] Particularly preferred are amylase variants comprising

[0631] (i) Amino acid substitutions (according to the numbering of SEQ ID NO: 2) combination:

[0632] X4Q+X25H+X176K+X186E+X251E+X405M+X482W,

[0633] (ii) has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity to SEQ ID NO: 1, and

[0634] (iii) a deletion of two amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of the amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0635] Most preferred is an amylase variant comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, with amino acid changes X4Q+X25H+X176K+X186E+X251E+X405M+X482W (according to the numbering of SEQ ID NO: 2).

[0636] Preferably, the present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0637] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 2,

[0638] (ii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of 116, 176, 181, 186, 195, 206, 225, 320 and 482 according to the numbering of SEQ ID NO: 2,

[0639] (iii) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or any one of SEQ ID NO: 3-15, preferably to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, and

[0640] (iv) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0641] Preferably, the present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0642] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 2,

[0643] (ii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of 116, 176, 181, 186, 195, 206, 225, 320 and 482 according to the numbering of SEQ ID NO: 2,

[0644] (iii) having at least 80% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or any one of SEQ ID NO: 3-15, preferably to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, preferably wherein the parent amylase of the amylase variant is an amylase according to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, and

[0645] (iv) the amylase variant of the present invention comprises a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0646] Preferably, the amylase variant comprises

[0647] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 2,

[0648] (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2,

[0649] (iii) with SEQ ID NO: 1 or SEQ ID NO: 2, or any one of SEQ ID NO: 3-15, preferably with SEQ ID NO: 1 or SEQ ID NO: 2, preferably with SEQ ID NO: 3-15 NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity, and

[0650] (iv) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0651] Preferably, the amylase variant comprises

[0652] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 2,

[0653] (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2,

[0654] (iii) with SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity, and

[0655] (iv) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0656] Preferably, the present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0657] (i) an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 2,

[0658] (ii) an amino acid substitution at either position 195 or 206 according to the numbering of SEQ ID NO: 2,

[0659] (iii) an amino acid substitution at one or more, preferably two or more, amino acid positions corresponding to the amino acid positions selected from the group consisting of: 116, 176, 181, 186, 225, 320 and 482 according to the numbering of SEQ ID NO: 2,

[0660] (iv) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 or any one of SEQ ID NO: 3-15, preferably to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, and

[0661] (v) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0662] Preferably, the present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0663] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 2,

[0664] (ii) any one of the amino acid substitutions X195F or X206Y according to the numbering of SEQ ID NO: 2,

[0665] (iii) one or more, two or more, three or more, four or more, five or more, six or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2,

[0666] (iv) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 or any one of SEQ ID NO: 3-15, preferably to SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, and

[0667] (v) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0668] Preferably, the present invention relates to amylase variants of a parent amylase, wherein the variant comprises

[0669] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 2,

[0670] (ii) any one of the amino acid substitutions X195F or X206Y according to the numbering of SEQ ID NO: 2,

[0671] (iii) one or more, two or more, three or more, four or more, five or more, six or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2,

[0672] (iv) has at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to SEQ ID NO: 1, and

[0673] (v) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0674] Preferably, the amylase variant comprises

[0675] (i) a combination of amino acid substitutions (according to the numbering of SEQ ID NO: 2) selected from the group consisting of:

[0676] X25H+X176K+X186E,

[0677] X25H+X176K+X186E+X195F,

[0678] X25H+X176K+X186E+X206Y,

[0679] X25H+X116K+X181T+X206Y+X225A+X320K,

[0680] X25H+X116K+X176K+X181T+X206Y+X225A+X320K,

[0681] X25H+X116K+X181T+X186E+X206Y+X225A+X320K,

[0682] X25H+X116K+X181T+X206Y+X225A+X320K+X482W,

[0683] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K,

[0684] X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W,

[0685] X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W,

[0686] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W,

[0687] X25H+X116K+X181T+X195F+X225A+X320K,

[0688] X25H+X116K+X176K+X181T+X195F+X225A+X320K,

[0689] X25H+X116K+X181T+X186E+X195F+X225A+X320K,

[0690] X25H+X116K+X181T+X195F+X225A+X320K+X482W,

[0691] X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K,

[0692] X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W,

[0693] X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W, and

[0694] X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W,

[0695] (ii) with SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity, and

[0696] (iii) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0697] Preferably, the amylase variant comprises

[0698] (i) a combination of amino acid substitutions (according to the numbering of SEQ ID NO: 2) selected from the group consisting of:

[0699] X25H+X176K+X186E+X195F,

[0700] X25H+X116K+X181T+X195F+X225A+X320K,

[0701] X25H+X116K+X176K+X181T+X195F+X225A+X320K,

[0702] X25H+X116K+X181T+X186E+X195F+X225A+X320K,

[0703] X25H+X116K+X181T+X195F+X225A+X320K+X482W,

[0704] X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K,

[0705] X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W,

[0706] X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W, and

[0707] X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W,

[0708] (ii) with SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity, and

[0709] (iii) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0710] Preferably, the amylase variant comprises

[0711] (i) a combination of amino acid substitutions (according to the numbering of SEQ ID NO: 2) selected from the group consisting of:

[0712] X25H+X176K+X186E+X206Y,

[0713] X25H+X116K+X181T+X206Y+X225A+X320K,

[0714] X25H+X116K+X176K+X181T+X206Y+X225A+X320K,

[0715] X25H+X116K+X181T+X186E+X206Y+X225A+X320K,

[0716] X25H+X116K+X181T+X206Y+X225A+X320K+X482W,

[0717] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K,

[0718] X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W,

[0719] X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W, and

[0720] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W,

[0721] (ii) with SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity, and

[0722] (iii) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (corresponding to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0723] Preferably, the amylase variant comprises

[0724] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 2,

[0725] (ii) one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X195F, X206Y, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2,

[0726] (iii) with SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity,

[0727] (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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2), and

[0728] (v) wherein the amylase variant exhibits one or more improved properties, preferably relative to the parent amylase, preferably relative to the parent amylase shown in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, preferably the amylase variant has an improvement in stability, thermostability, storage stability, storage stability in a detergent composition, wash performance, wash performance in a laundry detergent and / or wash performance in a dishwashing detergent, preferably the improved property is improved stability, preferably storage stability in a detergent composition, preferably wherein the improved property is expressed as an improvement factor (IF) > 1.0, and wherein preferably the improvement factor is equal to or greater than 1.1, preferably, equal to or greater than 1.2, more preferably, equal to or greater than 1.3.

[0729] Preferably, the amylase variant comprises

[0730] (i) amino acid substitution X25H according to the numbering of SEQ ID NO: 2,

[0731] (ii) any one of the amino acid substitutions X195F or X206Y according to the numbering of SEQ ID NO: 2,

[0732] (iii) one or more, two or more, three or more, four or more, five or more, six or more, or all of the amino acid substitutions selected from the group consisting of X116K, X176K, X181T, X186E, X225A, X320K and X482W according to the numbering of SEQ ID NO: 2,

[0733] (iv) the same as SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: NO:1 has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least up to 99.5% but less than 100% sequence identity,

[0734] (v) a deletion of one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184 (according to the numbering of SEQ ID NO: 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 a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2), and

[0735] (vi) wherein the amylase variant exhibits one or more improved properties, preferably relative to the parent amylase, preferably relative to the parent amylase shown in SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1, preferably the amylase variant has an improvement in stability, thermostability, storage stability, storage stability in detergent compositions, wash performance, wash performance in laundry detergents and / or wash performance in dishwashing detergents, preferably the improved property is improved stability, preferably wherein the improved property is expressed as an improvement factor (IF) > 1.0, and wherein preferably the improvement factor is equal to or greater than 1.1, preferably, equal to or greater than 1.2, more preferably, equal to or greater than 1.3.

[0736] Particularly preferably, the amylase variant comprises

[0737] (i) a combination of amino acid substitutions (according to the numbering of SEQ ID NO: 2) selected from the group consisting of:

[0738] X25H+X176K+X186E,

[0739] X25H+X176K+X186E+X195F,

[0740] X25H+X176K+X186E+X206Y,

[0741] X25H+X116K+X181T+X206Y+X225A+X320K,

[0742] X25H+X116K+X176K+X181T+X206Y+X225A+X320K,

[0743] X25H+X116K+X181T+X186E+X206Y+X225A+X320K,

[0744] X25H+X116K+X181T+X206Y+X225A+X320K+X482W,

[0745] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K,

[0746] X25H+X116K+X176K+X181T+X206Y+X225A+X320K+X482W,

[0747] X25H+X116K+X181T+X186E+X206Y+X225A+X320K+X482W,

[0748] X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W,

[0749] X25H+X116K+X181T+X195F+X225A+X320K,

[0750] X25H+X116K+X176K+X181T+X195F+X225A+X320K,

[0751] X25H+X116K+X181T+X186E+X195F+X225A+X320K,

[0752] X25H+X116K+X181T+X195F+X225A+X320K+X482W,

[0753] X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K,

[0754] X25H+X116K+X176K+X181T+X195F+X225A+X320K+X482W,

[0755] X25H+X116K+X181T+X186E+X195F+X225A+X320K+X482W, and

[0756] X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W,

[0757] (ii) has at least 85%, at least 85.5%, at least 86%, at least 86.5%, at least 87%, at least 87.5%, at least 88%, at least 88.5%, at least 89%, at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity to SEQ ID NO: 1, and

[0758] (iii) a deletion of two amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of the amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0759] Particularly preferred are amylase variants comprising

[0760] (i) Amino acid substitutions (according to the numbering of SEQ ID NO: 2) combination:

[0761] 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,

[0762] (ii) has at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% but less than 100% sequence identity to SEQ ID NO: 1, and

[0763] (iii) a deletion of two amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion of the amino acids corresponding to positions 181 and 182, 182 and 183, or 183 and 184, preferably a deletion of D183* and G184* (according to the numbering of SEQ ID NO: 2).

[0764] Most preferred are amylase variants comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1, having amino acid changes X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K, or X25H+X116K+X176K+X181T+X186E+X206Y+X225A+X320K+X482W, X25H+X116K+X176K+X181T+X186E+X195F+X225A+X320K+X482W (according to the numbering of SEQ ID NO: 2).

[0765] In one embodiment, the amylase variant of the enzyme composition comprises

[0766] (i) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO:9, or

[0767] (ii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO:7, or

[0768] (iii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO:3, or

[0769] (iv) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO: 13, or

[0770] (v) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity to SEQ ID NO: 15, or

[0771] (vi) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO:4, or

[0772] (vii) an amino acid substitution at one or more amino acid positions corresponding to the 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.

[0773] In one embodiment, the amylase variant comprises an enzyme 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 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%, An amino acid sequence having 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 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 selected from X183S, X243Q, X266Y and X475K (according to the numbering of SEQ ID NO: 9).

[0774] In one embodiment, the amylase variant comprises an amino acid sequence that is 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% identical to SEQ ID NO:9. , 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 amino acid substitutions at one or more amino acid positions corresponding to the amino acid positions selected from the group consisting of: 183, 243, 266 and 475, more preferably G183S, S243Q, F266Y, G475K (according to the numbering of SEQ ID NO: 9).

[0775] In one embodiment, the amylase variant comprises NO:9 has an amino acid sequence having 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, comprising two amino acid deletions in the regions of R180, G181, T182, G183, with an optional truncation of the C-terminal portion (amino acids 485-583), and further amino acid substitutions at one or more amino acid positions corresponding to amino acid positions selected from the group consisting of and 183, 243, 266 and 475, more preferably G183S, S243Q, F266Y and G475K (according to SEQ ID NO:9). NO:9 number) in its entirety.

[0776] In one embodiment, the amylase variant comprises an enzyme 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 92.5%, at least 93.5%, at least 94.5%, at least 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 99.6%, at least 100. %, 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 amino acid sequences having ...9.5% but less than 100% sequence identity in the sequence corresponding to the amino acid sequence selected from the group consisting of 38, 126, 129 , 153, 180, 187, 199, 203, 241, 362, 377, 458, 459, 460, 476 and 477, preferably one or more, preferably all selected from the group consisting of mutations: X38N, X126Y, X129I, X153W, X180D / H, X187P, X199L, X2 8N, X241Q, X362A, X377Y, X458N, X459S, X460T, X476K and X477E; more preferably all of X38N, X126Y, X129I, X153W, X178*, X179*, X180D, X187P, X203Y, X476K and X477E or all of X178*, X179*, X187P, X203Y X458N, X459S, X460T and X476K in combination (according to the numbering of SEQ ID NO: 7).

[0777] In one embodiment, the amylase variant comprises an amino acid sequence having 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 acid mutation at one or more amino acid positions of the amino acid position is preferably one or more, preferably all selected from the group consisting of: T38N, N126Y, T129I, F153W, T180D / H, E187P, M199L, I203Y, S241Q, S362A, R377Y, R458N, T459S, D460T, G476K and G477E; more preferably all of T38N, N126Y, T129I, F153W, R178*, G179*, T180D, E187P, I203Y, G476K and G477E or all of R178*, G179*, E187P, I203Y, G476K and G477E in combination. R458N, T459S, D460T and G476K (according to the numbering of SEQ ID NO: 7).

[0778] In one embodiment, the amylase variant comprises an amino acid sequence that is 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 95.5%, at least 96.5%, at least 97.5%, at least 98.5%, at least 99.6%, at least 100. An amino acid sequence having 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, comprising two amino acid deletions in the regions R181, G182, D183, G184, and further at one amino acid position corresponding to an amino acid selected from the group consisting of or amino acid substitutions at one or more amino acid positions: 1, 7, 51, 52, 54, 109, 140, 195, 206, 243, 260, 280, 284, 304, 391, 476 and 477; preferably one or more, preferably all, mutations selected from the group consisting of: X1*, X7A, X51I, X52Q, X54K, X109A, X140Y, X195F, X206Y, X243F, X260G, X X52Q, X54K, X109A, X140Y, X195F, X206Y, X243F, X260G, X280S, X284F / D / R, X304R, X391A, X476E / K and X477R / K / E in combination (according to the numbering of SEQ ID NO: 2).

[0779] In one embodiment, the amylase variant comprises an amino acid sequence having 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, comprising two amino acid deletions in the regions R181, G182, D183, G184, and further 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, 257 60, 280, 284, 304, 391, 476 and 477; preferably one or more, preferably all, mutations selected from the group consisting of: H1*, G7A, T51I, S52Q, N54K, G109A, W140Y, N195F, V206Y, Y243F, E260G, N280S, W284F / D / R, G304R, E391 A, G476E / K and G477R / K / E, preferably all 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 (according to the numbering of SEQ ID NO: 2).

[0780] In one embodiment, the amylase 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 96.5%, at least 97.5%, at least 98.5%, at least 99.6%, at least 100. An amino acid sequence having at least 6%, 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, comprising two amino acid deletions in the regions R181, G182, D183, G184, and further 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; preferably one or more, preferably all, mutations selected from the group consisting of: X9I / L, X118K, X149A, X182T, X186A, X195F, X202L, X246V, X257I, X295F, X299Y, X320K, X323T, X339S, X345R, X more preferably all mutations X118K, X183*, X184*, X195F, X320K and X458K or all of X9L, X118K, X149A, X182T, X183*, X184*, X186A, X195F, X202L, X257I, X295F, X299Y, X320K, X323T, X339S, X345R and X458K in combination (according to the numbering of SEQ ID NO: 13).

[0781] In one embodiment, the amylase variant comprises an amino acid sequence having 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, comprising two amino acid deletions in the regions R181, G182, D183, G184, and further 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, 330 39, 345, 458 and 471; preferably one or more, preferably all, selected from the group consisting of: M9I / L, R118K, G149A, G182T, G186A, N195F, M202L, T246V, T257I, Y295F, N299Y, R320K, M323T, A339S, E345R, R458K and N471E; more preferably all Mutations R118K, D183*, G184*, N195F, R320K and R458K or in combination M9L, R118K, G149A, G182T, D183*, G184*, G186A, N195F, M202L, T257I, Y295F, N299Y, R320K, M323T, A339S, E345R and R458K (according to the numbering of SEQ ID NO: 13).

[0782] An amylase having substitutions as described above is, for example, shown in SEQ ID NO:14.

[0783] 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.

[0784] In one embodiment, the amylase 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%, or at least 99.5% but less than 100% sequence identity, comprising two amino acid deletions in the regions of R181, G182, D183, G184, and further 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; preferably one or more, preferably all, mutations selected from the group consisting of: X1A, X54S, X56T, X72R, X109A, X113Q, X116Q, X149A, X167F, X172G, X174S, X182T, X195F, X202L, X206L / Y, X246V, X257I, X295F, X299Y, X320K, X339S, X345R, XK391A, X45 8K, X471E, X473R and X476K; preferably all of 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 in combination (according to the numbering of SEQ ID NO: 15).

[0785] In one embodiment, the amylase variant comprises an amino acid sequence having 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: 15, comprising at R181, G182, G183, G184, G185, G186, G187, G188, G189, G190, G191, G192, G193, G194, G195, G196, G197, G198, G199, G191, G199, G190, G191, G197, G199, G199, G190, G199, G190, G199, 82, D183, two amino acid deletions in the region G184, and further 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; preferably one or A plurality of, preferably all, mutations selected from the group consisting of: 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 G4 76K; preferably all of 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 in combination (according to the numbering of SEQ ID NO: 15).

[0786] 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%, or at least 99.5% but less than 100% sequence identity to SEQ ID NO:4, comprising two amino acid deletions in the regions R181, G182, H183, G184, and further at one or more amino acid positions corresponding to the amino acid positions selected from the group consisting of Amino acid substitutions at amino acid positions: 9, 118, 149, 182, 186, 195, 202, 246, 257, 295, 299, 320, 323, 339, 345, 405, 421, 422, 428, 458 and 471; preferably one or more, preferably all, mutations selected from the group consisting of: X9I / L, X118K, X149A, X182T, X186A , X195F, X202L, X246V, X257I, X295F, X299Y, X320K, X323T, X339S, X345R, X405L, X421H, X422P, X428T, X458K and X471E, more preferably all mutations X183*, X184*, X405L, X421H, X422P and X428T in combination (according to the numbering of SEQ ID NO: 4).

[0787] In one embodiment, the amylase variant comprises an amino acid sequence having 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, comprising two amino acid deletions in the regions R181, G182, H183, G184, and further 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; preferably one or more, preferably all mutations selected from the group consisting of: M9I / L, R118K, G149A, G182T, G186A, N195F, M202L, T246V, T257I, Y295F, N299Y, R320K, M323T, A339S, E345R, I405L, A421H, A422P, A428T, R458K and N471E, more preferably all mutations H183*, G184*, I405L, A421H, A422P and A428T (according to SEQ ID NO: 1) IDNO:4).

[0788] In one embodiment, the amylase variant comprises an enzyme 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% similar to SEQ ID NO:5.5% 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: 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 one or more, preferably all, mutations selected from the group consisting of: 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, X196 202L, X206L, X206Y, X246V, 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 one or more, preferably all 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 X1*, X54S, X56T, X72R, X109A, X113Q, X116Q, X167F, X172G, X174S, X182*, X183*, X184T, X195F, X206L, X391A, X473R and X476K in combination (numbering according to SEQ ID NO: 5).

[0789] In one embodiment, the amylase variant comprises an enzyme 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%, or at least 99.5% identical to SEQ ID NO:5.5% but less than 100% sequence identity, comprising two amino acid deletions in the regions R181, G182, D183, G184, and further 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; preferably one or more, preferably all, mutations selected from the group consisting of: H1*, N54S, V56T, K72R, G109A, F113Q, R116H, R116Q, V120L, T134E, W140Y, W167F, Q169E, Q172E, Q172G, Q172K, Q172N, Q172R, L173P, L173V, A174*, A174S, A 174K, G182T, N195F, M202L, 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 one or more, preferably all selected from the group consisting of: H1*, N54S, V 56T, K72R, G109A, F113Q, R116Q, W167F, Q172G, A174S, G182*, D183*, G184T, N195F, V206L, K391A, P473R and G476K, preferably all H1*, N54S, V56T, K72R, G109A, F113Q, R116Q, W167F, Q172G, A174S, G182*, D183*, G184T, N195F, V206L, K391A, P473R and G476K in combination (according to the numbering of SEQ ID NO: 5). .

[0790] An amylase having substitutions as described above is, for example, shown in SEQ ID NO:6.

[0791] 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.

[0792] Preferably, the composition further comprises an amylase variant comprising

[0793] a) the amino acid substitution X25H 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 (according to the numbering of SEQ ID NO: 2),

[0794] b) a deletion at one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion corresponding to two or more amino acids at positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion corresponding to amino acids at positions 181 and 182, 182 and 183, or 183 and 184, preferably a deletion corresponding to two or more, most preferably two amino acids at positions selected from the group consisting of R181, G182, D183 and G184, preferably D183* and G184*, wherein the numbering is according to the amino acid sequence shown in SEQ ID NO: 2, and

[0795] c) wherein 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.

[0796] Alternatively, preferably, the composition further comprises an amylase variant and wherein the amylase 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% or at least 99.5% identical to SEQ ID NO:5.5% but less than 100% sequence identity, and an amino acid substitution at one or more amino acid positions corresponding to an amino acid position 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, 2 99, 304, 320, 339, 345, 391, 395, 410, 422, 429, 444, 451, 458, 473, 474 and 476; preferred mutations X1*, X54S, X56T, X72R, X109A, X113Q, X116H, X116Q, X120L, X134E, X140Y, X167F, X169E, X172E, X172G, X172K, X172N, X1 72R, X173P, X173V, X174*, X174S, X174K, X182T, X260G, X265G, X295F, X299Y, X304R, X320A, X320K, X339S, , X444Q, X451W, X458K, X473R, X474V and X476K, more preferably one or more modifications 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 (according to SEQ ID NO: 1 ID NO: 5), preferably all modifications in combination, most preferably modifications H1*, N54S, V56T, K72R, G109A, F113Q, R116Q, W167F, Q172G, A174S, G182*, D183*, G184T, N195F, V206L, K391A, P473R and G476K in combination. Therefore, in another preferred alternative embodiment, the composition further comprises an amylase variant, and wherein the amylase variant comprises 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 96.5%, at least 97%, at least 98%, at least 99.5%, 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.

[0797] Preferably, the composition comprises protease, mannanase, cellulase and amylase, wherein

[0798] a. The protease is a protease variant comprising

[0799] (i) amino acid substitution X101E according to the numbering of SEQ ID NO: 17, and

[0800] (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 SEQ ID NO:16,

[0801] b. The mannanase is a mannanase variant comprising

[0802] (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

[0803] (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 according to positions 31-490 of SEQ ID NO:35, and

[0804] 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,

[0805] Wherein the amylase is an amylase variant comprising

[0806] a) the amino acid substitution X25H 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 (according to the numbering of SEQ ID NO: 2),

[0807] b) a deletion at one or more amino acids corresponding to positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion corresponding to two or more amino acids at positions selected from the group consisting of 181, 182, 183 and 184, preferably a deletion corresponding to amino acids at positions 181 and 182, 182 and 183, or 183 and 184, preferably a deletion corresponding to two or more, most preferably two amino acids at positions selected from the group consisting of R181, G182, D183 and G184, preferably D183* and G184*, wherein the numbering is according to the amino acid sequence shown in SEQ ID NO: 2, and

[0808] c) wherein 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.

[0809] Lipase

[0810] "Lipase", "lipolytic enzyme", "lipid esterase" all refer to enzymes of EC class 3.1.1 ("carboxylic ester hydrolase"). Lipase means an active protein having lipase activity (or lipolytic activity; triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74; enzymes having cutinase activity may be referred to herein as cutinases), sterol esterase activity (EC 3.1.1.13) and / or wax ester hydrolase activity (EC 3.1.1.50). Lipases include those of bacterial or fungal origin.

[0811] In one aspect of the invention, suitable lipases are selected from the group consisting of a lipase from Humicola (synonym Thermomyces) as described in EP 258068, EP 305216, WO 92 / 05249 and WO 2009 / 109500, e.g. a lipase from H. lanuginosa (T. lanuginosus), or a lipase from H. insolens as described in WO 96 / 13580; a lipase derived from Rhizomucor miehei as described in WO 92 / 05249; miehei); lipases from strains of Pseudomonas (some of which are now renamed Burkholderia), for example from Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (EP 218272, WO 94 / 25578, WO 95 / 30744, WO 95 / 35381, WO 96 / 00292), Pseudomonas cepacia (EP 331376), Pseudomonas stutzeri (P.stutzeri) (GB 1372034), Pseudomonas fluorescens, Pseudomonas sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012), Pseudomonas mendocina (WO 95 / 14783), Pseudomonas glumae (WO 95 / 35381, WO96 / 00292); lipases from Streptomyces griseus (WO 2011 / 150157) and Streptomyces spp.pristinaespiralis) (WO 2012 / 137147), a GDSL-type Streptomyces lipase (WO 2010 / 065455); a lipase from Thermobifida fusca as disclosed in WO 2011 / 084412; a lipase from Geobacillus stearothermophilus as disclosed in WO 2011 / 084417; a Bacillus lipase, for example as disclosed in WO 00 / 60063, as disclosed in Dartois et al. (1992), Biochemica et Biophysica Acta [Biochemistry and Biophysics Acta], 1131, 253-360 or WO 2011 / 084599 disclosed in Bacillus subtilis (B.subtilis), Bacillus stearothermophilus (B.stearothermophilus) (JP S64-074992) or Bacillus pumilus (B.pumilus) (WO 91 / 16422); as disclosed in WO 94 / 01541, a lipase from Candida antarctica; a cutinase from Pseudomonas mendocina (US 5389536, WO 88 / 09367); a cutinase from Magnaporthe grisea (WO 2010 / 107560); as disclosed in WO 90 / 09446, WO 00 / 34450 and WO 01 / 92502, a cutinase from Fusarum oleraceum (Fusarum oleraceum) (W00 / 09446, WO 00 / 34450 and WO 01 / 92502); as disclosed in WO 90 / 09446, WO 00 / 34450 and WO 01 / 92502, a cutinase from Pseudomonas mendocina (US 5389536, WO 88 / 09367 ... solani pisi); and a cutinase from Humicola lanuginosa as disclosed in WO 00 / 34450 and WO 01 / 92502.

[0812] Such suitable lipase variants are, for example, those developed by the methods as disclosed in WO 95 / 22615, WO 97 / 04079, WO 97 / 07202, WO 00 / 60063, WO 2007 / 087508, EP 407225 and EP 260105.

[0813] Commercially available lipases include, but are not limited to, those sold under the trade name Lipolase TM 、Lipex TM 、Lipolex TM and Lipoclean TM(Novozymes), Lumafast (originally from Genencor), Preferenz L (DuPont), Biotouch LL100 (AB Enzymes), and Lipomax (Gist-Brocades / now DSM).

[0814] In one embodiment, the lipase is selected from fungal triacylglycerol lipase (EC class 3.1.1.3). The fungal triacylglycerol lipase may be selected from the lipase of Thermomyces lanuginosus. In one embodiment, the Thermomyces lanuginosus lipase is selected from the triacylglycerol lipase of amino acids 1-269 of SEQ ID NO: 2 according to US 5869438 and variants thereof having lipolytic activity.

[0815] The Thermomyces lanuginosus lipase can be selected from a variant having lipolytic activity that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US 5869438.

[0816] The Thermomyces lanuginosus lipase can be selected from variants with lipolytic activity that contain only conservative mutations that do not involve the functional domain of amino acids 1-269 of SEQ ID NO: 2 of US 5869438. The lipase variants of this embodiment with lipolytic activity can be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US 5869438.

[0817] The Thermomyces lanuginosus lipase may be selected from variants having lipolytic activity comprising the following amino acid substitutions when compared to amino acids 1-269 of SEQ ID NO: 2 of US 5869438: T231R and N233R. When compared to amino acids 1-269 of SEQ ID NO: 2 of US 5869438, the lipase variant may further comprise one or more of the following amino acid exchanges: Q4V, V60S, A150G, L227G, P256K.

[0818] The Thermomyces lanuginosus lipase can be selected from variants with lipolytic activity comprising amino acid substitutions T231R, N233R, Q4V, V60S, A150G, L227G, P256K within the polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US 5869438 and being at least 95%, at least 96% or at least 97% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US 5869438.

[0819] The Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity comprising amino acid substitutions T231R and N233R within amino acids 1-269 of SEQ ID NO: 2 of US 5869438 and being at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US 5869438.

[0820] The Thermomyces lanuginosus lipase may be a variant of amino acids 1-269 of SEQ ID NO: 2 of US 5869438 having lipolytic activity, wherein the variant of amino acids 1-269 of SEQ ID NO: 2 of US 5869438 is characterized in containing amino acid substitutions T231R and N233R.

[0821] The Thermomyces lanuginosus lipase can be selected from a variant having lipolytic activity, preferably comprising at least one, preferably more than one, more preferably all of the following substitutions within the polypeptide sequence of amino acids 1-269 of SEQ ID NO: 1 of WO 2015 / 010009: N11K, A18K, G23K, K24A, V77I, D130A, V154I, V187T, T189Q and being at least 95%, at least 96% or at least 97% similar when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 1 of WO2015 / 010009.

[0822] Methods for obtaining enzymes and preparing enzyme compositions

[0823] Another embodiment of the present invention is a method of preparing an enzyme composition comprising a protease, a mannanase and / or a cellulase as described herein, the method preferably comprising the steps of:

[0824] a) obtaining the sequence of a variant of a protease, mannanase and / or cellulase as described herein,

[0825] b) using the sequence obtained under a) to produce said variant of a protease, mannanase or cellulase, as described herein, and

[0826] c) mixing in one or more steps two or more enzymes selected from the group consisting of proteases, mannanases and cellulases as described herein.

[0827] The method for obtaining specific enzyme variants is known to the technician. The method of introducing amino acid changes such as substitutions or deletions into protein sequences is well known in the art. Any mutagenesis program known in the art can be used to prepare variants of enzymes as described herein, and the mutagenesis program is such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, reorganization, etc. Site-directed mutagenesis is a technology that produces one or more (several) mutations at one or more determined sites of a polynucleotide. Site-directed mutagenesis can be completed in vitro by PCR, involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be carried out in vitro by cassette mutagenesis, which involves cutting by restriction endonucleases at sites in a plasmid containing a polynucleotide encoding a nucleotide to be mutated, and subsequently connecting oligonucleotides containing mutations in the polynucleotide. Usually, the restriction endonucleases that digest plasmids and oligonucleotides are identical, allowing the sticky ends of plasmids and inserts to be connected to each other. See, e.g., 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 accomplished in vivo by methods known in the art. See, e.g., U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16. Any site-directed mutagenesis method can be used in the present invention. There are many commercial kits available for preparing variants.

[0828] Synthetic gene construction requires in vitro synthesis of designed polynucleotide molecules to encode the target polypeptide. Gene synthesis can be performed using a variety of techniques, such as the multiplex microchip-based technology described in Tian et al. (2004, Nature 432: 1050-1054) and similar technologies in which oligonucleotides are synthesized and assembled on optically programmable microfluidic chips.

[0829] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known mutagenesis, recombination and / or shuffling methods followed by relevant 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-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0830] 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 can rapidly determine the importance of individual amino acid residues in a polypeptide.

[0831] Semisynthetic gene construction is to complete by combining synthetic gene construction and / or site-directed mutagenesis and / or random mutagenesis and / or the aspect of reorganization.The characteristic feature of semisynthetic construction is to utilize the polynucleotide fragment of synthesis and the method in conjunction with PCR technology.Therefore, the defined region of gene can therefore synthesize from the beginning, and other regions can use site-specific mutagenic primers to increase, and can carry out fallibility PCR or non-fallibility PCR amplification in other regions again.Then can polynucleotide subsequence be reorganized.

[0832] The enzyme obtained can be produced on an industrial scale and purified subsequently. The industrial production of the enzyme is usually accomplished by culturing a host cell (also referred to as fermentation) expressing the enzyme. Suitable host cells are as described below. The nucleic acid sequence encoding the corresponding enzyme as described herein can be transformed into a host cell, which is then cultured under conditions suitable for the host cell to produce the enzyme. In a preferred embodiment, the enzyme is purified from the host cell.

[0833] Producing the enzyme preferably comprises the following steps:

[0834] (a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding an enzyme as described herein by introducing a nucleic acid construct comprising a polynucleotide encoding an enzyme as described herein into the host cell;

[0835] (b) culturing the recombinant host cell of step (a) under conditions conducive to expression of the polynucleotide; and

[0836] (c) optionally, recovering the enzyme encoded by the polynucleotide.

[0837] The host cell culture is usually carried out in a suitable nutrient medium to allow the recombinant cells to grow and express the desired protein. At the end of the fermentation, the fermentation broth is collected and can be further processed, wherein the fermentation broth comprises a liquid portion and a solid portion. The target enzyme can be further purified from the fermentation broth.

[0838] Amylases, proteases, mannanases and cellulases as described herein may be secreted (enter the liquid portion of the fermentation broth), or may not be secreted from microbial cells (and therefore be contained in the cells of the fermentation broth). According to this, enzymes can be recovered from the liquid portion of the fermentation broth or from cell lysates. Preferably, the enzyme is secreted from the cells into the fermentation broth, preferably by a secretion signal peptide added to the end of the amino acid sequence of the corresponding enzyme. The recovery of the enzyme can be achieved by methods known to those skilled in the art. Suitable methods for recovering protein from the fermentation broth include, but are not limited to, collection, centrifugation, filtration, extraction and precipitation. If the target product precipitates or crystallizes in the fermentation broth or is at least partially combined with the particulate matter of the fermentation broth, additional processing steps may be required to release the target protein from the biomass or dissolve the target protein crystals and precipitates. WO 0043502 A1, WO 2008110498 A1 and WO 2017097869A1 describe methods for recovering target proteins from fermentation broth, which are precipitated and / or crystallized during fermentation. If the desired protein is contained in cells of the fermentation broth, it may be necessary to release the desired product from the cells. Release from the cells can be achieved, for example but not limited to, by cell lysis using techniques well known to those skilled in the art, such as lysozyme treatment, ultrasonic treatment, French press, or a combination thereof.

[0839] Proteases, mannanases and cellulases as described herein can be purified from fermentation broths by methods known in the art. For example, the corresponding enzymes can be separated from fermentation broths by conventional procedures, including but not limited to centrifugation, filtration, extraction, spray drying, evaporation or precipitation. The separated polypeptides can then be further purified by various procedures known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF)), differential solubility (e.g., ammonium sulfate precipitation) or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, eds., VCH Press, New York, 1989). The purified polypeptides can then be concentrated by procedures known in the art, including but not limited to ultrafiltration and evaporation, particularly thin film evaporation.

[0840] Preparations containing enzyme compositions

[0841] The enzyme composition described herein can be further processed to form a preparation containing the enzyme composition.Therefore, also claimed herein is a preparation comprising the enzyme composition described herein and at least one additional component.

[0842] Therefore, the present invention also relates to a method for preparing a formulation, which comprises the following steps: mixing

[0843] a) an enzyme composition as described herein; and

[0844] b) one or more components described herein.

[0845] The formulation may be a non-complex formulation, such as a formulation containing an enzyme composition, or a complex formulation, such as a detergent composition.

[0846] In one embodiment of the invention, the enzyme composition is formulated as a preparation containing the enzyme composition, preferably a preparation containing a concentrated enzyme composition. The preparation containing the enzyme composition can be solid or liquid. The protein formulation can be obtained by using technology known in the art. For example, but not limited to this, the solid enzyme formulation can be obtained by extruding or granulating. Suitable extrusion and granulation techniques are known in the art and are for example described in WO 94 / 19444A1 and WO 97 / 43482A1.

[0847] The liquid enzyme formulation may comprise an enzyme amount 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, all relative to the total weight of the enzyme formulation.

[0848] Preferably, the enzyme formulation comprises the enzyme of the present invention in an amount of 2-120 m...

Claims

1. A composition comprising two or more enzymes selected from the group consisting of proteases, mannanases and cellulases, wherein a. The protease is selected from I. A protease variant of a parent protease, wherein the protease variant comprises (i) compared to SEQ ID NO: 16, comprises at least two additional negatively charged amino acid sequences in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, and (ii) an amino acid sequence that is at least 60% but less than 100% identical to any one of SEQ ID NOs: 16-34, preferably SEQ ID NO: 16, or II. A protease variant of a parent protease, wherein the protease variant comprises (i) an amino acid substitution at one or more amino acid positions corresponding to the 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 substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO: 17, or (iii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO: 19, or (iv) an amino acid sequence that has 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, b. The mannanase is selected from I. A mannanase variant of a parent mannanase, wherein the mannanase variant comprises (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 according to positions 31 to 490 of SEQ ID NO: 35, or II. A mannanase variant of a parent mannanase, wherein the mannanase variant comprises (i) an amino acid substitution at one or more amino acid positions corresponding to the 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 at least 60% but less than 100% identical to SEQ ID NO:39, or (ii) an amino acid substitution at one or more amino acid positions corresponding to the 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, and amino acid sequences that are at least 60% but less than 100% identical to 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 substitution at one or more amino acid positions corresponding to the 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, and an amino acid sequence at least 60% but less than 100% identical to SEQ ID NO:40, c. The cellulase is selected from I. A cellulase variant comprising an amino acid sequence at least 60% identical to SEQ ID NO: 42, or II. A cellulase variant 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, and Wherein at least one enzyme is selected from the group consisting of aa. A protease variant of a parent protease, wherein the protease variant comprises (i) compared to SEQ ID NO: 16, comprises at least two additional negatively charged amino acid sequences in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 17, and (ii) an amino acid sequence at least 60% but less than 100% identical to SEQ ID NO: 16-34, preferably SEQ ID NO: 16, bb. a mannanase variant of a parent mannanase, wherein the mannanase variant comprises (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 according to positions 31-490 of SEQ ID NO:35, and cc. A cellulase variant comprising an amino acid sequence at least 60% identical to SEQ ID NO:

42.

2. A composition as claimed in 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 according to the numbering of SEQ ID NO: 17, preferably X101E.

3. The composition of claim 1 or claim 2, wherein the mannanase variant has at least 80% but less than 100% sequence identity to SEQ ID NO: 35 and comprises one or more amino acid substitutions selected from the group consisting of: NO:35 number X86N, X89V / L, X96D, X101T / V, X103Y / E / A, X107N, X108G, F、X231Q / K / L / P / Y、X233V、X235H / R / L / Q / N / Y、X244I / V / N、X254W、X255 Y / 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, X319D / E, X341F, X346T, X348S / R / N / M / G, X349T / S / G / D, X352N / G, X356Y / V / T / Q / H / C and X379V, preferably selected 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. A composition as described in any of the preceding claims, 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. A composition as claimed in any one of the preceding claims, wherein the composition further comprises an amylase, preferably selected from a) an amylase variant of a parent amylase, wherein the amylase variant (i) comprises an amino acid substitution at position 25 according to the numbering of SEQ ID NO: 2, (ii) comprises an amino acid substitution at one or more amino acid positions corresponding to the 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, and (iii) having at least 60% identity, preferably at least 91% identity, but less than 100% sequence identity to any one of SEQ ID NOs: 1-15, preferably SEQ ID NO: 1, and b) an amylase variant of a parent amylase, wherein the amylase variant comprises (i) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO:9, or (ii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO:7, or (iii) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO:3, or (iv) an amino acid substitution at one or more amino acid positions corresponding to the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO: 13, or (v) an amino acid substitution at one or more amino acid positions corresponding to the 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 having 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 the 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 having at least 60% but less than 100% sequence identity with SEQ ID NO:4, or (vii) an amino acid substitution at one or more amino acid positions corresponding to the 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.

6. The composition of claim 5, wherein the amylase variant has at least 80% but less than 100% sequence identity to SEQ ID NO: 1 and 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.

7. A composition as claimed in any one of the preceding claims, wherein the composition comprises one or more additional enzymes selected from the group consisting of lipases, hemicellulases, xylanases, DNases, dispersins, pectinases, oxidoreductases, cutinases, phosphodiesterases, lichenases, xanthan lyases and hexosaminidase, preferably lipases.

8. A composition as claimed in any preceding claim, wherein the composition is a detergent composition, preferably a laundry detergent composition or a hard surface cleaning detergent composition.

9. The composition of claim 8, wherein the detergent composition comprises detergent components selected from the group consisting of: enzyme stabilizing systems, surfactants, defoamers, builders, polymers, bleaching systems (bleaches), rheology modifiers, hydrotropes, softeners, desiccants, brighteners, buffers, foam inhibitors, preservatives, anti-corrosion additives, dyes and fragrances.

10. The composition of any one of the preceding claims, wherein the 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 preparing the composition according to any one of claims 1 to 10, comprising the step of mixing at least two enzymes selected from the group consisting of protease, mannanase and cellulase.

12. A method for cleaning, the method comprising the steps of: The textile or hard surface is contacted with a composition as claimed in any one of claims 1 to 10, preferably at low temperature.

13. The method of claim 12, wherein the method for cleaning is for cleaning complex stains from textiles or hard surfaces.

14. Use of a composition according to any one of claims 1 to 10 for providing a synergistic cleaning effect, preferably at low temperatures.

15. Use of a composition according to any one of claims 1 to 10 for removing complex stains, preferably at low temperatures.

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