Polypeptides having protease activity for use in detergent compositions

Abstract

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

Description

Technical Field

[0001] The present invention provides novel proteases. More specifically, it provides genetically engineered proteases, compositions comprising these enzymes, and methods of making and using these enzymes or compositions comprising these enzymes. Background Art

[0002] Enzymes are increasingly being used in various applications as sustainable alternatives to petrochemicals. Enzymes are biodegradable and can already function at lower temperatures, thus reducing energy consumption. In particular, in the detergent industry, enzymes are used in cleaning formulations to improve cleaning efficiency and / or reduce energy consumption during the washing step.

[0003] Proteases are enzymes that can hydrolyze proteins. Therefore, proteases have been used to remove protein stains and have been added to detergent compositions for this purpose. In detergent applications, proteases should be stable at elevated temperatures and / or within the denaturing conditions of detergents and wash liquors.

[0004] WO 2016 / 096711 and WO 2016 / 096714 describe subtilase variants and detergents containing the variants, which have improved stability and / or improved washing performance in liquid detergents compared to the parent subtilase. WO 2016 / 001450 and WO 2020 / 002255 disclose subtilase variants with increased stability. WO 2010 / 056640 also describes subtilisin variants. US 6,376,450 discloses multi-substituted protease variants that provide improved and enhanced cleaning power. US2020 / 172890 A1 discloses performance-enhanced and storage-stable protease variants. WO 2022 / 225696 A2 and WO 2018 / 069158 A1 also disclose protease variants.

[0005] Therefore, new proteases that meet these requirements are needed. Summary of the Invention

[0006] The present invention relates to a variant polypeptide having protease activity or a fragment of the polypeptide having protease activity, wherein:

[0007] (i) the polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, and

[0008] (ii) the polypeptide or fragment thereof comprises an amino acid substitution at amino acid residue 183, compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and with reference to the numbering of SEQ ID NO: 2, and comprises at least one additional amino acid substitution at amino acid residues selected from the group consisting of 43, 78 and 204.

[0009] The present invention further relates to polynucleotides encoding the variant polypeptides and compositions comprising the variant polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Shown are the residual activities (defined as the activity after storage divided by the activity at time point zero) of the variants of Table 5 (according to the presence of mutation N183D and at least one other mutation selected from N43K, S78N / D and N204D) after storage for 160 h at 37°C. DETAILED DESCRIPTION

[0011] 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.

[0012] Although the invention will be described with reference to specific embodiments, this description should not be construed in a limiting sense.

[0013] definition

[0014] Unless otherwise noted, terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0015] Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given. Unless otherwise indicated or obvious from the nature of the definitions, the definitions apply to all compounds, methods and uses described herein.

[0016] As used in this specification and the appended claims, the singular forms "a" and "an" include the corresponding plural forms as well, unless the context clearly dictates otherwise.

[0017] In the context of the present invention, the terms "about" and "approximately" denote an interval of accuracy that a person skilled in the art will understand still ensures the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5%.

[0018] In addition, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" and the like in this specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequential order or a chronological order. It is to be understood that the terms so used are interchangeable where appropriate and that the embodiments of the invention described herein are capable of operation 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, unless otherwise indicated in this application as described above or below, there is no temporal or temporal time interval coherence between such steps, i.e., the steps may be performed simultaneously or there may be a time interval of seconds, minutes, hours, days, weeks, months or even years between such steps.

[0019] 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.

[0020] It should be understood that the term "comprising" is not limiting. For the purposes of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising." If a group is defined hereinafter as comprising at least a certain number of members, this is meant to also encompass a group consisting only of these members.

[0021] " parent " sequence (also referred to as " parent enzyme " or " parent protein ") is the starting sequence for introducing a change (for example, by introducing one or more amino acid replacements) thereby producing a " variant " of the parent sequence in sequence. In the context of the present invention, the sequence according to any one of SEQ ID No:1 and 3 to 11 and the sequence with at least 60% homogeneity therewith are considered to be the parent sequence. The terms " enzyme variant " or " sequence variant " or " protein variant " or " variant polypeptide " or " protease variant " or " variant polypeptide with protease activity " are used interchangeably in this article, and are used with reference to the parent enzyme in the corresponding variant enzyme source. Therefore, the parent enzyme comprises wild-type enzyme and the variant of the wild-type enzyme for developing other variant. The amino acid sequence of variant enzyme is different to a certain extent from that of the parent enzyme.

[0022] In describing variant polypeptides of the invention, abbreviations for individual amino acids are used according to the generally accepted IUPAC single-letter or three-letter amino acid abbreviations.

[0023] "Amino acid substitutions" are described by providing the original amino acid, followed by the position number within the amino acid sequence, followed by the substituted amino acid. For example, a histidine at position 120 replaced by an alanine is represented as "His120Ala" or "H120A." A substitution can also be described by naming only the resulting amino acid in the variant, without specifying the parent amino acid at that position, for example, by using "X120A" or "120A" or "Xaa120Ala" or "120Ala."

[0024] Variants containing multiple substitutions are separated by "+", 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 substitutions can be separated by spaces or commas, for example, "R170Y G195E" or "R170Y,G195E". When different alternative substitutions can be introduced at a certain position, the different substitutions 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. Alternative substitutions at specific positions can also be expressed as "X120A,G,H", "120A,G,H", "X120A / G / H" or "120A / G / H". Alternatively, the different substitutions may be indicated by brackets, for example, "Arg170[Tyr,Gly]" or "Arg170{Tyr,Gly}" or abbreviated as "R170[Y,G]" or "R170{Y,G}".

[0025] The numbering of the amino acid residues of the proteases described herein is as commonly used in the art for proteases (see P.N. Bryan, Biochimica et Biophysica Acta 1543 (2000), 203-222, see page 204, left column, third paragraph), according to the numbering of BPN' subtilisin from Bacillus amyloliquefaciens, the sequence of which is shown in SEQ ID NO:2 (i.e., numbering according to SEQ ID NO:2 or according to "BPN' numbering").

[0026] Alternatively, amino acid positions may be described with reference to the numbering of SEQ ID NO: 1 or SEQ ID NO: 3 (i.e., numbering according to SEQ ID NO: 1 or numbering according to SEQ ID NO: 3). Table 1 below shows the amino acid numbering according to SEQ ID NO: 2 and the numbering of the corresponding amino acids in the sequence according to SEQ ID NO: 1 or 3:

[0027] Table 1

[0028]

[0029] The term "introducing at least two negative charges into a specific amino acid sequence" refers to increasing the net charge of the specific amino acid sequence by at least two negative charges. Such an increase in the net charge of the specific amino acid sequence by at least two negative charges is achieved by altering the amino acid sequence and can be achieved by one or more amino acid sequence alterations selected from the group consisting of substitutions, deletions, and insertions, preferably by one or more amino acid substitutions. The increase in the net charge of the specific amino acid sequence by at least two negative charges can be achieved by removing positive charges and / 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. Preferably, the introduction of at least two negative charges by amino acid sequence modification is evaluated under conditions typically present in the 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, most preferably at pH 7.0 or pH 8.0. In a preferred embodiment of the invention, at least two negative charges are introduced by replacing the arginine residue at position 101 (according to the numbering in SEQ ID NO: 2) with aspartic acid or glutamic acid.

[0030] 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).

[0031] The term "heterologous" (or exogenous or foreign or recombinant or non-native or non-natural) polypeptide is defined herein as a polypeptide that is not native to the host cell; a polypeptide that is native to the host cell in which structural modifications (e.g., deletions, substitutions, and / or insertions) have been made through recombinant DNA techniques to alter the native polypeptide; or a polypeptide that is native to the host cell in which the expression of the polypeptide is quantitatively altered or the expression of the polypeptide is directed from a genomic location that is different from that of the native host cell due to manipulation of the host cell DNA (e.g., a stronger promoter) through recombinant DNA techniques. Similarly, the term "heterologous" (or exogenous or foreign or recombinant or non-native or non-natural) polynucleotide refers to a polynucleotide that is not native to the host cell; a polynucleotide that is native to the host cell in which 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 the host cell in which expression is quantitatively altered due to manipulation of the polynucleotide's regulatory elements by recombinant DNA techniques (e.g., a stronger promoter); or a polynucleotide that is native to the 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 two or more amino acid sequences do not naturally occur in a specific combination with each other.

[0032] 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 introduced by man using genetic techniques. With respect to polynucleotides, "recombinant" includes all constructs produced by using genetic techniques / recombinant DNA technology, wherein

[0033] (a) a sequence of a polynucleotide or a portion thereof, or

[0034] (b) one or more genetic control sequences, including but not limited to a promoter, operably linked to the polynucleotide, or

[0035] (c) Both a) and b)

[0036] Not in its wild-type genetic environment or has been artificially modified.

[0037] "Synthetic" compounds are obtained by in vitro chemical and / or enzymatic synthesis.

[0038] Variant polynucleotide and variant polypeptide sequences can be defined by their sequence identity when compared to the parent sequence. Sequence identity is typically provided in the form of "% sequence identity" or "% identity." To calculate sequence identity, in a first step, a sequence alignment is generated. According to the present invention, a paired global alignment is generated, meaning that the two sequences are aligned over their entire length, typically using a mathematical method known as an alignment algorithm.

[0039] According to the present invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J.Mol.Biol. [Molecular Biology Journal] (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 (polynucleotides: gap opening = 10.0, gap extension = 0.5 and matrix = EDNAFULL; polypeptides: gap opening = 10.0, gap extension = 0.5, and matrix = EBLOSUM62) are used. 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 over its full length multiplied by 100: % identity = (identical residues / length of the alignment region showing the corresponding sequence of the present invention over its full length) * 100.

[0040] For calculating the percent identity of two nucleic acid sequences, the calculation and some explanations for the percent identity of two amino acid sequences are equally applicable. For the nucleic acid sequence of coding protein, pairwise comparison should be carried out on the complete length (from start codon to stop codon, excluding introns) 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 a second step, the identity values ​​are determined according to the comparison produced. Percent identity is calculated by: % identity=(identical residue / length of the comparison region of the sequence of the present invention from start codon to stop codon and excluding introns, shown on its complete length)*100.

[0041] Furthermore, a preferred nucleic acid sequence alignment program 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).

[0042] Variant polypeptides can also be defined by their sequence similarity when compared to the parent sequence. Sequence similarity is usually provided as "% sequence similarity" or "% similarity". The % sequence similarity takes into account that the defined set of amino acids shares similar properties, for example, by their size, by their hydrophobicity, by their charge or by other properties. In this article, the exchange of an amino acid with a similar amino acid 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, for example, the program "NEEDLE", which is one of the most commonly used amino acid similarity matrices for database searches and sequence alignments:

[0043] Amino acid A is similar to amino acid S. Amino acid D is similar to amino acids E and N. Amino acid E is similar to amino acids D, K, and Q. Amino acid F is similar to amino acids W and Y. Amino acid H is similar to amino acids N and Y. Amino acid I is similar to amino acids L, M, and V. Amino acid K is similar to amino acids E, Q, and R. Amino acid L is similar to amino acids I, M, and V. Amino acid M is similar to amino acids I, L, and V. Amino acid N is similar to amino acids D, H, and S. Amino acid Q is similar to amino acids E, K, and R. Amino acid R is similar to amino acids K and Q. Amino acid S is similar to amino acids A, N, and T. Amino acid T is similar to amino acid S. Amino acid V is similar to amino acids I, L, and M. Amino acid W is similar to amino acids F and Y.

[0044] Amino acid Y is similar to amino acids F, H, and W

[0045] 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 generated alignment. 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 aligned region showing the sequences of the invention over its entire length, multiplied by 100: % similarity = [(identical residues + similar residues) / length of the aligned region showing the sequences of the invention over its entire length] * 100.

[0046] For nucleic acids, similar sequences can also be determined by hybridization using corresponding stringent conditions. The term "high stringency conditions" means that a probe of at least 100 nucleotides in length is prehybridized and hybridized in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 50% formamide at 42°C for 12 to 24 hours according to standard Southern blotting procedures. The carrier material is finally washed three times with 2X SSC, 0.2% SDS at 65°C for 15 minutes each. The term "very high stringency conditions" means that a probe of at least 100 nucleotides in length is prehybridized and hybridized in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 50% formamide at 42°C for 12 to 24 hours according to standard Southern blotting procedures. The carrier material is finally washed three times with 2X SSC, 0.2% SDS at 70°C for 15 minutes each.

[0047] As used herein, " fragment " or " subsequence " refers to 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 respectively but still has the same or similar activity and / or function.Preferably, the functional fragment has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% identity, 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% of the length of the original full-length amino acid sequence. Preferably, the fragment comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide. Preferably, the functional fragment has at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% identity, 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% identity to the original full-length amino acid sequence. Also preferably, the functional fragment retains at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% identity, 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%, at least 99.5% or at least 100% of the enzymatic activity of the original full-length amino acid sequence. A functional fragment comprises consecutive nucleotides or amino acids compared to the original nucleic acid or original amino acid sequence, respectively. An "original full-length amino acid sequence" is an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3 or a variant polypeptide as claimed.

[0048] 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 operably linked to one or more control sequences (at least to a promoter) as described herein.

[0049] As used herein, the term "vector" includes any kind of construct suitable for carrying an exogenous polynucleotide sequence to be transferred to another cell or stably or transiently expressed 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 (phage)), phage (bacteriophage), baculovirus, clays, forsmids, artificial chromosomes, and any other vectors specific to a particular target host. 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.

[0050] The terms "introduction of a polynucleotide" or "transformation of a polynucleotide" as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, regardless of the method used for transfer. That is, as used herein, the term "transformation of a polynucleotide" is independent of vectors, shuttle systems, or host cells, and it not only relates to the polynucleotide transfer methods known in the art, such as transformation (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 also encompass any other type of polynucleotide transfer method, such as, but not limited to, transduction or transfection.

[0051] A polynucleotide encoding a polypeptide can be "expressed". The term "expression" or "gene expression" means the transcription of a particular gene(s) or a particular nucleic acid construct. The term "expression" or "gene expression" means the transcription of one or more genes or genetic constructs into structural RNA (e.g., rRNA, tRNA) or mRNA, which may or may not be subsequently translated into protein. This process includes DNA transcription and processing of the resulting mRNA product.

[0052] 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 a precipitate and a supernatant or a filter cake and a filtrate, respectively. The solution obtained after purification of the enzyme of interest from the fermentation broth is referred to herein as a "purified enzyme solution."

[0053] By "protein formulation" (or "enzyme preparation"), e.g., "protein variant formulation," is meant any non-complex formulation comprising minor ingredients, wherein these ingredients serve the purpose of stabilizing the protein contained in the protein formulation and / or the stability of the protein formulation itself. Preferably, non-complex protein formulations comprise a higher concentration of protein than complex formulations (e.g., than detergent formulations). Thus, preferably, non-complex protein formulations are concentrated protein variant formulations. Preferably, non-complex protein formulations comprise 20 to 120 mg / g active enzyme, whereas complex formulations, such as detergent compositions, comprise 0.002 to 10 mg / g active enzyme.

[0054] "Enzyme properties" include, but are not limited to, catalytic activity, substrate / cofactor specificity, product specificity, stability over time, thermostability, pH stability, and chemical stability. "Enzyme activity" or "catalytic activity" refers to the catalytic effect of an enzyme, expressed as units / mg of enzyme (specific activity) or converted substrate molecules / minute / enzyme molecules (molecular activity). Enzyme activity can be specified by the actual function of the enzyme, for example, proteases play proteolytic activity by catalyzing the hydrolytic cleavage of peptide bonds, lipases play lipolytic activity by the hydrolytic cleavage of ester bonds, and amylase activity relates to the hydrolysis of glycosidic linkages in polysaccharides, etc. According to the present invention, enzyme activity is proteolytic activity, which can be determined by using succinyl-Ala-Ala-Pro-Phe-p-nitroaniline (Suc-AAPF-pNA; see, for example, Del Mar et al. (1979), Analytical Biochem [analytical biochemistry] 99, 316-320) or Suc-AAPF-AMC (7-amido-4-methylcoumarin) as a substrate. Cleavage of pNA or AMC from the substrate molecule by proteolytic cleavage results in the release of a yellow color or a change in the fluorescence properties of the free pNA, which can be quantified by measuring the OD405 emission at 460 nm after excitation at 380 nm. Other methods using, for example, casein as a proteinaceous substrate are known to those skilled in the art.

[0055] The term "enzyme stability" according to the present invention refers 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.

[0056] 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 defined conditions. By comparing the measured values, the potential degree of loss of enzyme activity or the degree of residual enzyme activity can be determined. The degree of loss of enzyme activity or the degree of residual enzyme activity determines the stability or instability of the enzyme.

[0057] As used herein, an "enzyme inhibitor" is a compound that slows down or stops the activity of an enzyme. Enzyme inhibitors also typically stabilize the three-dimensional structure of the enzyme. Thus, enzyme inhibitors also typically act as "enzyme stabilizers."

[0058] "pH stability" refers to the ability of an enzyme to perform enzymatic activity after exposure to a certain pH value.

[0059] The terms "thermal stability" or "thermostability" or "temperature-dependent activity" refer to the ability of an enzyme to exert catalytic activity or wash performance after exposure to elevated temperatures. Preferably, the enzyme exerts catalytic activity or wash performance after exposure to a temperature of 40°C for 14 days (preferably in a detergent composition (preferably, in an ES1-C detergent as described herein)) or after exposure to a temperature of 92°C for at least 10 minutes.

[0060] The term "detergent stability" or "stability upon storage in a detergent composition" refers to the ability of an enzyme to exert catalytic activity or wash performance after storage in a detergent composition, preferably in a detergent composition (preferably, in an ES1-C detergent as described herein) at a temperature of 40°C or 50°C for 14 days.

[0061] 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 wash temperature, time, washing machinery, foam concentration, detergent type, and water hardness. The term "improved washing performance" is used to indicate that a better end result is obtained in stain removal under relevant washing conditions, or that less enzyme is required to obtain the same end result, based on weight, relative to corresponding control conditions.

[0062] As used herein, the term "specific performance" refers to the ability of each unit of active enzyme to clean and remove a specific stain or soil. In some embodiments, specific performance is determined using stains or soils such as eggs, egg yolks, milk, grass, minced meat, blood, chocolate sauce, baby food, sebum, etc.

[0063] "Detergent composition" or "detergent" means 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 as meaning a class of chemicals that can be used in detergent compositions. A typical detergent component is a surfactant. "Surfactant" (used synonymously with "surface active agent" in this article) means an organic chemical that changes the properties of a liquid at an interface when added to a liquid. Depending on its ionic charge, a surfactant is referred to as a nonionic surfactant, an anionic surfactant, a cationic surfactant, or amphoteric surfactant. 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, foaming capacity), the amount that effectively provides optical benefits (e.g., optical brightening, dye transfer inhibition, color care), and the amount of certain components that effectively aid processing (maintaining physical properties during processing, storage, and use; e.g., rheology modifiers, hydrotropes, desiccant). Detergent compositions typically have a protease concentration of from 0.002 to 10 mg / g active enzyme.

[0064] 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 laundry process can be carried out using technical devices such as domestic or industrial washing machines. Alternatively, the laundry process can be done manually.

[0065] The term "textile" means any textile material, including yarn (thread made from natural or synthetic fibers for knitting or weaving), yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and fabrics made from these materials, such as clothing, cloth, and other articles. As used herein, the term "fabric" (a textile made by weaving, knitting, or felting fibers) or "garment" (any article of clothing made from textiles) is also intended to include the broader term textiles.

[0066] The term "fiber" includes natural fibers, synthetic fibers and mixtures thereof. Examples of natural fibers are fibers of plant origin (such as flax, jute and cotton) or fibers of animal origin containing proteins like 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 can be single fibers or parts of textiles such as knitted, woven or nonwoven fabrics.

[0067] The term "hard surface cleaning" refers to both household 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 ware, glass, metal surfaces (including knives or cutlery) and medical instruments such as diagnostic instruments, trays, pans, holders, racks, tweezers, scissors, shears, saws (e.g., bone saws and their blades), hemostats, knives, chisels, rongeurs, files, pliers, drill bits, chisels, rasps, burrs, applicators, lithotripters, elevators, clamps, needle holders, carriers, clamps, hooks, gouges, curettes, retractors, straighteners, punches, extractors, spoons, keratomes, tongue depressors, squeezers, trocars, dilators, cages, glassware, pipes, catheters, cannulas, plugs, stents, endoscopes, arthroscopes and related equipment. A special form of hard surface cleaning is dishwashing, in particular automatic dishwashing (ADW).

[0068] The term "dishwashing" refers to all forms of dishwashing, whether manual or automatic. 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.

[0069] The cleaning performance is evaluated under relevant cleaning conditions. In this context, the term "relevant cleaning conditions" refers to the conditions actually used in washing machines, automatic dishwashers or during manual cleaning, in particular cleaning temperature, time, cleaning mechanics, foam concentration, detergent type and water hardness.

[0070] 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 / automated cleaning methods. Manual cleaning is used when mechanical devices are unavailable or when the medical device to be cleaned is too fragile or difficult to clean mechanically. Mechanical / automated cleaning methods remove dirt and microorganisms through automated cleaning and rinsing processes, which can include ultrasonic cleaning and washing.

[0071] In the field of soil removal, the term "stain" is generally used to refer to laundry, such as the cleaning of textiles, fabrics or fibers, while the term "soil" is generally used to refer to hard surface cleaning, such as the cleaning of dishes and cutlery. However, the terms "stain" and "soil" should be used interchangeably herein.

[0072] As used herein, "chelating builders" are distinguished from precipitating builders in that 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 effective chelating agents that are capable of strongly binding divalent cations (e.g., Ca2+), with the logarithmic stability constant (Log K) of the cation / chelating agent complex being 0.00175°C. Ca ) is greater than 4, in particular 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 two properties described above. Strong chelating builders include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetra(methylenephosphonic acid) (EDTMP), nitrilotrimethylenephosphonic acid (NTMP), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), methylglycinediacetic acid (MGDA), nitrilotriacetic acid (NTA), 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP), sodium tripolyphosphate (STPP), iminodisuccinic acid (IDS), N,N-diacetic acid tetrasodium salt (GLDA), pyrophosphates and ethylenediaminedisuccinic acid (EDDS).

[0073] "Antimicrobials" are chemical compounds that kill microorganisms or inhibit their growth or reproduction. Microorganisms can be bacteria, yeasts, or molds. "Preservatives" are antimicrobial agents that can be added to aqueous products and compositions to preserve the original properties, characteristics, and integrity of the products and compositions by killing contaminating microorganisms or inhibiting their growth.

[0074] A composition "substantially free" of a compound shall mean herein that the corresponding compound has not been intentionally added to the composition, meaning that at most an ineffective amount of the compound is present, most preferably 0% of the compound is present in the composition.

[0075] Detailed description

[0076] In the present invention, novel proteases are provided. More specifically, variants of parent proteases, methods of making variant proteases, compositions comprising the protease variants, and methods of using the variant protease variants or compositions comprising these variant proteases are provided.

[0077] Protease variants

[0078] The present invention relates to a variant polypeptide having protease activity or a fragment of the polypeptide having protease activity, wherein:

[0079] (i) the polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, and

[0080] (ii) the polypeptide or fragment thereof comprises an amino acid substitution at amino acid residue 183, and at least one additional substitution at amino acid residues selected from the group consisting of 43, 78, and 204, compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and with reference to the numbering of SEQ ID NO: 2.

[0081] The variant polypeptides of the present invention having protease activity are non-naturally occurring proteases. Preferably, the variant polypeptides of the present invention having protease activity are purified, isolated, synthesized and / or recombinant protease variants. Preferably, the variant polypeptides of the present invention having protease activity are purified and recombinant protease variants.

[0082] The proteases according to the present invention have "proteolytic activity" or "protease activity". "Proteolytic activity" or "protease activity" describes the ability to hydrolyze peptide bonds in polypeptides. Protease activity can be determined by assays known to those skilled in the art for measuring protease activity. Methods for analyzing proteolytic activity are well known in the literature (see, for example, Gupta et al. (2002), Appl. Microbiol. Biotechnol. 60: 381-395). For example, proteolytic activity can be determined by using succinyl-Ala-Ala-Pro-Phe-p-nitroaniline (Suc-AAPF-pNA; see, for example, DelMar et al. (1979), Analytical Biochem 99, 316-320) or Suc-AAPF-AMC (7-amido-4-methylcoumarin) as substrates. Cleavage of pNA or AMC from the substrate molecule by proteolytic cleavage results in the release of a yellow color or a change in the fluorescence properties of the free pNA, which can be quantified by measuring the OD405 emission at 460 nm after excitation at 380 nm.

[0083] In one embodiment, the variant polypeptides of the present invention having protease activity exhibit at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190% or at least 200% of the proteolytic activity of the parent protease (i.e., a protease according to SEQ ID NO: 1 or 3, preferably a protease according to SEQ ID NO: 3). In a preferred embodiment, the variant polypeptides of the present invention having protease activity exhibit the same proteolytic activity as the parent protease (i.e., a protease according to SEQ ID NO: 1 or 3, preferably a protease according to SEQ ID NO: 3). More preferably, the variant polypeptide having protease activity of the present invention exhibits increased proteolytic activity compared to the parent protease, ie the protease according to SEQ ID NO: 1 or 3, preferably the protease according to SEQ ID NO: 3.

[0084] Preferably, the parent protease of the variant polypeptide having protease activity of the present invention is a protease having at least 60% sequence identity with the protease according to SEQ ID NO: 1 or 3, preferably, the parent protease of the variant polypeptide having protease activity of the present invention is a protease according to SEQ ID NO: 1 or 3, and more preferably, the parent protease of the variant polypeptide having protease activity of the present invention is a protease according to SEQ ID NO: 3. Preferably, the parent protease of the variant polypeptide having protease activity of the present invention is a subtilisin (EC 3.4.21.62).

[0085] The present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein, with reference to the numbering of the amino acid sequence shown in SEQ ID NO: 2, said variant polypeptide comprises an amino acid substitution at amino acid residue 183 and at least one additional substitution at an amino acid residue selected from the group consisting of 43, 78 and 204, compared to the parent protease according to SEQ ID NO: 1 or 3.

[0086] The parent protease of the variant polypeptide having protease activity of the present invention is a protease having an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1 or 3. Preferably, the parent protease of the variant polypeptide having protease activity of the present invention is a protease according to SEQ ID NO: 1 or 3. Most preferably, the parent protease of the variant polypeptide having protease activity of the present invention is a protease according to SEQ ID NO: 3.

[0087] In a preferred embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, which comprises, according to the numbering of the amino acid sequence shown in SEQ ID NO: 2, amino acid substitutions X183D / E / C / Q / A / M and at least one additional substitution selected from the group consisting of X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q and X204D / E / C / G, compared to the parent sequence, preferably wherein the parent protease of the variant polypeptide having protease activity of the present invention is a protease according to SEQ ID NO: 1 or 3 or any protease having at least 60% sequence identity with SEQ ID NO: 1 or 3, more preferably, the parent protease of the variant polypeptide having protease activity is a protease according to SEQ ID NO: 1 or 3, and most preferably, the parent protease of the variant polypeptide having protease activity is a protease according to SEQ ID NO: 3.

[0088] In a preferred embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of the polypeptide having protease activity, which comprises, according to the numbering of the amino acid sequence shown in SEQ ID NO: 2, an amino acid substitution X183D / E and at least one additional substitution selected from X43K / R, X78N / D and X204D compared to the parent sequence, preferably wherein the parent protease of the variant polypeptide having protease activity of the present invention is a protease according to SEQ ID NO: 1 or 3 or any protease having at least 60% sequence identity with SEQ ID NO: 1 or 3, more preferably, the parent protease of the variant polypeptide having protease activity is a protease according to SEQ ID NO: 1 or 3, and most preferably, the parent protease of the variant polypeptide having protease activity is a protease according to SEQ ID NO: 3.

[0089] 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: 1 or 3 at the corresponding position (according to the numbering of SEQ ID NO: 2).

[0090] In a preferred embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, which comprises, according to the numbering of the amino acid sequence shown in SEQ ID NO: 2, the amino acid substitution N183D / E / C / Q / A / M and at least one additional substitution selected from the group consisting of N43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q and N204D / E / C / G, compared to the parent sequence, preferably wherein the parent protease of the variant polypeptide having protease activity of the present invention is a protease according to SEQ ID NO: 1 or 3 or any protease having at least 60% sequence identity with SEQ ID NO: 1 or 3, more preferably, the parent protease of the variant polypeptide having protease activity is a protease according to SEQ ID NO: 1 or 3, and most preferably, the parent protease of the variant polypeptide having protease activity is a protease according to SEQ ID NO: 3.

[0091] In a preferred embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of the polypeptide having protease activity, which comprises, according to the numbering of the amino acid sequence shown in SEQ ID NO: 2, an amino acid substitution N183D / E and at least one additional substitution selected from N43K / R, S78N / D and N204D compared to the parent sequence, preferably wherein the parent protease of the variant polypeptide having protease activity of the present invention is a protease according to SEQ ID NO: 1 or 3 or any protease having at least 60% sequence identity with SEQ ID NO: 1 or 3, more preferably, the parent protease of the variant polypeptide having protease activity is a protease according to SEQ ID NO: 1 or 3, and most preferably, the parent protease of the variant polypeptide having protease activity is a protease according to SEQ ID NO: 3.

[0092] Preferably, the present invention relates to a variant polypeptide having protease activity, wherein the variant polypeptide comprises an amino acid substitution at amino acid residue N183, compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and with reference to the numbering of the amino acid sequence shown in SEQ ID NO: 2, and comprises at least one additional substitution at an amino acid residue selected from the group consisting of N43, S78 and N204.

[0093] Preferably, the present invention relates to a variant polypeptide having protease activity, wherein the variant polypeptide comprises an amino acid substitution at amino acid residue N183D / E, compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and with reference to the numbering of the amino acid sequence shown in SEQ ID NO: 2, and comprises at least one additional substitution at an amino acid residue selected from the group consisting of N43K / R, S78N / D and N204D.

[0094] Preferably, the present invention relates to a variant polypeptide having protease activity, wherein the variant polypeptide comprises an amino acid substitution at amino acid residue N183, compared to the amino acid sequence shown in SEQ ID NO: 3 and with reference to the numbering of the amino acid sequence shown in SEQ ID NO: 2, and comprises at least one additional substitution at an amino acid residue selected from the group consisting of N43, S78 and N204.

[0095] Preferably, the present invention relates to a variant polypeptide having protease activity or a fragment of the polypeptide having protease activity, wherein the variant polypeptide comprises one of the following combinations of amino acid substitutions:

[0096] (a) X183D / E / C / Q / A / M and X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I;

[0097] (b) X183D / E / C / Q / A / M and X78N / D / R / W / F / H / K / E / L / Y / M / C / Q;

[0098] (c) X183D / E / C / Q / A / M and X204D / E / C / G;

[0099] (d) X183D / E / C / Q / A / M, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I and

[0100] (e) X183D / E / C / Q / A / M, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I and X204D / E / C / G;

[0101] (f)X183D / E / C / Q / A / M, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, and X204D / E / C / G; or

[0102] (g) X183D / E / C / Q / A / M, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I,

[0103] The polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0104] Also preferably, the present invention relates to a variant polypeptide having protease activity or a fragment of the polypeptide having protease activity, wherein the variant polypeptide comprises one of the following combinations of amino acid substitutions:

[0105] (a) N183D / E and N43K / R;

[0106] (b) N183D / E and S78N / D;

[0107] (c) N183D / E and N204D;

[0108] (d) N183D / E, N43K / R, and S78N / D;

[0109] (e) N183D / E, N43K / R, and N204D;

[0110] (f) N183D / E, S78N / D and N204D; or

[0111] (g) N183D / E, N43K / R, S78N / D and N204D, and

[0112] The polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0113] Also preferably, the present invention relates to a variant polypeptide having protease activity or a fragment of the polypeptide having protease activity, wherein the variant polypeptide comprises one of the following combinations of amino acid substitutions:

[0114] (a) N183D and N43K;

[0115] (b) N183D and S78N;

[0116] (c) N183D and N204D;

[0117] (d) N183D, N43K, and S78N;

[0118] (e) N183D, N43K and N204D;

[0119] (f) N183D, S78N and N204D; or

[0120] (g) N183D, N43K, S78N and N204D, and

[0121] The polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0122] In one embodiment, with reference to the numbering of SEQ ID NO: 2, the variant polypeptide further comprises an amino acid substitution at amino acid residue 76. Preferably, the amino acid substitution at amino acid residue 76 is X76D, and more preferably, the substitution at amino acid residue 76 is N76D.

[0123] Accordingly, the present invention also relates to a variant polypeptide having protease activity or a fragment of the polypeptide having protease activity, wherein the variant polypeptide comprises one of the following combinations of amino acid substitutions:

[0124] (a) X183D / E / C / Q / A / M, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I and X76D;

[0125] (b) X183D / E / C / Q / A / M, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q and X76D;

[0126] (c) X183D / E / C / Q / A / M, X204D / E / C / G, and X76D;

[0127] (d) X183D / E / C / Q / A / M,

[0128] (e) X183D / E / C / Q / A / M, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X204D / E / C / G and X76D;

[0129] (f)X183D / E / C / Q / A / M, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X204D / E / C / G, and X76D; or

[0130] (g) X183D / E / C / Q / A / M, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I,

[0131] The polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0132] Also preferably, the present invention relates to a variant polypeptide having protease activity or a fragment of the polypeptide having protease activity, wherein the variant polypeptide comprises one of the following combinations of amino acid substitutions:

[0133] (a) N183D / E, N43K / R, and N76D;

[0134] (b) N183D / E, S78N / D, and N76D;

[0135] (c) N183D / E, N204D, and N76D;

[0136] (d) N183D / E, N43K / R, S78N / D and N76D;

[0137] (e) N183D / E, N43K / R, N204D and N76D;

[0138] (f) N183D / E, S78N / D, N204D and N76D; or

[0139] (g) N183D / E, N43K / R, S78N / D, N204D and N76D, and

[0140] The polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0141] Also preferably, the present invention relates to a variant polypeptide having protease activity or a fragment of the polypeptide having protease activity, wherein the variant polypeptide comprises one of the following combinations of amino acid substitutions:

[0142] (a) N183D, N43K, and N76D;

[0143] (b) N183D, S78N, and N76D;

[0144] (c) N183D, N204D, and N76D;

[0145] (d) N183D, N43K, S78N, and N76D;

[0146] (e) N183D, N43K, N204D and N76D;

[0147] (f) N183D, S78N, N204D and N76D; or

[0148] (g) N183D, N43K, S78N, N204D and N76D, and

[0149] The polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0150] More preferably, the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein said variant polypeptide comprises substitutions N183D, N43K, S78N and N204D, and wherein the polypeptide or its fragment has an amino acid sequence that is at least 60% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 3.

[0151] More preferably, the present invention relates to a variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein said variant polypeptide comprises substitutions N183D, N43K, S78N, N204D and N76D, and wherein the polypeptide or its fragment has an amino acid sequence that is at least 60% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 3.

[0152] In one embodiment, with reference to the numbering of SEQ ID NO: 2, the variant polypeptide with protease activity or the fragment of the polypeptide with protease activity having an amino acid substitution at position 183 and one or more of positions 43, 78 and 204, and optionally position 76 as described above further comprises at least one amino acid substitution at an amino acid residue selected from the group consisting of: 18, 24, 56, 109, 144, 182, 237, 240, 248, 256 and 260.

[0153] In one embodiment, with reference to the numbering of SEQ ID NO: 2, the variant polypeptide with protease activity or the fragment of the polypeptide with protease activity having an amino acid substitution at position 183 and one or more of positions 43, 78 and 204, and optionally position 76 as described above further comprises at least one amino acid substitution selected from the group consisting of X18A / D / C / E / Q, X24K, X56D, X109K / A, X144N / R, X182K / R / E, X237R, X240E / N, X248Q / R, X256E / T / D / R / P and X260D / K.

[0154] In one embodiment, with reference to the numbering of SEQ ID NO: 2, the variant polypeptide with protease activity or the fragment of the polypeptide with protease activity having an amino acid substitution at position 183 and one or more of positions 43, 78 and 204, and optionally position 76 as described above further comprises at least one amino acid substitution selected from the group consisting of X18A / D / Q, X24K, X56D, X109K / A, X144N / R, X182K / R / E, X237R, X240E / N, X248Q / R, X256E / T / D / R / P and X260D / K.

[0155] Preferably, with reference to the numbering of SEQ ID NO: 2, the variant polypeptide with protease activity having amino acid substitutions at position 183 and one or more of positions 43, 78 and 204, and optionally position 76 as described above, or a fragment of the polypeptide with protease activity, further comprises at least one amino acid substitution selected from the group consisting of N18A / D / Q, S24K, S56D, Q109K / A, S144N / R, Q182K / R / E, K237R, S240E / N, N248Q / R, S256E / T / D / R / P and T260D / K.

[0156] More preferably, with reference to the numbering of SEQ ID NO: 2, the variant polypeptide with protease activity having an amino acid substitution at position 183 and one or more of positions 43, 78 and 204, and optionally position 76 as described above, or a fragment of the polypeptide with protease activity further comprises at least one amino acid substitution selected from the group consisting of N18D, S24K, S56D, Q109K, S144N, Q182K, K237R, S240E, N248Q, S256D and T260D.

[0157] More preferably, with reference to the numbering of SEQ ID NO: 2, the variant polypeptide with protease activity having an amino acid substitution at position 183 and one or more of positions 43, 78 and 204, and optionally position 76 as described above, or a fragment of the polypeptide with protease activity further comprises at least one amino acid substitution selected from the group consisting of N18Q, S24K, S56D, Q109K, S144N, Q182K, K237R, S240E, N248Q, S256D and T260D.

[0158] In one embodiment, the variant polypeptide having protease activity or a fragment of the polypeptide having protease activity having amino acid substitutions at position 183 and one or more of positions 43, 78 and 204, and optionally position 76 as described above does not contain the amino acid substitutions S3T, V4I and V199I.

[0159] In one embodiment, the variant polypeptide with protease activity or the fragment of the polypeptide with protease activity having an amino acid substitution at position 183 and one or more of positions 43, 78 and 204, and optionally position 76 as described above further comprises one of the combinations of amino acid substitutions according to Table 2.

[0160] Preferably, the variant polypeptide having protease activity or the fragment of the polypeptide having protease activity comprises one of the following combinations of amino acid substitutions:

[0161] (a) X183D / E / C / Q / A / M and X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I;

[0162] (b) X183D / E / C / Q / A / M and X78N / D / R / W / F / H / K / E / L / Y / M / C / Q;

[0163] (c) X183D / E / C / Q / A / M and X204D / E / C / G;

[0164] (d) X183D / E / C / Q / A / M, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I and

[0165] (e) X183D / E / C / Q / A / M, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I and X204D / E / C / G;

[0166] (f)X183D / E / C / Q / A / M, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, and X204D / E / C / G; or

[0167] (g)X183D / E / C / Q / A / M, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I,

[0168] and further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0169] Also preferably, the variant polypeptide having protease activity or the fragment of the polypeptide having protease activity comprises one of the following combinations of amino acid substitutions:

[0170] (a) N183D / E and N43K / R;

[0171] (b) N183D / E and S78N / D;

[0172] (c) N183D / E and N204D;

[0173] (d) N183D / E, N43K / R, and S78N / D;

[0174] (e) N183D / E, N43K / R, and N204D;

[0175] (f) N183D / E, S78N / D and N204D; or

[0176] (g) N183D / E, N43K / R, S78N / D, and N204D

[0177] and further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0178] Also preferably, the variant polypeptide having protease activity or the fragment of the polypeptide having protease activity comprises one of the following combinations of amino acid substitutions:

[0179] (a) N183D and N43K;

[0180] (b) N183D and S78N;

[0181] (c) N183D and N204D;

[0182] (d) N183D, N43K, and S78N;

[0183] (e) N183D, N43K and N204D;

[0184] (f) N183D, S78N and N204D; or

[0185] (g) N183D, N43K, S78N and N204D

[0186] and further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0187] Also preferably, the variant polypeptide having protease activity or the fragment of the polypeptide having protease activity comprises one of the following combinations of amino acid substitutions:

[0188] (a) X183D / E / C / Q / A / M, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I and X76D;

[0189] (b) X183D / E / C / Q / A / M, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q and X76D;

[0190] (c) X183D / E / C / Q / A / M, X204D / E / C / G, and X76D;

[0191] (d) X183D / E / C / Q / A / M,

[0192] (e) X183D / E / C / Q / A / M, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X204D / E / C / G and X76D;

[0193] (f)X183D / E / C / Q / A / M, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X204D / E / C / G, and X76D; or

[0194] (g)X183D / E / C / Q / A / M, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I,

[0195] and further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0196] Also preferably, the variant polypeptide having protease activity or the fragment of the polypeptide having protease activity comprises one of the following combinations of amino acid substitutions:

[0197] (a) N183D / E, N43K / R, and N76D;

[0198] (b) N183D / E, S78N / D, and N76D;

[0199] (c) N183D / E, N204D, and N76D;

[0200] (d) N183D / E, N43K / R, S78N / D and N76D;

[0201] (e) N183D / E, N43K / R, N204D and N76D;

[0202] (f) N183D / E, S78N / D, N204D and N76D; or

[0203] (g) N183D / E, N43K / R, S78N / D, N204D, and N76D

[0204] and further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0205] Also preferably, the variant polypeptide having protease activity or the fragment of the polypeptide having protease activity comprises one of the following combinations of amino acid substitutions:

[0206] (a) N183D, N43K, and N76D;

[0207] (b) N183D, S78N, and N76D;

[0208] (c) N183D, N204D, and N76D;

[0209] (d) N183D, N43K, S78N, and N76D;

[0210] (e) N183D, N43K, N204D and N76D;

[0211] (f) N183D, S78N, N204D and N76D; or

[0212] (g) N183D, N43K, S78N, N204D, and N76D

[0213] and further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

[0214] Also preferably, the variant polypeptide having protease activity or a fragment of the polypeptide having protease activity comprises the substitutions N183D, N43K, S78N and N204D, and further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or fragment thereof has an amino acid sequence that is at least 60% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 3.

[0215] Also preferably, the variant polypeptide having protease activity or a fragment of the polypeptide having protease activity comprises the substitutions N183D, N43K, S78N, N204D and N76D, and further comprises one of the combinations of amino acid substitutions according to Table 2, wherein the polypeptide or fragment thereof has an amino acid sequence that is at least 60% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 3.

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291] Preferably, the variant polypeptides of the present invention comprise a combination of substitutions selected from the group consisting of:

[0292] (a)X24K;

[0293] (b)X24K; X43K; X78N;

[0294] (c)X24K;

[0295] (d)X24K;

[0296] (e)X24K;X43K;X78N;X183D;X204D;X240E;X248R;X260K

[0297] (f)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X248Q

[0298] (g)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q

[0299] (h)X24K;X43K;X56D;X144N;X182K;X183D;X204D;X240E;X248Q

[0300] (i)X24K;X43K;X56D;X144N;X182K;X183D;X204D;X248Q;X260D

[0301] (j)X24K;X43K;X78N;X156D;X183D;X204D;X240E;X248R;X260K

[0302] (k)X24K;X43K;X78N;X182E;X183D;X204D;X237R;X248R;X260K

[0303] (l)X24K;X43K;X78N;X182E;X183D;X204D;X240E;X248R;X260K

[0304] (m)X24K;X43K;X78N;X183D;X204D;X237R;X240E;X248R;X260K

[0305] (n)X24K;X43K;X56D;X78N;X109K;X144N;X182E;X183D;X204D;X248Q

[0306] (o)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X248Q;X260K

[0307] (p)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X237R;X248Q

[0308] (q)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q;X260D

[0309] (r)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q;X260K

[0310] (s)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X240E;X248Q

[0311] (t)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X248Q;X260D

[0312] (u)X24K;X43K;X56D;X144N;X182K;X183D;X204D;X237R;X248Q;X260D

[0313] (v)X24K;X43K;X56D;X78N;X109K;X144N;X182E;X183D;X204D;X248Q;X260D

[0314] (w)X24K;X43K;X56D;X78N;X109K;X144N;X182K;X183D;X204D;X248Q;X260D

[0315] (x)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X237R;X240E;X248Q

[0316] (y)X24K;X43K;X56D;X78N;X109K;X144N;X182K;X183D;X204D;X248Q;X260D

[0317] (z)X24K;X43K;X56D;X78N;X109K;X144N;X182K;X183D;X204D;X240E;X248Q;X260K

[0318] (aa)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X237R;X240E;X248Q;X260D

[0319] (bb)X26I;

[0320] (cc)X26I;

[0321] (dd)X26I;

[0322] (ee)X26I;

[0323] (ff)X26I;

[0324] (gg)X26I;X43K;X56D;X78D;X103S;X109A;X116E;X130G;X144R;X183D;X204D;X240N;X248R, and

[0325] (hh)X26I;

[0326] Also preferably, the variant polypeptides of the present invention comprise a combination of substitutions selected from the group consisting of:

[0327] (a)S24K; N43K; S56D; S78N; S144N; Q182K; N183D; N204D; N248Q

[0328] (b)S24K; N43K; S78N; N183D; N204D; N248R; T260K

[0329] (c)S24K; N43K; S78N; Q182E; N183D; N204D; N248R; T260K

[0330] <h2 style=";text-align:left;direction:ltr">(d)S24K;N43K;S78N;N183D;N204D;K237R;N248R;T260K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0331] <h2 style=";text-align:left;direction:ltr"> (e)S24K;N43K;S78N;N183D;N204D;S240E;N248R;T260K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0332] <h2 style=";text-align:left;direction:ltr"> (f)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;N248Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0333] <h2 style=";text-align:left;direction:ltr"> (g)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0334] <h2 style=";text-align:left;direction:ltr"> (h)S24K;N43K;S56D;S144N;Q182K;N183D;N204D;S240E;N248Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0335] <h2 style=";text-align:left;direction:ltr"> (i)S24K;N43K;S56D;S144N;Q182K;N183D;N204D;N248Q;T260D<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0336] <h2 style=";text-align:left;direction:ltr"> (j)S24K;N43K;S78N;S156D;N183D;N204D;S240E;N248R;T260K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0337] <h2 style=";text-align:left;direction:ltr"> (k)S24K;N43K;S78N;Q182E;N183D;N204D;K237R;N248R;T260K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0338] <h2 style=";text-align:left;direction:ltr"> (l)S24K;N43K;S78N;Q182E;N183D;N204D;S240E;N248R;T260K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0339] <h2 style=";text-align:left;direction:ltr"> (m)S24K;N43K;S78N;N183D;N204D;K237R;S240E;N248R;T260K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0340] <h2 style=";text-align:left;direction:ltr"> (n)S24K;N43K;S56D;S78N;Q109K;S144N;Q182E;N183D;N204D;N248Q<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0341] <h2 style=";text-align:left;direction:ltr"> (o)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;N248Q;T260K<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0342] (p)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;K237R;N248Q

[0343] (q)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q;T260D

[0344] (r)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q;T260K

[0345] (s)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;S240E;N248Q

[0346] (t)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;N248Q;T260D

[0347] (u)S24K;N43K;S56D;S144N;Q182K;N183D;N204D;K237R;N248Q;T260D

[0348] (v)S24K;N43K;S56D;S78N;Q109K;S144N;Q182E;N183D;N204D;N248Q;T260D

[0349] (w)S24K;N43K;S56D;S78N;Q109K;S144N;Q182K;N183D;N204D;N248Q;T260D

[0350] (x)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;K237R;S240E;N248Q

[0351] (y)S24K;N43K;S56D;S78N;Q109K;S144N;Q182K;N183D;N204D;N248Q;T260D

[0352] (z)S24K; N43K; S56D; S78N; Q109K; S144N; Q182K; N183D; N204D; S240E; N248Q; T260K (aa) S24K; N43K; S56D ;S78N; S144N; Q182K; N183D; N204D; K237R; S240E; N248Q; T260D(bb)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R(cc)V26I; S56D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D ;N204D;S240N;N248R(dd)V26I;N43K;S56D;S78D;A103S;Q109A;N116E;S130G;N183D;N204D;S240N;N248R

[0353] (ee)V26I; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237R; S240N; N248R

[0354] (ff)V26I; S56D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R

[0355] (gg)V26I;N43K;S56D;S78D;A103S;Q109A;N116E;S130G;S144R;N183D;N204D;S240N;N248R, and

[0356] (hh)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237A; S240E; N248R

[0357] Preferably, the variant polypeptides of the present invention comprise a combination of substitutions selected from the group consisting of:

[0358] (a)X24K;

[0359] (b)X24K; X43K; X78N;

[0360] (c)X24K;X43K;X78N;X182E;X183D;X204D;X248R;X260K

[0361] (d)X24K;X43K;X78N;X183D;X204D;X237R;X248R;X260K

[0362] (e)X24K;X43K;X78N;X183D;X204D;X240E;X248R;X260K

[0363] (f)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X248Q

[0364] (g)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q

[0365] (h)X24K;X43K;X56D;X144N;X182K;X183D;X204D;X240E;X248Q

[0366] (i)X24K;X43K;X56D;X144N;X182K;X183D;X204D;X248Q;X260D

[0367] (j)X24K;X43K;X78N;X156D;X183D;X204D;X240E;X248R;X260K

[0368] (k)X24K;X43K;X78N;X182E;X183D;X204D;X237R;X248R;X260K

[0369] (l)X24K;X43K;X78N;X182E;X183D;X204D;X240E;X248R;X260K

[0370] (m)X24K;X43K;X78N;X183D;X204D;X237R;X240E;X248R;X260K

[0371] (n)X24K;X43K;X56D;X78N;X109K;X144N;X182E;X183D;X204D;X248Q

[0372] (o)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X248Q;X260K

[0373] (p)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X237R;X248Q

[0374] (q)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q;X260D

[0375] (r)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q;X260K

[0376] (s)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X240E;X248Q

[0377] (t)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X248Q;X260D

[0378] (u)X24K;X43K;X56D;X144N;X182K;X183D;X204D;X237R;X248Q;X260D

[0379] (v)X24K;X43K;X56D;X78N;X109K;X144N;X182E;X183D;X204D;X248Q;X260D

[0380] (w)X24K;X43K;X56D;X78N;X109K;X144N;X182K;X183D;X204D;X248Q;X260D

[0381] (x)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X237R;X240E;X248Q

[0382] (y)X24K;X43K;X56D;X78N;X109K;X144N;X182K;X183D;X204D;X248Q;X260D

[0383] (z)X24K;X43K;X56D;X78N;X109K;X144N;X182K;X183D;X204D;X240E;X248Q;X260K

[0384] (aa)X24K;

[0385] (bb)X18Q;

[0386] (cc)X18Q;

[0387] (dd)X18Q;

[0388] (ee)X18Q;

[0389] (ff)X18Q;

[0390] (gg)X18Q;X26I;X43K;X56D;X78D;X103S;X109A;X116E;X130G;X144R;X183D;X204D;X240N;X248R, and

[0391] (hh)X18Q;

[0392] Also preferably, the variant polypeptides of the present invention comprise a combination of substitutions selected from the group consisting of:

[0393] (a)S24K; N43K; S56D; S78N; S144N; Q182K; N183D; N204D; N248Q

[0394] (b)S24K;N43K;S78N;N183D;N204D;N248R;T260K

[0395] (c)S24K;N43K;S78N;Q182E;N183D;N204D;N248R;T260K

[0396] (d)S24K;N43K;S78N;N183D;N204D;K237R;N248R;T260K

[0397] (e)S24K;N43K;S78N;N183D;N204D;S240E;N248R;T260K

[0398] (f)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;N248Q

[0399] (g)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q

[0400] (h)S24K;N43K;S56D;S144N;Q182K;N183D;N204D;S240E;N248Q

[0401] (i)S24K;N43K;S56D;S144N;Q182K;N183D;N204D;N248Q;T260D

[0402] (j)S24K;N43K;S78N;S156D;N183D;N204D;S240E;N248R;T260K

[0403] (k)S24K;N43K;S78N;Q182E;N183D;N204D;K237R;N248R;T260K

[0404] (l)S24K;N43K;S78N;Q182E;N183D;N204D;S240E;N248R;T260K

[0405] (m)S24K;N43K;S78N;N183D;N204D;K237R;S240E;N248R;T260K

[0406] (n)S24K;N43K;S56D;S78N;Q109K;S144N;Q182E;N183D;N204D;N248Q

[0407] (o)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;N248Q;T260K

[0408] (p)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;K237R;N248Q

[0409] (q)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q;T260D

[0410] (r)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q;T260K

[0411] (s)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;S240E;N248Q

[0412] (t)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;N248Q;T260D

[0413] (u)S24K;N43K;S56D;S144N;Q182K;N183D;N204D;K237R;N248Q;T260D

[0414] (v)S24K;N43K;S56D;S78N;Q109K;S144N;Q182E;N183D;N204D;N248Q;T260D

[0415] (w)S24K;N43K;S56D;S78N;Q109K;S144N;Q182K;N183D;N204D;N248Q;T260D

[0416] (x)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;K237R;S240E;N248Q

[0417] (y)S24K;N43K;S56D;S78N;Q109K;S144N;Q182K;N183D;N204D;N248Q;T260D

[0418] (z)S24K; N43K; S56D; S78N; Q109K; S144N; Q182K; N183D; N204D; S240E; N248Q; T260K

[0419] (aa)S24K; N43K; S56D; S78N; S144N; Q182K; N183D; N204D; K237R; S240E; N248Q; T260D

[0420] (bb)N18Q; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R

[0421] (cc)N18Q; V26I; S56D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R

[0422] (dd)N18Q; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R

[0423] (ee)N18Q; V26I; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237R; S240N; N248R

[0424] (ff)N18Q; V26I; S56D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R

[0425] (gg)N18Q;V26I;N43K;S56D;S78D;A103S;Q109A;N116E;S130G;S144R;N183D;N204D;S240N;N248R, and

[0426] (hh)N18Q; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237A; S240E; N248R

[0427] Preferably, the variant polypeptides of the present invention comprise a combination of substitutions selected from the group consisting of:

[0428] (a)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q

[0429] (b)X24K;X43K;X78N;X183D;X204D;X248R;X260K

[0430] (c)X24K;X43K;X78N;X182E;X183D;X204D;X248R;X260K

[0431] (d)X24K;X43K;X78N;X183D;X204D;X237R;X248R;X260K

[0432] (e)X24K;X43K;X78N;X183D;X204D;X240E;X248R;X260K

[0433] (f)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X248Q

[0434] (g)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q

[0435] (h)X24K;X43K;X56D;X144N;X182K;X183D;X204D;X240E;X248Q

[0436] (i)X24K;X43K;X56D;X144N;X182K;X183D;X204D;X248Q;X260D

[0437] (j)X24K;X43K;X78N;X156D;X183D;X204D;X240E;X248R;X260K

[0438] (k)X24K;X43K;X78N;X182E;X183D;X204D;X237R;X248R;X260K

[0439] (l)X24K;X43K;X78N;X182E;X183D;X204D;X240E;X248R;X260K

[0440] (m)X24K;X43K;X78N;X183D;X204D;X237R;X240E;X248R;X260K

[0441] (n)X24K;X43K;X56D;X78N;X109K;X144N;X182E;X183D;X204D;X248Q

[0442] (o)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X248Q;X260K

[0443] (p)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X237R;X248Q

[0444] (q)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q;X260D

[0445] (r)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q;X260K

[0446] (s)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X240E;X248Q

[0447] (t)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X248Q;X260D

[0448] (u)X24K;X43K;X56D;X144N;X182K;X183D;X204D;X237R;X248Q;X260D

[0449] (v)X24K;X43K;X56D;X78N;X109K;X144N;X182E;X183D;X204D;X248Q;X260D

[0450] (w)X24K;X43K;X56D;X78N;X109K;X144N;X182K;X183D;X204D;X248Q;X260D

[0451] (x)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X237R;X240E;X248Q

[0452] (y) X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X248Q; X260D

[0453] (z) X24K; X43K; X56D; X78N; X109K; X144N; X182K; X183D; X204D; X240E; X248Q; X260K

[0454] (aa) X24K; X43K; X56D; X78N; X144N; X182K; X183D; X204D; X237R; X240E; X248Q; X260D

[0455] (bb) X18D; X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R

[0456] (cc) X18D; X26I; X56D; X78D; X103S; X109A; X116E; X130G; X144R; X183D; X204D; X240N; X248R

[0457] (dd) X18D; X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X240N; X248R

[0458] (ee) X18D; X26I; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X237R; X240N; X248R

[0459] (ff) X18D; X26I; X56D; X78D; X103S; X109A; X116E; X130G; X144R; X183D; X204D; X240N; X248R

[0460] (gg) X18D; X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X144R; X183D; X204D; X240N; X248R, and

[0461] (hh) X18D; X26I; X43K; X56D; X78D; X103S; X109A; X116E; X130G; X183D; X204D; X237A; X240E; X248R.

[0462] Also preferably, the variant polypeptides of the present invention comprise a combination of substitutions selected from the group consisting of:

[0463] (a)S24K; N43K; S56D; S78N; S144N; Q182K; N183D; N204D; N248Q

[0464] (b)S24K; N43K; S78N; N183D; N204D; N248R; T260K

[0465] (c)S24K; N43K; S78N; Q182E; N183D; N204D; N248R; T260K

[0466] (d)S24K; N43K; S78N; N183D; N204D; K237R; N248R; T260K

[0467] (e)S24K; N43K; S78N; N183D; N204D; S240E; N248R; T260K

[0468] (f)S24K; N43K; S56D; S78N; S144N; Q182E; N183D; N204D; N248Q

[0469] (g)S24K; N43K; S56D; S78N; S144N; Q182K; N183D; N204D; N248Q

[0470] (h)S24K; N43K; S56D; S144N; Q182K; N183D; N204D; S240E; N248Q

[0471] (i)S24K; N43K; S56D; S144N; Q182K; N183D; N204D; N248Q; T260D

[0472] (j)S24K; N43K; S78N; S156D; N183D; N204D; S240E; N248R; T260K

[0473] (k)S24K; N43K; S78N; Q182E; N183D; N204D; K237R; N248R; T260K

[0474] (l)S24K; N43K; S78N; Q182E; N183D; N204D; S240E; N248R; T260K

[0475] (m)S24K;N43K;S78N;N183D;N204D;K237R;S240E;N248R;T260K

[0476] (n)S24K;N43K;S56D;S78N;Q109K;S144N;Q182E;N183D;N204D;N248Q

[0477] (o)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;N248Q;T260K

[0478] (p)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;K237R;N248Q

[0479] (q)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q;T260D

[0480] (r)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q;T260K

[0481] (s)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;S240E;N248Q

[0482] (t)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;N248Q;T260D

[0483] (u)S24K;N43K;S56D;S144N;Q182K;N183D;N204D;K237R;N248Q;T260D

[0484] (v)S24K;N43K;S56D;S78N;Q109K;S144N;Q182E;N183D;N204D;N248Q;T260D

[0485] (w)S24K;N43K;S56D;S78N;Q109K;S144N;Q182K;N183D;N204D;N248Q;T260D

[0486] (x)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;K237R;S240E;N248Q

[0487] (y) S24K; N43K; S56D; S78N; Q109K; S144N; Q182K; N183D; N204D; N248Q; T260D

[0488] (z) S24K; N43K; S56D; S78N; Q109K; S144N; Q182K; N183D; N204D; S240E; N248Q; T260K

[0489] (aa) S24K; N43K; S56D; S78N; S144N; Q182K; N183D; N204D; K237R; S240E; N248Q; T260D

[0490] (bb) N18D; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R

[0491] (cc) N18D; V26I; S56D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R

[0492] (dd) N18D; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R

[0493] (ee) N18D; V26I; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237R; S240N; N248R

[0494] (ff) N18D; V26I; S56D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R

[0495] (gg) N18D; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R, and

[0496] (hh)N18D; V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237A; S240E; N248R

[0497] Preferably, the variant polypeptides of the present invention comprise a combination of substitutions selected from the group consisting of:

[0498] (a)X24K;

[0499] (b)X24K;

[0500] (c)X24K;

[0501] (d)X24K;

[0502] (e)X24K;

[0503] (f)X24K;

[0504] (g)X24K;

[0505] (h)X24K;

[0506] (i)X24K;

[0507] (j)X24K;

[0508] (k)X24K;X43K;X76D;X78N;X182E;X183D;X204D;X237R;X248R;X256D;X260K

[0509] (l)X24K;X43K;X76D;X78N;X182E;X183D;X204D;X240E;X248R;X256D;X260K

[0510] (m)X24K;X43K;X76D;X78N;X183D;X204D;X237R;X240E;X248R;X256D;X260K

[0511] (n)X24K;X43K;X56D;X76D;X78N;X109K;X144N;X182E;X183D;X204D;X248Q;X256D

[0512] (o)X24K;X43K;X56D;X76D;X78N;X144N;X182E;X183D;X204D;X248Q;X256D;X260K

[0513] (p)X24K;X43K;X56D;X76D;X78N;X144N;X182K;X183D;X204D;X237R;X248Q;X256D

[0514] (q)X24K;X43K;X56D;X76D;X78N;X144N;X182K;X183D;X204D;X248Q;X256D;X260D

[0515] (r)X24K;X43K;X56D;X76D;X78N;X144N;X182K;X183D;X204D;X248Q;X256D;X260K

[0516] (s)X24K;X43K;X56D;X76D;X78N;X144N;X182E;X183D;X204D;X240E;X248Q;X256D

[0517] (t)X24K;X43K;X56D;X76D;X78N;X144N;X182E;X183D;X204D;X248Q;X256D;X260D

[0518] (u)X24K;X43K;X56D;X76D;X144N;X182K;X183D;X204D;X237R;X248Q;X256D;X260D

[0519] (v)X24K;X43K;X56D;X76D;X78N;X109K;X144N;X182E;X183D;X204D;X248Q;X256D;X260D

[0520] (w)X24K;X43K;X56D;X76D;X78N;X109K;X144N;X182K;X183D;X204D;X248Q;X256D;X260D

[0521] (x)X24K;X43K;X56D;X76D;X78N;X144N;X182K;X183D;X204D;X237R;X240E;X248Q;X256D

[0522] (y)X24K;X43K;X56D;X76D;X78N;X109K;X144N;X182K;X183D;X204D;X248Q;X256D;X260D

[0523] (z)X24K;X43K;X56D;X76D;X78N;X109K;X144N;X182K;X183D;X204D;X240E;X248Q;X256D;X260K

[0524] (aa)X24K;X43K;X56D;X76D;X78N;X144N;X182K;X183D;X204D;X237R;X240E;X248Q;X256D;X260D

[0525] (bb)X18D;X26I;X43K;X56D;X76D;X78D;X103S;X109A;X116E;X130G;X183D;X204D;X240N;X248R;X256D

[0526] (cc)X18D;X26I;X56D;X76D;X78D;X103S;X109A;X116E;X130G;X144R;X183D;X204D;X240N;X248R;X256D

[0527] (dd)X18D;

[0528] (ee)X18D;

[0529] (ff)X18D;

[0530] (gg)X18D;X26I;X43K;X56D;X76D;X78D;X103S;X109A;X116E;X130G;X144R;X183D;X204D;X240N;X248R;X256D, and

[0531] (hh)X18D;

[0532] Also preferably, the variant polypeptides of the present invention comprise a combination of substitutions selected from the group consisting of:

[0533] (a)S24K; N43K; S56D; N76D; S78N; S144N; Q182K; N183D; N204D; N248Q

[0534] (b)S24K; N43K; N76D; S78N; N183D; N204D; N248R; S256D; T260K

[0535] (c)S24K; N43K; N76D; S78N; Q182E; N183D; N204D; N248R; S256D; T260K

[0536] (d)S24K; N43K; N76D; S78N; N183D; N204D; K237R; N248R; S256T; T260K

[0537] (e)S24K;N43K;N76D;S78N;N183D;N204D;S240E;N248R;S256T;T260K

[0538] (f)S24K;N43K;S56D;N76D;S78N;S144N;Q182E;N183D;N204D;N248Q;S256D

[0539] (g)S24K;N43K;S56D;N76D;S78N;S144N;Q182K;N183D;N204D;N248Q;S256D

[0540] (h)S24K;N43K;S56D;N76D;S144N;Q182K;N183D;N204D;S240E;N248Q;S256D

[0541] (i)S24K;N43K;S56D;N76D;S144N;Q182K;N183D;N204D;N248Q;S256D;T260D

[0542] (j)S24K;N43K;N76D;S78N;S156D;N183D;N204D;S240E;N248R;S256D;T260K

[0543] (k)S24K;N43K;N76D;S78N;Q182E;N183D;N204D;K237R;N248R;S256D;T260K

[0544] (l)S24K;N43K;N76D;S78N;Q182E;N183D;N204D;S240E;N248R;S256D;T260K

[0545] (m)S24K;N43K;N76D;S78N;N183D;N204D;K237R;S240E;N248R;S256D;T260K

[0546] (n)S24K;N43K;S56D;N76D;S78N;Q109K;S144N;Q182E;N183D;N204D;N248Q;S256D

[0547] (o)S24K;N43K;S56D;N76D;S78N;S144N;Q182E;N183D;N204D;N248Q;S256D;T260K

[0548] (p)S24K;N43K;S56D;N76D;S78N;S144N;Q182K;N183D;N204D;K237R;N248Q;S256D

[0549] (q)S24K;N43K;S56D;N76D;S78N;S144N;Q182K;N183D;N204D;N248Q;S256D;T260D

[0550] (r)S24K;N43K;S56D;N76D;S78N;S144N;Q182K;N183D;N204D;N248Q;S256D;T260K

[0551] (s)S24K;N43K;S56D;N76D;S78N;S144N;Q182E;N183D;N204D;S240E;N248Q;S256D

[0552] (t)S24K;N43K;S56D;N76D;S78N;S144N;Q182E;N183D;N204D;N248Q;S256D;T260D

[0553] (u)S24K;N43K;S56D;N76D;S144N;Q182K;N183D;N204D;K237R;N248Q;S256D;T260D

[0554] (v)S24K;N43K;S56D;N76D;S78N;Q109K;S144N;Q182E;N183D;N204D;N248Q;S256D;T260D

[0555] (w)S24K;N43K;S56D;N76D;S78N;Q109K;S144N;Q182K;N183D;N204D;N248Q;S256D;T260D

[0556] (x)S24K;N43K;S56D;N76D;S78N;S144N;Q182K;N183D;N204D;K237R;S240E;N248Q;S256D

[0557] (y)S24K;N43K;S56D;N76D;S78N;Q109K;S144N;Q182K;N183D;N204D;N248Q;S256D;T260D

[0558] (z)S24K; N43K; S56D; N76D; S78N; Q109K; S144N; Q182K; N183D; N204D; S240E; N248Q; S256D; T260K

[0559] (aa)S24K; N43K; S56D; N76D; S78N; S144N; Q182K; N183D; N204D; K237R; S240E; N248Q; S256D; T260D

[0560] (bb)N18D; V26I; N43K; S56D; N76D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R; S256D

[0561] (cc)N18D; V26I; S56D; N76D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R; S256D

[0562] (dd)N18D; V26I; N43K; S56D; N76D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R; S256D

[0563] (ee)N18D; V26I; S56D; N76D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237R; S240N; N248R; S256D

[0564] (ff)N18D; V26I; S56D; N76D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R; S256D

[0565] (gg)N18D; V26I; N43K; S56D; N76D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R; S256D, and

[0566] (hh)N18D; V26I; N43K; S56D; N76D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237A; S240E; N248R; S256D.

[0567] Preferably, the variant polypeptide of the invention comprises at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, compared to SEQ ID NO: 1, preferably, the protease comprises the amino acid substitution X101E or X101D according to the numbering of SEQ ID NO: 2. Most preferably, the variant polypeptide of the invention comprises the amino acid substitution X101E according to the numbering of SEQ ID NO: 2, compared to SEQ ID NO: 1.

[0568] In an alternative embodiment, the protease variant does not have an amino acid substitution at position 101 according to the numbering of SEQ ID NO: 2, preferably, the protease variant comprises amino acid R101.

[0569] In a preferred embodiment, with reference to the numbering of SEQ ID NO: 2, the variant polypeptide of the present invention comprises amino acid residue D or E at position 101, preferably, comprises E at position 101.

[0570] Preferably, the one or more amino acid changes (preferably substitutions) are at amino acid positions located on the surface of the protease. Preferably, according to the numbering of SEQ ID NO: 2, the one or more substitutions at amino acid positions located on the surface of the protease are selected from the group consisting of 24, 43, 56, 76, 78, 109, 144, 182, 183, 204, 237, 240, 248, 256 and 260. Preferably, the protease of the invention comprises one or more amino acid substitutions at an amino acid position located on the surface of the protease, according to the numbering of SEQ ID NO: 2, wherein the substitutions at an amino acid position located on the surface of the protease are selected from the group consisting of: X24K, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X56D, X76D, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X204D / E / C / G, X237R / A, X240E / N, X248Q / R, X256E / T / D / R / P and X260D / K. Whether an amino acid residue is located on the surface of a protease can be determined by determining the relative accessible surface area or relative solvent accessibility (RSA) of the protein residue, which is a measure of the solvent exposure of the residue. It can be calculated by dividing the ASA (solvent accessible surface area) by the MaxASA (the maximum possible solvent accessible surface area for a given residue). The RSA of surface-exposed amino acids is preferably greater than 0.4, more preferably greater than 0.5 or 0.6.

[0571] Preferably, the protease variant comprises 2 to 15 amino acid substitutions compared to the parent protease according to SEQ ID NO: 1 or 3. Preferably, the protease variant comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14 of the above amino acid substitutions compared to the parent protease according to SEQ ID NO: 1 or 3.

[0572] Variant polypeptides of the present invention comprising the amino acid substitutions described herein and having protease activity preferably have a sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97 ...1%, at least 97.1%, at least 97.1%, at least 97.1%, at least 97.1%, at least 97.1%, at least 97.1%, at least 97.1%, at least 97.1%, at least 7%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, but less than 100% sequence identity.

[0573] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 12. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0574] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 13. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0575] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 14. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0576] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 15. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0577] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 16. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0578] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 17. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0579] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 18. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0580] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 19. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 183D and 204D.

[0581] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 20. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 183D and 204D.

[0582] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 21. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0583] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 22. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0584] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 23. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0585] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 24. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0586] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 25. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0587] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 26. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0588] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 27. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0589] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 28. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0590] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 29. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0591] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 30. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0592] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 31. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0593] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 32. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 183D and 204D.

[0594] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 33. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0595] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 34. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0596] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 35. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0597] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 36. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0598] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 37. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0599] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 38. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78N, 183D and 204D.

[0600] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 39. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 76D, 183D and 204D.

[0601] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 40. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 78D, 183D and 204D.

[0602] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 41. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78D, 183D and 204D.

[0603] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 42. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 78D, 183D and 204D.

[0604] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 43. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 78D, 183D and 204D.

[0605] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 44. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78D, 183D and 204D.

[0606] In one embodiment, the amino acid sequence of a variant polypeptide having protease activity of the present invention has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.1%, at least 98.2%, or at least 98.3% of the amino acid sequence of SEQ ID NO: 45. 2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% identical, and preferably, with reference to the numbering of SEQ ID NO: 2, comprises amino acid residues 43K, 78D, 183D and 204D.

[0607] The invention still further relates to the fragment of variant polypeptide, wherein this fragment has protease activity.Compared with full-length variant polypeptide, this fragment lacks at least one amino acid.In one embodiment, the fragment of variant polypeptide comprises 100 to 259 continuous amino acids of full-length variant polypeptide, preferably, the fragment of variant polypeptide comprises 130 to 259 or 150 to 259 continuous amino acids of full-length variant polypeptide, more preferably, the fragment of variant polypeptide comprises 180 to 259 or 200 to 259 continuous amino acids of full-length variant polypeptide, and most preferably, the fragment of variant polypeptide comprises 210 to 259, 220 to 259, 230 to 259, 240 to 259 or 250 to 259 continuous amino acids of full-length variant polypeptide.

[0608] In one embodiment, the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide and has at least 65% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3; preferably, the fragment of the variant polypeptide comprises 130 to 259 or 150 to 259 consecutive amino acids of the full-length variant polypeptide and has at least 65% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3; more preferably, the fragment of the variant polypeptide comprises 180 to 259 or 200 to 259 consecutive amino acids of the full-length variant polypeptide and has at least 65% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. NO:3 has at least 65% but less than 100% sequence identity; and most preferably, the fragment of the variant polypeptide comprises 210 to 259, 220 to 259, 230 to 259, 240 to 259 or 250 to 259 consecutive amino acids of the full-length variant polypeptide and has at least 65% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0609] In one embodiment, the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and with reference to the numbering of SEQ ID NO: 2, comprises amino acid residue 183 and at least one other amino acid residue selected from 43, 78 and 204; preferably, the fragment of the variant polypeptide comprises 130 to 259 or 150 to 259 consecutive amino acids of the full-length variant polypeptide, and with reference to the numbering of SEQ ID NO: 2, comprises amino acid residue 183 and at least one other amino acid residue selected from 43, 78 and 204; more preferably, the fragment of the variant polypeptide comprises 180 to 259 or 200 to 259 consecutive amino acids of the full-length variant polypeptide, and with reference to the numbering of SEQ ID NO:2, comprising amino acid residue 183 and at least one other amino acid residue selected from 43, 78 and 204; and most preferably, the fragment of the variant polypeptide comprises 210 to 259, 220 to 259, 230 to 259, 240 to 259 or 250 to 259 consecutive amino acids of the full-length variant polypeptide, and with reference to the numbering of SEQ ID NO:2, comprising amino acid residue 183 and at least one other amino acid residue selected from 43, 78 and 204.

[0610] In one embodiment, a fragment of a variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, has at least 65% but less than 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, and comprises amino acid residue 183 and at least one additional amino acid residue selected from 43, 78 and 204, with reference to the numbering of SEQ ID NO: 2; preferably, a fragment of a variant polypeptide comprises 130 to 259 or 150 to 259 consecutive amino acids of the full-length variant polypeptide, has at least 65% but less than 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, and comprises amino acid residue 183 and at least one additional amino acid residue selected from 43, 78 and 204, with reference to the numbering of SEQ ID NO: 2; more preferably, a fragment of a variant polypeptide comprises 180 to 259 or 200 to 259 consecutive amino acids of the full-length variant polypeptide, NO:3 has at least 65% but less than 100% sequence identity with the amino acid sequence shown in NO:3, and comprises amino acid residue 183 and at least one additional amino acid residue selected from 43, 78 and 204, with reference to the numbering of SEQ ID NO:2; and most preferably, the fragment of the variant polypeptide comprises 210 to 259, 220 to 259, 230 to 259, 240 to 259 or 250 to 259 consecutive amino acids of the full-length variant polypeptide, has at least 65% but less than 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and comprises amino acid residue 183 and at least one additional amino acid residue selected from 43, 78 and 204, with reference to the numbering of SEQ ID NO:2.

[0611] In one embodiment, the fragment of the variant polypeptide retains at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% identity, 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% of the protease activity of the full-length variant polypeptide. Protease activity can be determined as described above.

[0612] The full-length variant polypeptide is 269 amino acids in length.

[0613] In one embodiment, the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide according to any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45. Preferably, the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide according to any one of SEQ ID NO: NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45. More preferably, a fragment of a variant polypeptide comprises 130 to 259 or 150 to 259 consecutive amino acids of a full-length variant polypeptide of any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45. NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45, and most preferably, a fragment of a variant polypeptide comprises 180 to 259 or 200 to 259 consecutive amino acids of a full-length variant polypeptide of any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45, and most preferably, a fragment of a variant polypeptide comprises NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45. 210 to 259, 220 to 259, 230 to 259, 240 to 259, or 250 to 259 consecutive amino acids of a full-length variant polypeptide of any one of NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,

[0614] In one embodiment the protease variants of the present invention exhibit one or more improved properties compared to a parent protease, preferably compared to the protease according to SEQ ID NO: 3.

[0615] Variant polypeptides having protease activity according to the present invention exhibit increased stability, in particular storage stability, compared to the parent protease according to SEQ ID NO: 3. Increased stability is preferably expressed as residual activity after stability stimulation. Preferably, storage stability is expressed as residual activity after storage under corresponding storage conditions, preferably in a detergent composition (preferably in a laundry detergent or dishwashing detergent, preferably a laundry detergent). Preferably, after storage, the residual activity of the protease variant is increased compared to the residual activity of the parent protease according to SEQ ID NO: 3. Preferably, the residual activity of the protease variant after storage is increased by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190% or at least 200% compared to the residual activity of the parent protease, preferably compared to the protease according to SEQ ID NO: 3.

[0616] Preferably, the improved properties are one or more properties selected from the group consisting of: increased stability, thermostability, performance in detergent, performance in laundry detergent, performance in ADW detergent. Preferably, the improved activity is improved wash performance, laundry detergent wash performance, and / or ADW detergent wash performance. Preferably, the improved stability is improved thermostability, thermostability in a detergent composition (preferably in a laundry detergent composition or dishwashing detergent composition, preferably a laundry detergent composition), stability under storage conditions. Preferably, the improved properties are improved thermostability, improved thermostability in a detergent composition, improved stability under storage in a detergent composition, and / or improved wash performance. Preferably, the improved properties are improved thermostability, preferably improved thermostability in a detergent composition, improved stability under storage in a detergent composition, and / or improved wash performance. Preferably, the improved property is improved thermal stability, improved thermal stability in a detergent composition, improved stability under storage conditions, preferably improved stability under storage in a detergent composition, preferably improved stability under storage in a laundry detergent and / or improved stability under storage in an ADW detergent, improved wash performance, preferably improved wash performance of a laundry detergent and / or improved wash performance of an ADW detergent.

[0617] Preferably, the improved property is improved stability in detergent compositions, preferably laundry detergent compositions.

[0618] Nucleic acid constructs

[0619] The present invention also relates to polynucleotides encoding variant polypeptides having protease activity of the present invention. Preferably, the polynucleotide is a codon-optimized polynucleotide for improved expression in a specific host cell, preferably a Bacillus cell.

[0620] The present invention also relates to a nucleic acid construct (preferably an expression cassette) comprising a polynucleotide as described herein.

[0621] Typically, an expression cassette comprises three elements: a promoter sequence, an open reading frame, and a 3' untranslated region, which, in eukaryotes, usually contains a polyadenylation site. Additional regulatory elements may include transcriptional and translational enhancers. Intron sequences may also be added to the 5' untranslated region (UTR) or the coding sequence to increase the amount of the mature messenger accumulated in the cytosol. The expression cassette may be a part of a vector or may be integrated into the genome of a host cell and replicated together with the genome of its host cell. Expression cassettes are generally capable of increasing or decreasing expression.

[0622] The present invention also relates to an expression vector comprising a polynucleotide or nucleic acid construct as described herein.The expression vector may be a low copy number vector or a high copy number vector.

[0623] In another embodiment, carrier can be used for the transcribing and translating of foreign polynucleotides.After being transformed into host cell or host cell organelle, carrier as used herein can provide the section for transcribing and translating of foreign polynucleotides.Such other section can comprise regulating nucleotide sequence, maintaining and / or duplicating required one or more replication origins, one or more selectable markers, polyadenylation signal, the suitable site for the insertion of foreign coding sequence, such as multiple cloning site etc. in specific cell type.An example is when needing carrier as additional genetic element (for example, plasmid or clay molecule) to be maintained in bacterial cell.The limiting examples of suitable replication origin comprises f1-ori and colE1.

[0624] A vector may replicate without integrating into the host cell genome, for example, as a plasmid in a bacterial host cell, or it may integrate part or all of its DNA into the host cell genome and thereby result in the replication and expression of its DNA.

[0625] The polynucleotide encoding the variant polypeptide can be introduced into a vector by standard recombinant DNA techniques. Once introduced into a vector, the polynucleotide comprising the coding sequence can be suitable for introduction into (transformation, transduction, transfection, etc.) a host cell or host cell organelle. A cloning vector suitable for expression of the polynucleotide sequence in a host cell or a host cell organelle can be selected.

[0626] host cells

[0627] The present invention also relates to a host cell comprising a polynucleotide encoding a variant polypeptide of the present invention, a nucleic acid construct as described herein, or an expression vector as described herein. In one embodiment of the present invention, the vector is used for transformation of a host cell.

[0628] The polynucleotide encoding protease variants as described herein can be transiently or stably introduced into the host cell, and non-integration can be kept, for example, as a plasmid. Usually, stable transformation is due to the nucleic acid comprising an external coding sequence being integrated into the host cell chromosome or as an episome (independent nuclear DNA fragment). Usually, transient transformation is due to the nucleic acid comprising an external nucleic acid sequence being integrated into the host cell chromosome or not as an episome.

[0629] Introduction of nucleic acids into host cells can be achieved, for example, but not limited to, by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829 or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5271-5278). Specific transformation protocols are known in the art for each type of host cell (see, eg, Hanahan, 1983, J. Mol. Biol. 166:557-580 for protoplast transformation of E. coli).

[0630] A variety of host cells can be used to express the nucleic acid constructs described herein. Host cells containing the nucleic acid constructs described herein can be obtained by one of the methods described herein for introducing a polynucleotide into such a host cell. The host cells of the present invention do not naturally express the protease variant. Thus, the host cell is a recombinant host cell; the nucleic acid constructs described herein are heterologous to the host cell.

[0631] In one embodiment, the host cell is a prokaryotic or eukaryotic organism. In another embodiment, the host cell is a bacterium, an archaea, a fungal cell, a yeast cell, or a eukaryotic cell. In another embodiment, the host cell is a non-human host cell.

[0632] In one embodiment, the host cell is a bacterial cell. The bacterial host cell can be any gram-positive bacteria or gram-negative bacteria. Gram-positive bacteria include but are not limited to Bacillus, Brevibacterium, Corynebacterium, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus and Oceanobacillus. Gram-negative bacteria include, but are not limited to, Escherichia, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Acetobacter, Flavobacterium, Fusobacterium, and Gluconobacter. In particular embodiments, the bacterial host cell is an Escherichia coli cell. In one embodiment, the host cell is a bacterial cell. In particular embodiments, the host cell is a cell of the genus Escherichia or Bacillus.

[0633] In the methods of the present invention, the bacterial host cell may be any Bacillus cell. Bacillus cells useful in the practice of the present invention include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus methylotrophicus, Bacillus cereus, and Bacillus thunbergii. cereus), Bacillus paralicheniformis, Bacillus subtilis, and Bacillus thuringiensis cells. In one embodiment, the bacterial host cell is a Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell. In a preferred embodiment, the bacterial host cell is a Bacillus licheniformis cell, Bacillus pumilus, or Bacillus subtilis cell. Preferably, the bacterial host cell is a Bacillus licheniformis cell.

[0634] Preparation method

[0635] Another embodiment of the present invention is a method for obtaining a protease variant of a parent protease, the method comprising the steps of:

[0636] a) introducing into a parent protease, preferably into a protease according to SEQ ID NO: 1 or SEQ ID NO: 3, preferably into a protease according to SEQ ID NO: 3, an amino acid substitution at amino acid residue 183 and at least one further amino acid substitution at an amino acid residue selected from the group consisting of 43, 78 and 204, with reference to the numbering of the amino acid sequence shown in SEQ ID NO: 2, thereby providing a variant polypeptide of said parent protease,

[0637] wherein the variant has at least 65% but less than 100% sequence identity with the amino acid sequence of the polypeptide of SEQ ID NO: 1 or 3, preferably SEQ ID NO: 3, and wherein the variant polypeptide has protease activity, and preferably wherein the variant polypeptide has improved properties relative to the parent protease.

[0638] The manner in which amino acid substitutions are introduced into protein sequences is well known in the art. Variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, and the like.

[0639] The variant polypeptides obtained by the above procedures can be produced on an industrial scale and subsequently purified. The industrial production of enzymes is typically accomplished by cultivating host cells (also referred to as fermentations) expressing the enzyme. Suitable host cells are described herein. The nucleic acid sequence encoding the variant polypeptides as described herein can be transformed into a host cell, which is then cultured under conditions suitable for host cell production of protease variants. In a preferred embodiment, the variant polypeptides are purified from the host cell with protease activity.

[0640] Therefore, in yet another embodiment, the present invention relates to a method for producing a variant polypeptide having protease activity, the method comprising the steps of:

[0641] (a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding a variant polypeptide having protease activity as described herein;

[0642] (b) cultivating the recombinant host cell of step (a) under conditions conducive to expression of the polynucleotide; and

[0643] (c) optionally, recovering a variant polypeptide having protease activity encoded by the polynucleotide.

[0644] Host cell culture is typically carried out in a suitable nutrient medium that allows the recombinant cells to grow and express the desired protein. At the end of the fermentation, the fermentation broth comprising a liquid portion and a solid portion is collected and can be further processed. Variant polypeptides having protease activity can be further purified from the fermentation broth.

[0645] Variant polypeptides with protease activity as herein described can be secreted (enter the liquid portion of fermented liquid), or can not be secreted from microbial cells (and therefore be contained in the cells of fermented liquid).According to this, the protease variants can be reclaimed from the liquid portion of fermented liquid or from cell lysates.Preferably, the protease variants are secreted into the fermented liquid from the cells, preferably by adding a secretion signal peptide to the N-terminal of the amino acid sequence, thereby forming the propeptide of the variant polypeptide.After the propeptide cutting, the variant polypeptide is folded, and mature protease is released as active variant.The recovery of variant polypeptides with protease activity can be achieved by methods well known to those skilled in the art. Suitable methods for recovering protein from fermented liquid include but are not limited to collection, centrifugation, filtration, extraction and precipitation.If the target protein precipitates or crystallizes in the fermented liquid or is at least partially combined with the particulate matter of the fermented liquid, then other processing steps may be needed to discharge the target protein or dissolve the target protein crystals and precipitate from biomass. WO 00 / 43502A1, WO 2008 / 110498A1 and WO 2017 / 097869A1 describe methods for recovering a target protein from a fermentation broth that precipitates and / or crystallizes during fermentation. If the desired protein is contained in cells of the fermentation broth, it may be necessary to release the target protein 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, ultrasonication, a French press, or a combination thereof.

[0646] Variant polypeptides with protease activity can be purified from fermentation broth by methods known in the art. For example, protease variants can be separated from fermentation broth by conventional procedures, including but not limited to centrifugation, filtration, extraction, spray drying, evaporation or precipitation. The polypeptide separated can then be further purified by multiple procedures known in the art, including but not limited to chromatography (for example, ion exchange, affinity, hydrophobic, chromatofocusing and size exclusion), electrophoresis procedures (for example, preparative isoelectric focusing (IEF)), differential solubility (for example, ammonium sulfate precipitation) or extraction (see, for example, Protein Purification [protein purification], J.-C. Janson and Lars Ryden, editors, VCH Publishers [VCH press], New York, 1989). The polypeptide purified can then be concentrated by procedures known in the art, including but not limited to ultrafiltration and evaporation, particularly thin film evaporation.

[0647] Protease composition

[0648] Purified solutions of the protease variants described herein can be further processed to form compositions containing the protease.Thus, also claimed herein are compositions comprising the protease variants described herein and at least one additional component.

[0649] Therefore, the present invention also relates to a process for preparing a composition comprising the steps of: mixing

[0650] i. a protease variant as described herein; and

[0651] ii. one or more components described herein.

[0652] Furthermore, the present invention relates to a method for improving the stability of a protease in a composition, comprising the steps of: mixing

[0653] (a) a protease variant as described herein; and

[0654] (b) one or more components described herein.

[0655] The composition may be a non-complex formulation (eg, a protease variant formulation with one or some other components) or a complex formulation (eg, a detergent composition).

[0656] In one embodiment of the invention, the protease variant is formulated as a protease variant formulation, preferably a concentrated protease variant formulation. The protease variant formulation can be solid or liquid. The protein formulation can be obtained using techniques known in the art. For example, but not limited to, solid enzyme formulations can be obtained by extrusion or granulation. Suitable extrusion and granulation techniques are known in the art and are described, for example, in WO 94 / 19444A1 and WO 97 / 43482A1.

[0657] Liquid protease variant formulations may comprise enzyme amounts ranging from 2% to 40%, 2% to 30%, 2% to 25%, 2% to 12%, or preferably 2%-6% by weight, all relative to the total weight of the enzyme formulation.

[0658] In one embodiment, the protease variant formulation, particularly the liquid enzyme formulation, further comprises one or more additional compounds selected from the group consisting of: solvents, salts, pH adjusters, preservatives, enzyme stabilizers, and thickeners. Preferably, the protease variant formulation is substantially free of surfactants, i.e., the protease variant formulation comprises less than 1% surfactant, preferably less than 0.5% surfactant. The solvent may be water and / or an organic solvent. The aqueous protease variant formulation of the present invention may comprise water in an amount of more than about 30% by weight, more than about 40% by weight, more than about 50% by weight, or more than about 60% by weight, all relative to the total weight of the enzyme formulation. The protease variant formulation of the present invention may comprise an organic solvent in an amount of more than 30% by weight, more than 40% by weight, more than about 50% by weight, more than about 60% by weight, more than about 70% by weight, or more than about 80% by weight, all relative to the total weight of the enzyme formulation. The organic solvent may be a water-miscible solvent. The organic solvent may be one or more selected from the group consisting of glycerin, propylene glycol, polypropylene glycol, and polyethylene glycol.

[0659] In one embodiment, the protease variant formulation includes at least one preservative. Preferably, preservative means a substance added to the liquid composition for the purpose of preservation, meaning that more preferably, the known compound with antiseptic characteristics contained in the liquid composition formed during the production process is excluded from the term preservative. In one embodiment, the preservative is selected from the group consisting of: 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol, formic acid (in acid form or its salt form) and 4,4'-dichloro-2-hydroxydiphenyl ether. Typically, the liquid composition of the present invention includes at least one preservative, and its amount is less than 10ppm, such as relative to the gross weight of the liquid composition, and its amount ranges from 2ppm to 5% by weight. Preferably, the protease variant formulation does not contain preservative, which means that the content of the preservative is less than 1ppm, preferably 0ppm.

[0660] Preferably, the protease variant formulation comprises an enzyme stabilization system. Preferably, the enzyme stabilization system comprises at least one compound selected from the group consisting of a polyol (preferably 1,3-propylene glycol, ethylene glycol, glycerol, 1,2-propylene glycol or sorbitol), an inorganic salt (preferably CaCl2, MgCl2 or NaCl), a short chain (preferably C1-C3) carboxylic acid or a salt thereof (preferably formic acid, formate (preferably sodium formate), acetic acid, acetate or lactate), a borate, boric acid, boronic acid (preferably 4-formylphenylboronic acid (4-FPBA)), a peptide aldehyde (preferably benzyloxycarbonyl-VAL-H (Z-VAL-H or Cbz-VAL-H) ​​or benzyloxycarbonyl-GAY-H (Z-GAY-H or Cbz-GAY-H)), a peptide acetal and a peptide aldehyde bisulfite adduct. Preferably, the enzyme stabilization system comprises a combination of at least two compounds selected from the group consisting of salts, polyols, and short-chain carboxylic acids, and preferably one or more compounds selected from the group consisting of borates, boric acid, boronic acid (preferably 4-formylphenylboronic acid (4-FPBA)), peptide aldehydes, peptide acetals, and peptide aldehyde bisulfite adducts. In a particularly preferred embodiment, the stabilization system comprises a protease inhibitor, preferably selected from borates, boric acid, boronic acid (preferably 4-FPBA), peptide aldehydes (preferably peptide aldehydes like Z-VAL-H or Z-GAY-H), peptide acetals, and peptide aldehyde bisulfite adducts, preferably the protease inhibitor is a peptide aldehyde, preferably Z-VAL-H or Z-GAY-H. In one embodiment, the stabilization system does not comprise a protease inhibitor. Preferably, the composition does not contain boron. Preferably, the protease variant formulation comprises a calcium salt, preferably calcium chloride.

[0661] In one embodiment, the protease variant formulation comprises a protease inhibitor, preferably a peptide aldehyde, in an amount in the range of about 0.05% to 0.8% by weight relative to the total weight of the liquid protease variant formulation. Preferably, the peptide aldehyde is present in an amount in the range of about 0.1% to 0.6% by weight, about 0.1% to 0.5% by weight, about 0.1% to 0.4% by weight, or about 0.1% to 0.35% by weight, all relative to the total weight of the liquid protease variant formulation.

[0662] Preferably, the liquid protease variant formulation comprises or consists of a protease variant, a solvent, an enzyme stabilization system, and optionally a preservative and optionally a second enzyme different from the protease variant. Preferably, the protease variant formulation is substantially free of surfactant, i.e., the protease variant formulation comprises less than 1% surfactant, preferably less than 0.5% surfactant.

[0663] Therefore, the present invention also relates to a method for preparing a protease variant formulation, preferably a concentrated protease variant formulation, comprising the steps of: mixing

[0664] a) a protease variant as described herein; and

[0665] b) one or more components selected from the group consisting of a solvent, an enzyme stabilization system, a preservative, and a second enzyme different from the protease variant.

[0666] Furthermore, the present invention relates to a method for improving the stability of a protease in a composition, the method comprising the steps of: mixing

[0667] a) a protease variant as described herein; and

[0668] b) one or more components selected from the group consisting of a solvent, an enzyme stabilization system, a preservative, and a second enzyme different from the protease variant.

[0669] In one embodiment, the protease variants described herein are part of a microorganism (live, attenuated or killed), a probiotic or a prebiotic.

[0670] Second enzyme

[0671] In another embodiment, the composition comprising a protease variant as described herein further comprises one or more second enzymes different from the protease variant. Preferably, the second enzyme is selected from the group consisting of amylases, one or more proteases other than the protease variants described herein, lipases, cellulases, mannanases, hemicellulases, phospholipases, esterases, pectinases, lactases, peroxidases, xylanases, cutinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, beta-glucanases, arabinosidases, hyaluronidases, chondroitins. Preferably, the second enzyme is an enzyme selected from the group consisting of: an amylase, a lipase, a cellulase, a mannanase, a xylanase, a DNA enzyme, a dispersin, a pectinase, an oxidoreductase, and a cutinase, and a combination of at least two of the foregoing types. Most preferably, the second enzyme is an amylase, preferably an α-amylase.

[0672] The compositions of the present invention may comprise more than one enzyme of a different type (e.g., an amylase and a protease), or more than one enzyme of the same type (e.g., two or more different proteases), or a mixture thereof (e.g., an amylase and two different proteases, one of which is a protease variant described herein).

[0673] Protease

[0674] Proteases other than the protease variants of the present invention may comprise an amino acid sequence having, preferably, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but less than 100% sequence identity to SEQ ID NO: 1 and comprising an amino acid substitution at one or more of the following positions: 3, 4, 9, 15, 27, 33, 36, 57, 68, 77, 87, 9 5,96,97,98,99,100,101,102,103,104,106,118,120,123,128,129,130,131,154,160,167,170,194,199,205,206,217,218,222,224,232,235,236,245,252 and 274 (according to BPN 'numbering), it has proteolytic activity. In one embodiment, such protease has no mutation at position Asp32, His64 and Ser221 (with reference to SEQ ID NO:2 numbering). Preferably, the protease used in combination with the protease variants described herein comprises an amino acid sequence having, preferably increasing sequence identity to SEQ ID NO: 1, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, but less than 100%, and is further characterized by having an amino acid glutamic acid (E), or aspartic acid (D), or asparagine (N), or glutamine (Q), or alanine (A), or glycine (G) or serine (S), preferably glutamic acid (E), at position 101 (with reference to the numbering of SEQ ID NO: 2), and having proteolytic activity. Most preferred is a protease having at least 80% but less than 100% sequence identity to SEQ ID NO: 1, and characterized by having an amino acid glutamic acid (E) at position 101 (with reference to the numbering of SEQ ID NO: 2), and having proteolytic activity. The protease may comprise an amino acid substitution at position 101 (e.g., R101E), alone or in combination with one or more substitutions at positions 3, 4, 9, 15, 27, 33, 36, 57, 68, 77, 87, 95, 96, 97, 98, 99, 100, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 252, and / or 274 (with reference to the numbering of SEQ ID NO: 2), and have proteolytic activity.In one embodiment, the protease comprises one or more additional substitutions: (a) threonine at position 3 (3T), (b) isoleucine at position 4 (4I), (c) alanine, threonine or arginine at position 63 (63A, 63T or 63R), (d) aspartic acid or glutamic acid at position 156 (156D or 156E), (e) proline at position 194 (194P), (f) methionine at position 199 (199M), (g) isoleucine at position 205 (205I), (h) aspartic acid, glutamic acid or glycine at position 217 (217D, 217E or 217G), (i) a combination of two or more amino acids according to (a) to (h), numbering with reference to SEQ ID NO: 2. Suitable proteases may be at least 80% identical to SEQ ID NO: 1 and characterized in that they comprise one amino acid (according to (a) to (h)) or a combination according to (i) and amino acids 101E, 101D, 101N, 101Q, 101A, 101G or 101S (with reference to the numbering of SEQ ID NO: 2), and have proteolytic activity. In one embodiment, the protease is at least 80% identical to SEQ ID NO: 1 and is characterized in that it comprises the mutations (with reference to the numbering of SEQ ID NO: 2) R101E, or S3T+V4I+V205I, or S3T+V4I+R101E+V205I or S3T+V4I+V199M+V205I+L217D, and has proteolytic activity. In another embodiment, the protease comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 1, and is further characterized in that it comprises S3T+V4I+S9R+A15T+V68A+D99S+R101S+A103S+I104V+N218D (with reference to the numbering of SEQ ID NO: 2), and has proteolytic activity. In another embodiment, the protease may have an amino acid sequence having at least 80% identity to SEQ ID NO: 1. NO:1 amino acid sequence that is at least 80% identical, and is further characterized by comprising R101E and one or more substitutions selected from the group consisting of: S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61D,R, S87E, G97S, A98D,E,R, S106A,W, N117E, H120V,D,K,N, S125M, P129D, E136Q, S144W, S161T, S163A,G, Y171L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D, and L262N,Q,D (referring to SEQ ID NO:1). ID NO: 2), and has proteolytic activity.

[0675] lipase

[0676] "Lipase," "lipolytic enzyme," and "lipid esterase" all refer to enzymes of EC class 3.1.1 ("carboxylic ester hydrolases"). 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.

[0677] In one aspect of the invention, suitable lipases (component (b)) 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. 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 these 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 / 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, WO 96 / 00292); lipases from Streptomyces griseus (WO 2011 / 150157) and Streptomyces spp.pristinaespiralis) (WO 2012 / 137147), GDSL-type Streptomyces lipase (WO 2010 / 065455); lipase from Thermobifida fusca as disclosed in WO 2011 / 084412; lipase from Geobacillus stearothermophilus as disclosed in WO 2011 / 084417; Bacillus lipases, for example as disclosed in WO 00 / 60063, lipase from Bacillus subtilis as disclosed in Dartois et al. (1992), Biochemica et Biophysica Acta, 1131, 253-360 or WO 2011 / 084599, lipase from Bacillus stearothermophilus (JP S64-074992) or Bacillus pumilus (WO 91 / 16422); a lipase from Candida antarctica as disclosed in WO 94 / 01541; a cutinase from Pseudomonas mendocina (US 5389536, WO 88 / 09367); a cutinase from Magnaporthe grisea (WO 2010 / 107560); a cutinase from Fusarum solani pisi as disclosed in WO 90 / 09446, WO 00 / 34450 and WO 01 / 92502; and a cutinase from Humicola lanuginosa as disclosed in WO 00 / 34450 and WO 01 / 92502.

[0678] 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.

[0679] 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 A / S), Lumafast (originally from Genencor), Preferenz L (DuPont), and Lipomax (Gist-Brocades / now DSM).

[0680] 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 lanuginosa lipase is selected from the triacylglycerol lipase of amino acids 1-269 of SEQ ID NO: 2 of US 5869438 and variants thereof having lipolytic activity.

[0681] The Thermomyces lanuginosus lipase may be selected from variants having lipolytic activity that are 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.

[0682] The Thermomyces lanuginosus lipase can be selected from variants having 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 having lipolytic activity can have at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US 5869438.

[0683] The Thermomyces lanuginosus lipase may be selected from variants having lipolytic activity comprising the following amino acid substitutions: T231R and N233R when compared to amino acids 1-269 of SEQ ID NO: 2 of US 5869438. The lipase variant may further comprise one or more of the following amino acid exchanges: Q4V, V60S, A150G, L227G, P256K when compared to amino acids 1-269 of SEQ ID NO: 2 of US 5869438.

[0684] The Thermomyces lanuginosus lipase can be selected from variants having 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 having at least 95%, at least 96% or at least 97% similarity when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US 5869438.

[0685] The Thermomyces lanuginosus 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 having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US 5869438.

[0686] 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 by containing amino acid substitutions T231R and N233R.

[0687] The Thermomyces lanuginosus lipase may be selected from the following variants having lipolytic activity, which variant preferably comprises 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 has at least 95%, at least 96% or at least 97% similarity when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 1 of WO 2015 / 010009.

[0688] amylase

[0689] "Amylases" (alpha-amylases and / or beta-amylases) include amylases of bacterial or fungal origin (EC 3.2.1.1 and 3.2.1.2, respectively). Preferably, the amylase is selected from the group of alpha-amylases (EC 3.2.1.1).

[0690] Amylases have "amylolytic activity" or "amylase activity" which involves the (endo)hydrolysis of glycosidic linkages in polysaccharides.

[0691] Amylases may be derived from Bacillus licheniformis having SEQ ID NO: 2 as described in WO 95 / 10603 and variants thereof having at least 95% identity. Suitable variants are described in WO 95 / 10603 comprising one or more substitutions at the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408 and 444, which have amylolytic activity. Variants are described in SEQ ID NO: 4 of WO 94 / 02597, WO 94 / 018314, WO 97 / 043424 and WO 99 / 019467.

[0692] The amylase may further be from Bacillus stearothermophilus having SEQ ID NO: 6 as disclosed in WO 02 / 10355, or an amylase optionally having a C-terminal truncation relative to the wild-type sequence. Suitable variants of SEQ ID NO: 6 include variants comprising a deletion at position 179 and / or 181 and / or 182 and / or a substitution at position 193.

[0693] The amylase may further be from Bacillus sp. 707 having SEQ ID NO: 6 as disclosed in WO 99 / 19467 and variants thereof having at least 95% identity. Preferred variants of SEQ NO: 6 as disclosed in WO 99 / 19467 are those having substitutions, deletions or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.

[0694] The amylase may further be from Bacillus halmapalus having SEQ ID NO: 2 or SEQ ID NO: 7 as described in WO 96 / 23872, also described herein as SP-722. Preferred variants are described in WO 97 / 3296, WO 99 / 194671 and WO 2013 / 001078.

[0695] The amylase may further be from Bacillus sp. DSM 12649 having SEQ ID NO: 4 as disclosed in WO 00 / 22103 and variants thereof having at least 95% identity thereto.

[0696] The amylase may further be from Bacillus sp. A 7-7 (DSM 12368) having an amino acid sequence at least 95% identical to SEQ ID NO: 2, particularly with respect to the region from amino acids 32 to 516 according to SEQ ID NO: 2, as disclosed in WO 02 / 10356.

[0697] The amylase may further be from Bacillus strain TS-23 having SEQ ID NO: 2 and variants thereof as disclosed in WO 2009 / 061380.

[0698] The amylase may further be from Cytophaga sp. having SEQ ID NO: 1 as disclosed in WO 2013 / 184577 and variants thereof having at least 95% identity.

[0699] The amylase may further be from Bacillus megaterium DSM 90 having SEQ ID NO: 1 as disclosed in WO 2010 / 104675 and variants thereof having at least 95% identity thereto.

[0700] The amylase may further be from Bacillus sp. comprising amino acids 1 to 485 of SEQ ID NO: 2 as described in WO 00 / 60060 and variants thereof having at least 95% identity thereto.

[0701] The amylase may further be from Bacillus amyloliquefaciens or a variant thereof, preferably selected from the amylase according to SEQ ID NO: 3 as described in WO 2016 / 092009.

[0702] The amylase may have an amino acid sequence according to SEQ ID NO: 12 as described in WO 2006 / 002643, or may be an amylase variant thereof comprising the substitutions Y295F and M202LITV within said SEQ ID NO: 12.

[0703] The amylase may have an amino acid sequence according to SEQ ID NO: 6 as described in WO 2011 / 098531, or may be an amylase variant comprising a substitution within said SEQ ID NO: 6 at one or more positions selected from the group consisting of: 193G,A,S,T,M, 195F,W,Y,L,I,V, 197F,W,Y,L,I,V, 198Q,N, 200F,W,Y,L,I,V, 203F,W,Y,L,I,V, 206F,W,Y,N,L,I,V,H,Q,D,E, 210F,W,Y,L,I,V, 212F,W,Y,L,I,V, 213G,A,S,T,M, and 243F,W,Y,L,I,V.

[0704] The amylase may have an amino acid sequence according to SEQ ID NO: 1 as described in WO 2013 / 001078, or amylase variants comprising alterations at two or more (several) positions corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476 and G477 within said SEQ ID NO: 1.

[0705] The amylase may have an amino acid sequence according to SEQ ID NO: 2 as described in WO 2013 / 001087, or may be an amylase variant comprising a deletion of positions 181+182, or 182+183, or 183+184 within said SEQ ID NO: 2, optionally comprising one, two or more modifications at any of the positions corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476 and G477 within said SEQ ID NO: 2.

[0706] The amylase may be a hybrid alpha-amylase derived from the above amylases, eg, as described in WO 2006 / 066594.

[0707] According to WO 2014 / 183920, the hybrid amylase may have A and B domains that are at least 90% identical to SEQ ID NO: 2 of WO 2014 / 183920 and a C domain that is at least 90% identical to SEQ ID NO: 6 of WO 2014 / 183920, wherein the hybrid amylase has amylolytic activity; preferably, the hybrid α-amylase has at least 95% identity to SEQ ID NO: 23 of WO 2014 / 183920 and has amylolytic activity.

[0708] According to WO 2014 / 183921, the hybrid amylase may have A and B domains and a C domain, the A and B domains being at least 75% identical to SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 26, SEQ ID NO: 32 and SEQ ID NO: 39 as disclosed in WO 2014 / 183921, and the C domain being at least 90% identical to SEQ ID NO: 6 of WO 2014 / 183921, wherein the hybrid amylase has amylolytic activity; preferably, the hybrid alpha-amylase is at least 95% identical to SEQ ID NO: 30 as disclosed in WO 2014 / 183921 and has amylolytic activity;

[0709] According to WO 2021 / 032881, the hybrid amylase may comprise A and B domains derived from an alpha amylase derived from Bacillus species A7-7 (DSM 12368), and a C domain derived from an alpha amylase from Bacillus cereus; preferably, the A and B domains are at least 75% identical to the amino acid sequence of SEQ ID NO: 42, and the C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44 - both sequences are as disclosed in WO 2021 / 032881; more preferably, the hybrid amylase is at least 80% identical to SEQ ID NO: 54 as disclosed in WO 2021 / 032881.

[0710] In one embodiment, the at least one amylase is selected from commercially available amylases including but not limited to those sold under the trade name Duramyl TM Termamyl TM 、Fungamyl TM 、Stainzyme TM 、Stainzyme Plus TM、Natalase TM , Liquozyme X and BAN TM 、Amplify TM 、Amplify Prime TM (from Novozymes), and Rapidase TM 、Purastar TM 、Powerase TM 、Effectenz TM (M100 from DuPont), Preferenz TM (S1000, S110 and F1000; from DuPont), PrimaGreen TM (ALL; DuPont), Optisize TM Products sold by (DuPont).

[0711] Mannanase

[0712] As used herein, a "mannanase" is an enzyme selected from the group of mannan degrading enzymes. The mannan degrading enzyme may be selected from β-mannosidase (EC 3.2.1.25), endo-1,4-β-mannosidase (EC 3.2.1.78) and 1,4-β-mannobiosidase (EC 3.2.1.100). Preferably, the mannan degrading enzyme is selected from the group of endo-1,4-β-mannosidase (EC 3.2.1.78), which may be referred to herein as endo-β-1,4-D-mannanase, β-mannanase or mannanase.

[0713] 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 enzymatic activity of at least 40% of its maximum activity at a given pH of 7 to 12, preferably 7.5 to 10.5.

[0714] The mannanase may be selected from the group consisting of mannanases derived from Bacillus organisms, such as those described in JP-0304706 [beta-mannanase from Bacillus sp.], JP-63056289 [alkaline, thermostable beta-mannanase], JP-63036774 [Bacillus microorganism FERM P-8856 producing beta-mannanase and beta-mannosidase at alkaline pH], JP-08051975 [alkaline beta-mannanase from alkalophilic Bacillus sp. AM-001], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 91 / 18974 [mannanase active at extreme pH and temperature], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 91 / 18974 [mannanase active at extreme pH and temperature], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 91 / 18974 [mannanase active at extreme pH and temperature], WO 97 / 11164 [mannanase from Bacillus amyloliquefaciens], WO 2014 / 100018 [endo-(3-mannanase 1 (Bleman 1; see US 5,476,775) cloned from Bacillus circulans or Bacillus lentus strain CMG1240]. Suitable mannanases are described in WO 99 / 064619.

[0715] The mannanase may be selected from mannanases derived from Trichoderma organisms, such as those disclosed in WO 93 / 24622.

[0716] Mannanase can be selected from commercially available mannanases, such as (Novozymes) or (M100)(DuPont).

[0717] Cellulase

[0718] "Cellulase" is an enzyme that can hydrolyze cellulose. Cellulase can be selected from cellobiohydrolases (1,4-PD-glucan cellobiohydrolase, EC 3.2.1.91), endo-ss-1,4-glucanases (EC 3.2.1.4) and ss-glucosidases (EC 3.2.1.21). Endoglucanases of EC class 3.2.1.4 can be designated as endoglucanases, endo-1,4-ss-D-glucan 4-glucanohydrolases, endo-1,4-β-glucanases, carboxymethyl cellulases and β-1,4-glucanases.

[0719] Endoglucanases can be classified by amino acid sequence similarity in Family 5 which contains more than 20 endoglucanases of EC 3.2.1.4 (Henrissat, B., accessed UniProt 10 / 26 / 2011). Reference is also made to T.-M. Enveri, "Microbial Cellulases," in WM Fogarty, Microbial Enzymes and Biotechnology, Applied Science Publishers, pp. 183-224 (1983); Methods in Enzymology, (1988) Vol. 160, pp. 200-391 (edited by Wood, W. A. ​​and Kellogg, S. T.); 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, 1990. Reviews [FEMS Microbiology Reviews] 13 (1994) pp. 25-58; Henrissat, B., "Cellulases and their interaction with cellulose", Cellulose [Cellulose] (1994), vol. 1, pp. 169-196.

[0720] Preferably, the cellulase is selected from the glycosyl hydrolase family 7 (GH7, pfam00840), preferably from the endoglucanase (EC 3.2.1.4).

[0721] Preferably, alkaline cellulases are used, wherein "alkaline cellulases" is intended to cover cellulases having enzymatic activity at a given pH in the range of 7 to 12, preferably 7.5 to 10.5.

[0722] In one embodiment, the cellulase is selected from cellulases comprising a cellulose binding domain. In another embodiment, the cellulase comprises a catalytic domain but does not comprise a cellulose binding domain.

[0723] In one embodiment the formulation of the invention comprises at least one endoglucanase of EC class 3.2.1.4 derived from

[0724] Bacillus, for example Bacillus species CBS 670.93 and CBS 669.93

[0725] Melanocarpus, for example Melanocarpus albomyces as disclosed in WO 97 / 14804

[0726] Clostridium species, such as Clostridium thermocellum

[0727] Humicola, for example, EP 0495257, EP 0531315, EP 0531372, US 4435307, US 5648263, US 5776757, WO 89 / 09259, WO 91 / 17244, WO 94 / 07998 (sequences shown in Figure 1 and its 43 kd human variant), Humicola insolens (DSM 1800) disclosed in WO 95 / 24471, WO 96 / 11262 and WO 98 / 12307.

[0728] Fusarium, for example Fusarium oxysporum, for example strain J79 (DSM 2672) as disclosed in EP 0 495 257, EP 0 531 315, EP 0 531 372, US 5 648 263, US 5 776 757, WO 89 / 09259, WO 91 / 17244, WO 95 / 24471 and WO 96 / 11262

[0729] Thielavia, for example Thielavia terrestris or Myceliophthora thermophila strain CBS11765 as disclosed in EP 0531315, US 5648263, US 5776757, WO 89 / 09259, WO 91 / 17244, WO 95 / 24471, WO 96 / 11262, WO 96 / 29397 (SEQ ID NO: 9 and variants thereof) and WO 98 / 12307.

[0730] • Trichoderma, for example Trichoderma reesei, Trichoderma longibrachiatum or Trichoderma harzianum as disclosed in EP 1305432, EP 1240525, WO 92 / 06165, WO 94 / 21801, WO 94 / 26880, WO 95 / 02043, WO 95 / 24471 and WO 02 / 099091.

[0731] • Aspergillus, for example Aspergillus aculeatus as disclosed in WO 93 / 17244.

[0732] • Erwinia spp., for example, Erwinia chrysanthermi as described by MH Boyer et al., European Journal of Biochemistry, Vol. 162, pp. 311-316 (1987).

[0733] Acremonium, e.g., Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pinkertoniae, Acremonium roseogriseum, Acremonium incoloratum, and Acremonium furatum as disclosed in WO 96 / 11262 and WO 96 / 29397 (SEQ ID NO: 5 and variants thereof).

[0734] Cellvibrio genus, for example Cellvibrio mixtus DSM 11683, Cellvibrio mixtus DSM 11684, Cellvibrio mixtus DSM 11685, Cellvibrio mixtus ACM 2601, Cellvibrio mixtus DSM 1523 and Cellvibrio gilvus DSM 11686, as disclosed in WO 98 / 08940.

[0735] • Cephalosporium, for example Cephalosporium sp. RYM-202 as disclosed in WO 96 / 11262.

[0736] Suitable cellulases also include those that are variants of the above-mentioned cellulases having cellulolytic activity. In one embodiment, the cellulase variants include variants having at least 40% to 100% identity when compared to the full-length polypeptide sequence of the parent enzyme as disclosed above. In one embodiment, the cellulase variants having cellulolytic activity have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity and / or identity to the full-length polypeptide sequence of the parent enzyme as disclosed above.

[0737] The cellulase may be a Humicola insolens DSM 1800 cellulase complex having endoglucanase, cellobiohydrolase and beta-glucosidase activities.

[0738] The cellulase may be a Humicola insolens DSM 1800 endoglucanase (EC 3.2.1.4), preferably having a polypeptide sequence according to positions 21-435 of SEQ ID NO: 2 as disclosed in WO 2018 / 224544, or a variant thereof having at least 95% identity.

[0739] The cellulase may be a Humicola insolens endoglucanase (EC 3.2.1.4) having 43 kD, preferably prepared according to e.g. WO 94 / 07998. Figure 1 a) or a variant thereof (preferably at least 90% identical thereto), preferably those disclosed in WO 94 / 07998.

[0740] The cellulase may be a Bacillus species cellulase (EC 3.2.1.4) selected from a polypeptide having at least 80% similarity and / or identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO: 2 of WO 2004 / 053039 or a catalytically active fragment thereof. In one embodiment, the cellulase is a mature polypeptide having at least 95% identity to SEQ ID NO: 1 of WO 2018 / 224544.

[0741] The cellulase may be a Thielavia terrestris cellulase (EC 3.2.1.4) having a polypeptide having at least 80% similarity and / or identity to the amino acid sequence of position 1 to position 299 of SEQ ID NO: 4 of WO 2004 / 053039, or a catalytically active fragment thereof. In one embodiment, the cellulase is a mature polypeptide having at least 95% identity to SEQ ID NO: 4 of WO 2018 / 224544.

[0742] The cellulase may be a mature Sordaria fimicola cellulase, preferably having a polypeptide sequence according to SEQ ID NO: 5 of WO 2018 / 224544 or a variant thereof having at least 95% identity thereto.

[0743] The cellulase may be selected from and (Novozymes), Clazinase TM and Puradax HA TM (Genencor Int. Inc.) and KAC-500(B) TM (Kao Corporation).

[0744] Detergent composition

[0745] In one embodiment, the present invention relates to the use of a protease variant in a detergent composition. Accordingly, the present invention also relates to a detergent composition comprising a protease variant as described herein and one or more detergent components.

[0746] Therefore, the present invention also relates to a process for preparing a detergent composition, the process comprising the steps of: mixing

[0747] a) a protease variant as described herein; and

[0748] b) one or more detergent components as described herein.

[0749] The present invention also relates to a method for preparing a detergent composition having improved protease stability and / or for providing a detergent composition having improved cleaning performance, the method comprising the steps of: mixing

[0750] a) a protease variant as described herein; and

[0751] b) one or more detergent components as described herein.

[0752] Addition of the liquid protease variant formulation to a detergent composition, preferably a liquid detergent composition, is typically carried out at a weight ratio of about 1:1000, 1:500, 1:100, 1:50, 1:30, 1:25, 1:20 or 1:10 liquid protease variant formulation:detergent composition.

[0753] The one or more detergent components may be selected from the group consisting of an additional enzyme other than the protease variant, an enzyme stabilizing system, a surfactant, an antifoaming agent, a builder, a polymer, a bleaching system (bleaching agent), a rheology modifier, a hydrotrope, a softener, a drying agent, a whitening agent, a buffer, a preservative, an anti-corrosion additive, a dye, and a fragrance.

[0754] Preferably, at least one component of the detergent is selected from the group consisting of a surfactant, a builder, a polymer, a preservative and a second enzyme different from the protease variant. Preferably, one or more detergent components, preferably surfactants and / or builders are biodegradable and / or bio-based.

[0755] Detergent component may have more than one function in the final application of detergent composition, so any detergent component mentioned in the context of a specific function herein may also have another function in the final application of detergent composition. The function of a specific detergent component in the final application of detergent composition generally depends on its amount in detergent composition, i.e. the effective amount of detergent component. The type and / or amount of detergent component in detergent composition varies according to desired application (such as laundry washing for white textiles, colored textiles and wool). Selected one or more components also depend on the physical form (liquid, solid, gel, provided in a sachet or provided as a tablet, etc.) of detergent composition. For example, one or more components selected for laundry preparations further depend on regional conventions, which themselves are related to the following aspects: such as the washing temperature used, the mechanical principle of the washing machine (vertical axis compared to horizontal axis machine), the water consumption of each wash cycle, etc., and geographical characteristics like the average hardness of water.

[0756] In one embodiment, the detergent composition is a formulation of more than two detergent components, wherein at least one component is effective to remove stains, at least one component is effective to provide optimal cleaning conditions, and at least one component is effective to maintain the physical properties of the detergent.

[0757] The detergent composition may be a liquid or solid detergent composition or a combination of liquid and solid detergent compositions. The liquid detergent composition is preferably a gel detergent composition. The solid detergent composition may be a soap bar or a powder detergent composition, preferably a powder detergent composition, wherein the powder detergent composition may be compressed into tablets.

[0758] The detergent composition can be a unit dose or multi-dose composition. The detergent composition can be in the form of a pouch (including a multi-compartment pouch). The detergent composition can be a laundry or dishwashing detergent composition suitable for home care and / or industrial and institutional (I&I) cleaning. Both the laundry and dishwashing compositions can be in the form of hand wash compositions or automatic wash compositions. Preferably, the dishwashing composition is an automatic dishwashing (ADW) composition.

[0759] The detergent pouch can be any form, shape and material that is suitable for accommodating composition, for example, does not allow composition to be discharged from pouch before contacting with water.Pouch is made of water-soluble film, and this film surrounds internal volume.Described internal volume can be divided into the compartment of pouch.Preferred film is polymeric material, preferably forms the polymer of film or thin sheet, for example polyvinyl alcohol copolymer and hydroxypropyl methylcellulose (HPMC).Pouch can comprise solid laundry detergent composition or part component and / or liquid detergent composition or part component separated by water-soluble film.The compartment of liquid component can be different from the compartment containing solid (referring to for example US2009 / 0011970) in composition.

[0760] In one embodiment, the pH of the detergent composition is in the range of 5-12, preferably in the range of 6-11, more preferably in the range selected from 6-10, 7-9 and 7.5-8.5. In one embodiment, the formulation is a detergent composition, preferably a liquid detergent composition.

[0761] In one embodiment, the detergent composition according to the present invention comprises one or more surfactants.Depending on their ionic charge, surfactants are referred to as nonionic surfactants, anionic surfactants, cationic surfactants or amphoteric surfactants.

[0762] The detergent composition of the present invention can comprise one or more surfactants, and these one or more surfactants can be anionic surfactants and / or cationic surfactants and / or nonionic surfactants and / or semi-polar surfactants and / or zwitterionic surfactants or its mixture.In a preferred embodiment, the detergent composition of the present invention comprises at least one surfactant.In a specific embodiment, the detergent composition of the present invention comprises a mixture of one or more nonionic surfactants and one or more anionic surfactants.One or more surfactants typically exist with the level of about 0.1wt.-% to 60wt.-% (for example 1wt.-% to 40wt.-%, 3wt.-% to 20wt.-% or 3wt.-% to 10wt.-%). One or more surfactants are selected based on the desired cleaning application, and include one or more any conventional surfactants known in the art. Any surfactant known in the art for detergents can be used. Non-limiting examples of surfactants are disclosed in McCutcheon's 2016 Detergents and Emulsifiers, and McCutcheon's 2016 Functional Materials, both North American and International Editions, MC Publishing Co., 2016. Other useful examples are disclosed in earlier editions of the same publications known to those skilled in the art.

[0763] When included therein, the detergent typically comprises from about 1 wt.-% to 40 wt.-% (e.g., from 5 wt.-% to 30 wt.-%, from 5 wt.-% to 15 wt.-%, or from 20 wt.-% to 25 wt.-%) of anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, alkene sulfonates (ALS), olefin ... sulfonate), alkane-2,3-diyl bis(sulfates), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates), secondary alkane sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfo fatty acid methyl esters (α-SFMe or SES) including methyl ester sulfonate (MES), alkyl or alkenyl succinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinic acid or soaps, and combinations thereof.

[0764] When included therein, the detergent typically contains from about 0 to 10 wt.-% of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyldimethylethanolamine quaternary ammonium salts (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearyl ammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

[0765] When included therein, the detergent typically comprises about 0.2 wt.-% to 40 wt.-% of nonionic surfactant, e.g. 0.5 wt.-% to 30 wt.-%, in particular 1 wt.-% to 20 wt.-%, 3 wt.-% to 10 wt.-%, 3 wt.-% to 5 wt.-% or 8 wt.-% to 12 wt.-%. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (e.g., ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polyglycosides (APG), alkoxylated amines, fatty acid monoethanolamide (FAM), fatty acid diethanolamide (FADA), ethoxylated fatty acid monoethanolamide (EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamide (GA) or fatty acid glucamide (FAGA)), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

[0766] When included therein, the detergent typically contains from about 0 to 10 wt.-% of a semi-polar surfactant. Non-limiting examples of semi-polar surfactants include amine oxides (AOs), such as alkyldimethylamine oxide, N-(coconut alkyl)-N,N-dimethylamine oxide, and N-(tallow-alkyl)-N,N-bis-(2-hydroxyethyl)amine oxide, fatty acid alkanolamides, and ethoxylated fatty acid alkanolamides, and combinations thereof.

[0767] When included therein, detergents typically contain about 0 to 10 wt.-% of zwitterionic surfactants.Non-limiting examples of zwitterionic surfactants include betaines, alkyldimethylbetaines, sulfobetaines, and combinations thereof.

[0768] The detergent composition according to the present invention may contain one or more compounds selected from complexing agents (chelating agents, chelants, sequestrating agents), precipitants and ion exchange compounds, which can form water-soluble complexes with calcium and magnesium. Such compounds may be referred to herein as "builders" or "building agents", but this does not mean that such compounds are limited to this function in the final application of the detergent composition.

[0769] In one embodiment, the detergent composition of the present invention comprises at least one builder selected from non-phosphate-based builders, such as sodium gluconate, one or more citrates, one or more silicates, one or more carbonates, one or more phosphonates, one or more aminocarboxylates, one or more polycarboxylates, one or more polysulfonates and one or more polyphosphonates. In one embodiment, the detergent composition of the present invention comprises a strong chelating builder. Preferably, the detergent composition of the present invention is phosphate-free, meaning substantially free of phosphate-based builders. In this context, "substantially free of phosphate" is understood to mean that the sum of the content of phosphate and polyphosphate, as determined gravimetrically and with reference to the corresponding detergent composition of the present invention, is in the range of 10 ppm to 1% by weight. In another preferred embodiment, the detergent composition comprises a phosphonate, wherein the phosphonate is preferably DTPMP and / or HEDP.

[0770] In one embodiment, the detergent composition of the present invention comprises at least one "citrate" selected from monoalkali metal and dialkali metal salts of citric acid, in particular monosodium and preferably trisodium citric acid, ammonium or substituted ammonium salts of citric acid, and citric acid itself. Citrate can be used as an anhydrous compound or as a hydrate, for example as sodium citrate dihydrate. The total content of citrate can be in the range of 0% to about 20% by weight, in the range of about 0.5% to about 10% by weight, or in the range of 1%-5% by weight, all relative to the total weight of the detergent composition. In one embodiment, the detergent composition of the present invention comprises a total amount of citrate in the range of about 1%-3% relative to the total weight of the detergent composition.

[0771] The detergent compositions of the present invention may contain one or more silicates. "One or more silicates" in the context of the present invention include in particular sodium disilicate and sodium metasilicate, aluminosilicates such as sodium aluminosilicates like zeolite A (i.e. Na 12 (AlO2) 12 (SiO2) 12 *27H2O) and sheet silicates, in particular those of the formula α-Na2Si2O5, β-Na2Si2O5 and δ-Na2Si2O5.

[0772] The detergent composition of the present invention may contain one or more carbonates. The term "one or more carbonates" includes alkali metal carbonates and alkali metal bicarbonates, preferably sodium salts. Particularly suitable is sodium carbonate (Na2CO3).

[0773] The detergent compositions of the present invention may contain one or more phosphonates. "Phosphonates" include, but are not limited to, 2-phosphinobutane-1,2,4-tricarboxylic acid (PBTC); ethylenediaminetetrakis(methylenephosphonic acid) (EDTMPA); 1-hydroxyethane-1,1-diphosphonic acid (HEDP), CH2C(OH)[PO(OH)2]2; aminotris(methylenephosphonic acid) (ATMP), N[CH2PO(OH)2]3; aminotris(methylenephosphonic acid) sodium salt (ATMP), N[CH2PO(ONa)2]3; 2-hydroxyethyliminobis(methylenephosphonic acid), HOCH2CH2N[CH2PO(OH)2]2; diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), (HO)2POCH2N[CH2CH2N[CH2PO(OH)2]2]2; diethylenetriaminepenta(methylenephosphonate) sodium salt, C9H (28-x) N3Na x O 15 P5 (x = 7); Hexamethylenediamine (tetramethylenephosphonic acid) potassium salt, C 10 H (28-x) N2K x O 12 P4 (x = 6); and bis(hexamethylene)triamine (pentamethylenephosphonic acid), (HO2)POCH2N[(CH2)2N[CH2PO(OH)2]2]2. Salts thereof may also be suitable.

[0774] The detergent compositions of the present invention may comprise one or more aminocarboxylates. Non-limiting examples of suitable "aminocarboxylates" include, but are not limited to, diethanolglycine (DEG), dimethylglycine (DMG), nitrilotriacetic acid (NTA), N-hydroxyethylaminodiacetic acid, ethylenediaminetetraacetic acid (EDTA), N-(2-hydroxyethyl)iminodiacetic acid (HEIDA), hydroxyethylenediaminetriacetic acid, N-hydroxyethyl-ethylenediaminetriacetic acid (HEDTA), hydroxyethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid (DTPA) and methylglycinediacetic acid (MGDA), glutamic acid-diacetic acid (GLDA), iminodisuccinic acid (IDS), hydroxyiminodisuccinic acid, ethylenediaminedisuccinic acid (EDDS), aspartic acid-diacetic acid and their alkali metal or ammonium salts. Also suitable are aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamate (SMGL), N-(2-sulfoethyl)glutamate (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), para-aminobenzenesulfonic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and sulfomethyl-N,N-diacetic acid (SMDA) and their alkali metal or ammonium salts. Preferred are MGDA or EDDS. The term "ammonium salt" as used in this context refers to a salt having at least one cation carrying a nitrogen atom that is permanently or temporarily quaternized. Examples of cations carrying at least one nitrogen atom that is permanently quaternized include tetramethylammonium, tetraethylammonium, dimethyldiethylammonium and nC 10 -C 20 -alkyltrimethylammonium. Examples of cations carrying at least one nitrogen atom which is temporarily quaternized include protonated amines and ammonia, such as monomethylammonium, dimethylammonium, trimethylammonium, monoethylammonium, diethylammonium, triethylammonium, nC 10 -C 20 -alkyldimethylammonium 2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium, tris(2-hydroxyethyl)ammonium, N-methyl 2-hydroxyethylammonium, N,N-dimethyl-2-hydroxyethylammonium, and especially NH4 + .

[0775] In one embodiment, the detergent composition of the present invention comprises more than one builder. Preferably, the detergent composition of the present invention contains less than 0.2% by weight of nitrilotriacetic acid (NTA), or 0.01% to 0.1% by weight of NTA relative to the total weight of the detergent composition.

[0776] In one embodiment, the detergent composition of the present invention comprises at least one aminocarboxylate selected from methylglycine diacetate (MGDA), glutamic acid diacetate (GLDA) and corresponding salts thereof, for example, alkali metal (e.g., sodium) salts thereof, in an amount in the range of 0.1% to 25.0% by weight, in the range of 1.0% to 18.0% by weight, in the range of 3.0% to 15.0% by weight, in the range of 3.0% to 10.0% by weight, or in the range of 5.0% to 8.0% by weight, relative to the total weight of the detergent composition.

[0777] The detergent composition of the present invention may include one or more hydrotropes. One or more hydrotropes may be selected from organic solvents such as ethanol, isopropyl alcohol, ethylene glycol, 1,2-propylene glycol, and other organic solvents known in the art that are miscible with water under normal conditions, but are not limited thereto. In one embodiment, the detergent composition of the present invention includes 1,2-propylene glycol in an amount of 5% to 10% by weight, preferably about 6% by weight, all relative to the gross weight of the detergent composition. Other non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium isopropylbenzenesulfonate (SCS), sodium cymenesulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0778] In one embodiment, detergent composition includes at least one preservative. Preferably, preservative means a substance added to the liquid composition for antiseptic purposes, meaning that more preferably, the known compound with antiseptic characteristics contained in the liquid composition formed during the production process is excluded from the term preservative. In one embodiment, preservative is selected from the group consisting of: 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol, formic acid (in acid form or its salt form) and 4,4'-dichloro-2-hydroxydiphenyl ether. Typically, the liquid composition of the present invention includes at least one preservative, and its amount is less than 10ppm, such as relative to the gross weight of the liquid composition, and the scope of its amount is 2ppm to 5% by weight. Preferably, the liquid composition does not contain preservative, which means that the content of the preservative is less than 1ppm.

[0779] In one embodiment, the detergent composition comprising a protease variant as described herein further comprises one or more second enzymes different from the protease variant. Preferably, the second enzyme is selected from the group consisting of an amylase, a second protease, a lipase, a cellulase, a mannanase, a hemicellulase, a phospholipase, an esterase, a pectinase, a lactase, a peroxidase, a xylanase, a cutinase, a pectate lyase, a keratinase, a reductase, an oxidase, a phenoloxidase, a lipoxygenase, a ligninase, a pullulanase, a tannase, a pentosanase, a maranase, a β-glucanase, an arabinosidase, a hyaluronidase, a chondroitinase, a laccase, a nuclease, , DNA enzyme, phosphodiesterase, phytase, carbohydrase, galactanase, xanthanase, xyloglucanase, oxidoreductase, perhydrolase, aminopeptidase, asparaginase, carbohydrase, carboxypeptidase, catalase, chitinase, cyclodextrin glycosyltransferase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β-glucosidase, invertase, ribonuclease, transglutaminase and dispersin, and a combination of at least two of the foregoing types. More preferably, the second enzyme is selected from the group consisting of amylase, lipase, cellulase, mannanase, xylanase, DNA enzyme, dispersin, pectinase, oxidoreductase and cutinase, and a combination of at least two of the foregoing types. Most preferably, the second enzyme is an amylase, preferably an α-amylase.

[0780] Particularly preferred additional enzymes are disclosed elsewhere herein, and that description is also incorporated by reference into this part of the specification.

[0781] The composition of the invention may comprise more than one enzyme of different types (eg an amylase and a protease), or more than one enzyme of the same type (eg two or more different proteases) or a mixture thereof (eg an amylase and two different proteases).

[0782] The detergent composition may comprise a water-soluble source of calcium and / or magnesium ions. In one embodiment, the detergent composition comprises an enzyme stabilization system as described herein. Preferably, in particular in the case of liquid detergent compositions, the detergent composition may comprise at least one protease inhibitor as described herein, preferably selected from boronic acid derivatives (preferably 4-FPBA) and peptide aldehydes (preferably Z-VAL-H or Z-GAY-H). Preferably, the detergent composition does not contain boron.

[0783] In one embodiment, the present invention is directed to a method of providing a detergent composition, preferably a liquid detergent composition, more preferably a liquid laundry detergent composition, comprising the steps of combining in one or more steps:

[0784] (a) at least one protease variant according to the present invention, preferably wherein the protease is provided within a protease variant formulation as described herein; and

[0785] (b) at least one detergent component, preferably selected from surfactants, builders, polymers, preservatives, and a second enzyme different from the protease variant, present in an amount effective for cleaning performance and / or maintaining physical properties of the detergent.

[0786] In one embodiment, the present invention relates to a detergent composition comprising

[0787] a) a protease variant as described herein;

[0788] b) one or more surfactants, preferably in a concentration of 0.2% to 65%, preferably 0.2% to 40%,

[0789] c) one or more builders, preferably at a concentration of 0.01% to 25%, and

[0790] d) optionally one or more additional compounds selected from the group consisting of: additional enzymes different from the protease in a), defoamers, polymers, bleaching systems (bleaches), rheology modifiers, hydrotropes, softeners, drying agents, brighteners, buffers, preservatives, anti-corrosion additives, dyes and fragrances; preferably wherein the detergent composition is a liquid, powder, sachet or capsule detergent composition.

[0791] Preferably, the detergent composition, preferably a powder detergent composition, of the present invention comprises, in addition to the protease variant as described herein, one or more compounds selected from the group consisting of: alcohol ethoxylate 7EO, coconut fatty acid C 12-18 、C 12 -C 14 - fatty alcohol ether sulfate (1-3EO, preferably 2EO), linear alkylbenzene sulfonic acid, sodium acetate, sodium citrate, sodium silicate, sodium carbonate, sodium phosphate, sodium bicarbonate, zeolite 4A, HEDP, MGDA, sodium sulfate, sodium chloride, optical brighteners, and polymers, and optionally bleach activators and percarbonates.

[0792] Preferably, the detergent composition, preferably a powder detergent composition, of the present invention comprises, in addition to a protease variant as described herein,

[0793] b) one or more surfactants selected from the group consisting of: alcohol ethoxylate 7EO, coconut fatty acid C 12-18 、C 12 -C 14- fatty alcohol ether sulfate (1-3EO, preferably 2EO), linear alkylbenzene sulfonic acid, preferably, in a concentration of 0.2%-65%,

[0794] c) one or more builders selected from the group consisting of HEDP, MGDA, GLDA and DTPMP, preferably in a concentration of 0.01% to 25%, and

[0795] d) one or more compounds selected from the group consisting of sodium acetate, sodium citrate, sodium silicate, sodium carbonate, sodium phosphate, sodium bicarbonate, zeolite 4A, sodium sulfate, sodium chloride, optical brighteners, and polymers, and optionally bleach activators and percarbonates.

[0796] Preferably, the detergent composition, preferably a liquid detergent composition, of the present invention comprises, in addition to the protease variant as described herein, one or more compounds selected from the group consisting of: alcohol ethoxylate 7EO, coconut fatty acid C 12-18 、C 12 -C 14 - fatty alcohol ether sulfate (1-3EO, preferably 2EO), linear alkylbenzenesulfonic acid, sulfonic acid, 1,2-propylene glycol, triethanolamine, monoethanolamine, NaOH, glycerol, ethanol, sodium citrate and polymers.

[0797] Preferably, the detergent compositions, preferably liquid detergent compositions, of the present invention comprise, in addition to the protease variant as described herein,

[0798] b) one or more surfactants selected from the group consisting of: alcohol ethoxylate 7EO, coconut fatty acid C 12-18 、C 12 -C 14 - fatty alcohol ether sulfate (1-3EO, preferably 2EO), linear alkylbenzene sulfonic acid, preferably, in a concentration of 0.2%-65%,

[0799] c) one or more builders selected from the group consisting of HEDP, MGDA, GLDA and DTPMP, preferably in a concentration of 0.01% to 25%, and

[0800] d) one or more compounds selected from the group consisting of sulfonic acid, 1,2 propylene glycol, triethanolamine, monoethanolamine, NaOH, glycerol, ethanol, sodium citrate, and polymers.

[0801] Preferred formulations

[0802] In one embodiment, the protease variants described herein are included in a formulation comprising one or more, preferably all, compounds selected from the group consisting of (all percentages are w / w):

[0803] 0.05% to 0.2% of a protease variant described herein;

[0804] Anionic detersive surfactants (e.g., alkylbenzene sulfonates, alkyl ethoxylated sulfates, and mixtures), 8% to 15%;

[0805] Nonionic detersive surfactant (e.g., alkyl ethoxylated alcohol), 0.5% to 4%;

[0806] Cationic detersive surfactants (e.g., quaternary ammonium compounds), 0 to 4%;

[0807] Other detersive surfactants (e.g., zwitterionic detersive surfactants, amphoteric surfactants, and mixtures thereof), 0% to 4%;

[0808] Carboxylate polymers (e.g., copolymers of maleic acid and acrylic acid), 1% to 4%;

[0809] polyethylene glycol polymers (e.g., polyethylene glycol polymers containing polyvinyl acetate side chains), 0.5% to 4%;

[0810] Polyester soil release polymers (such as Repel-o-tex and / or Texcare polymers), 0.1% to 2%;

[0811] Cellulosic polymers (e.g., carboxymethylcellulose, methylcellulose, and combinations thereof), 0.5% to 2%;

[0812] other polymers (e.g., amine polymers, dye transfer inhibitor polymers, hexamethylenediamine derivative polymers, and mixtures thereof), 0% to 4%;

[0813] Zeolite builders and phosphate builders (e.g., zeolite 4A and / or sodium tripolyphosphate), 0% to 4% by weight;

[0814] Other builders (such as sodium citrate and / or citric acid), 0% to 3%;

[0815] Carbonates (e.g., sodium carbonate and / or sodium bicarbonate), 15% to 30%;

[0816] Silicates (such as sodium silicate), 0% to 10%;

[0817] Fillers (e.g., sodium sulfate and / or biofillers), 10% to 40%;

[0818] Available oxygen sources (such as sodium percarbonate), 10% to 20%;

[0819] bleach activators (e.g., tetraacetylethylenediamine (TAED) and / or nonanoyloxybenzenesulfonate (NOBS)), 2% to 8%;

[0820] bleach catalysts (e.g. quaternary oxaziridinium-based bleach catalysts and / or transition metal bleach catalysts), 0% to 0.1%;

[0821] Other bleaching agents (e.g., reducing bleach and / or preformed peracids), 0% to 10%;

[0822] Chelating agents (e.g., ethylenediamine-N'N'-disuccinic acid (EDDS) and / or hydroxyethanediphosphonic acid (HEDP)), 0.2% to 1%;

[0823] Photobleach (e.g., sulfonated zinc phthalocyanine and / or sulfonated aluminum phthalocyanine), 0% to 0.1%;

[0824] Colorants (e.g., Direct Violet 99, Acid Red 52, Acid Blue 80, Direct Violet 9, Solvent Violet 13, and any combination thereof), 0% to 1%;

[0825] Brightener (e.g., Brightener 15 and / or Brightener 49), 0.1% to 0.4%;

[0826] fabric softeners (e.g., montmorillonite clay and / or polydimethylsiloxane (PDMS)), 0% to 4%;

[0827] Flocculants (such as polyethylene oxide), 0% to 1%;

[0828] Foam suppressors (e.g., silicones and / or fatty acids), 0% to 0.1%;

[0829] Fragrance (e.g., fragrance microcapsules, spray fragrance, starch encapsulated fragrance notes, fragrance-loaded zeolites, and any combination thereof), 0.1% to 1%; and

[0830] Aesthetic agents (e.g., colored soap rings and / or colored spots / strips), 0% to 1%; and

[0831] Optionally, an additional protease other than the protease variants described herein (e.g., Savinase, Coronase, Ovozyme, Kannase, Liquanase, Polarzyme, Purafect, Purafast, Properase, Excellase, FN3, FN4, Effectenz P, Preferenz P, Progress Uno, Progress Excel, Blaze, Excellenz P), about 0.05 wt % to about 0.2 wt %;

[0832] Optionally, an amylase (e.g., Termamyl(R), Termamyl Ultra(R), Natalase(R), OptisizeHT Plus(R), Purastar, Powerase(R), Stainzyme(R), Preferenz S, Effectenz S, Amplify, Amplify Prime, Achieve alpha, Excellenz S, and any combination thereof), about 0.05 wt % to about 0.2 wt %;

[0833] Optionally, cellulase (e.g., Carezyme, Celluclean, Puradax, Biotouch, Whitezyme, Revitalenz, and combinations thereof), 0.05% to 0.2%;

[0834] Optionally, lipase (e.g., Lipex, Lipolex, Lipoclean, Preferenz L, and any combination thereof), 0.05% to 0.2%;

[0835] Optionally, other enzymes (e.g., xyloglucanases, cutinases, pectate lyases (e.g., Xpect), mannanases (e.g., Mannanway, Mannastar, Marvellenz, Effectenz M, Preferenz M, Preferenz F, and combinations thereof) bleaching enzymes, and combinations thereof), 0.05% to 0.2%;

[0836] Other margin.

[0837] In another embodiment, the protease variants described herein are included in a formulation comprising one or more, preferably all, compounds selected from the group consisting of (all percentages are w / w):

[0838] 0.05% to 0.2% of a protease variant described herein;

[0839] A carboxyl group-containing polymer (comprising about 60% to about 70% by mass of an acrylic acid-based monomer (A); and about 30% to about 40% by mass of a sulfonic acid group-containing monomer (B); and wherein the average molecular weight is about 23,000 to about 50,000, preferably in the range of about 25,000 to about 38,000 (as described in WO 2014032269)), about 0.5 wt% to about 1.5 wt%;

[0840] Anionic detersive surfactant (e.g., alkylbenzene sulfonates, alkyl ethoxylated sulfates, and mixtures thereof), about 8 wt% to about 15 wt%;

[0841] Nonionic detersive surfactant (e.g., alkyl ethoxylated alcohol), about 0.5 wt% to 4 wt%;

[0842] Cationic detersive surfactant (e.g., quaternary ammonium compound), about 0 wt % to about 4 wt %;

[0843] Other detersive surfactants (e.g., zwitterionic detersive surfactants, amphoteric surfactants, and mixtures thereof), about 0 wt % to 4 wt %;

[0844] Carboxylate polymer (e.g., copolymer of maleic acid and acrylic acid), about 1 wt% to about 4 wt%;

[0845] Polyethylene glycol polymer (e.g., polyethylene glycol polymer comprising polyvinyl acetate side chains), about 0 wt % to about 4 wt %;

[0846] Polyester soil release polymer (e.g., Repel-O-Tex(R) and / or Texcare(R) polymer), about 0.1 wt % to about 2 wt %;

[0847] Cellulosic polymers (e.g., carboxymethyl cellulose, methyl cellulose, and combinations thereof), from about 0.5 wt % to about 2 wt %;

[0848] Other polymers (e.g., amine polymers, dye transfer inhibitor polymers, hexamethylenediamine derivative polymers, and mixtures thereof), from about 0 wt % to about 4 wt %;

[0849] Zeolite builder and phosphate builder (e.g., zeolite 4A and / or sodium tripolyphosphate), from about 0 wt% to about 4 wt%;

[0850] Other builders (such as sodium citrate and / or citric acid), from about 0 wt % to about 3 wt %;

[0851] Carbonate (such as sodium carbonate and / or sodium bicarbonate), about 15 wt% to about 30 wt%;

[0852] Silicate (e.g., sodium silicate), from about 0 wt % to about 10 wt %;

[0853] Filler (such as sodium sulfate and / or biological filler), about 10 wt % to about 40 wt %;

[0854] An oxygen source (such as sodium percarbonate) can be used, from about 10 wt% to about 20 wt%;

[0855] bleach activators (e.g., tetraacetylethylenediamine (TAED) and / or nonanoyloxybenzenesulfonate (NOBS)), from about 2 wt% to about 8 wt%;

[0856] bleach catalyst (e.g., quaternary oxaziridinium-based bleach catalyst and / or transition metal bleach catalyst), from about 0 wt% to about 0.1 wt%;

[0857] Other bleaching agents (e.g., reducing bleach and / or preformed peracids), from about 0 wt % to about 10 wt %;

[0858] Chelating agents (e.g., ethylenediamine-N'N'-disuccinic acid (EDDS) and / or hydroxyethanediphosphonic acid (HEDP)), about 0.2 wt% to about 1 wt%;

[0859] Photobleach (e.g., sulfonated zinc phthalocyanine and / or sulfonated aluminum phthalocyanine), from about 0 wt% to about 0.1 wt%;

[0860] toner (e.g., Direct Violet 99, Acid Red 52, Acid Blue 80, Direct Violet 9, Solvent Violet 13, and any combination thereof), from about 0 wt% to about 0.5 wt%;

[0861] Brightener (e.g., Brightener 15 and / or Brightener 49), about 0.1 wt% to about 0.4 wt%;

[0862] Fabric softener (e.g., montmorillonite clay and / or polydimethylsiloxane (PDMS)), 0 wt% to 15 wt%;

[0863] Flocculant (such as polyethylene oxide), 0wt% to 1wt%;

[0864] Foam inhibitors (e.g., silicones and / or fatty acids), 0 wt% to 0.1 wt%;

[0865] Fragrance (e.g., fragrance microcapsules, spray fragrance, starch encapsulated fragrance notes, fragrance-loaded zeolites, and any combination thereof), 0.1 wt% to 1 wt%; and

[0866] Aesthetic agents (e.g., colored soap rings and / or colored spots / strips), 0 wt% to 1 wt%; and

[0867] Optionally, an additional protease different from the protease variants described herein (e.g., Savinase, Coronase, Ovozyme, Kannase, Liquanase, Polarzyme, Purafect, Purafast, Properase, Excellase, FN3, FN4, Effectenz P, Preferenz P, Progress Uno, Progress Excel, Blaze, Excellenz P), about 0.05 wt % to about 0.2 wt %,

[0868] Optionally, an amylase (e.g., Termamyl(R), Termamyl Ultra(R), Natalase(R), OptisizeHT Plus(R), Purastar, Powerase(R), Stainzyme(R), Preferenz S, Effectenz S, Amplify, Amplify Prime, Achieve alpha, Excellenz S), about 0.05 wt % to about 0.2 wt %,

[0869] Optionally, cellulase (e.g., Carezyme(R), Celluzyme(R), Puradax, Celluclean(R), Biotouch, Whitezyme, Revitalenz, and combinations thereof, typically having an enzyme activity of about 10 to 50 mg active enzyme / g), about 0.05 wt% to 0.5 wt%

[0870] Optionally, lipase (e.g., Lipex(R), Lipolex(R), Lipoclean(R), Preferenz L, and any combination thereof, typically having an enzyme activity of about 10 mg to about 50 mg active enzyme / g), about 0.2 wt% to about 1 wt%

[0871] Optionally, other enzymes (e.g., xyloglucanases (e.g., Whitezyme(R)), cutinases, pectate lyases (e.g., Xpect), mannanases (e.g., Mannanway, Mannastar, Marvellenz, Effectenz M, Preferenz M, Preferenz F, and combinations thereof), bleaching enzymes, typically having an enzyme activity of about 10 mg to about 50 mg active enzyme / g), 0 wt % to 2 wt %,

[0872] Other margin.

[0873] Further preferred detergent formulations comprise the components listed below (all percentages are w / w):

[0874] - Water, alcohol ethoxysulfate, alcohol ethoxylate, amino oxide, citric acid, C12-18 topped palm kernel fatty acids, amylase, glycosidase, ethanol, 1,2 propylene glycol, sodium formate, calcium chloride, sodium hydroxide, silicone emulsion, trans-sulfated EHDQ, a protease variant as described herein;

[0875] - sodium linear alkylbenzene sulfonate 8.8%, ethoxylated fatty alcohol C12-18 (7EO) 4.7%, sodium soap 3.2%, defoamer DC2-4248S 3.9%, sodium aluminosilicate zeolite 4A 28.3%, sodium carbonate 11.6%, sodium salt of acrylic acid and maleic acid copolymer (Sokalan CP5) 2.4%, sodium silicate 3.0%, carboxymethyl cellulose 1.2%, Dequest 2066 2.8%, optical brightener 0.2%, sodium sulfate 6.5%, amylase 0.4%, a protease variant as described herein;

[0876] -12% LAS, 11% AEO Biosoft N25-7 (NI), 7% AEOS (SLES), 6% MPG (monopropylene glycol), 3% ethanol, 3% TEA, 2.75% cocoa soap, 2.75% soy soap, 2% glycerin, 2% sodium hydroxide, 2% sodium citrate, 1% sodium formate, 0.2% DTM PA and 0.2% PCA, a protease variant as described herein;

[0877] -5%-15% anionic surfactants; <5% nonionic surfactants, phosphonates, soaps; enzymes, optical brighteners, benzisothiazolinone, methylisothiazolinone, fragrances, α-isomethylionone, citronellol, geraniol, linalool, protease variants as described herein;

[0878] - Water, Sodium Dodecylbenzenesulfonate, C14-C15 Pareth-7, Sodium Citrate, Propylene Glycol, Sodium Palm Kernel Oleate, Sodium Laureth Sulfate, MEA Dodecylbenzenesulfonate, Quaternized Sulfated Ethoxylated Hexamethylenediamine, Sodium Cumenesulfonate, Fragrance, PEG / Vinyl Acetate Copolymer, Sodium Formate, Hydrogenated Castor Oil, Sodium Diethylenetriamine Pentamethylenephosphonate, PEG / PPG-10 / 2 Propylheptyl Ether, Butylphenyl Methylpropional, Polyvinylpyridine-N-oxide, Sorbitol, Glycerin, Ethanolamine, Sodium Hydroxide, α-Isomethyl Ionone, Amylase, Calcium Chloride, Geraniol, Linalool, Citronellol, Tripropylene Glycol, Glycosidase, Benzisothiazolinone, Dimethicone, Glycosidase, Sodium Acetate, Cellulase, Colorant, Glyceryl Stearate, Hydroxyethylcellulose, Silica, Protease Variants as Described herein;

[0879] - Water, sodium laureth sulfate, propylene glycol, C14-C15 pareth-7, sodium citrate, sodium palm kernel oleate, ethanol, sodium formate, quaternized sulfated ethoxylated hexamethylenediamine, sodium hydroxide, fragrance, polyvinylpyridine-N-oxide, sorbitol, calcium chloride, amylase, glycerol, glucosidase, glycosidase, sodium acetate, colorant, cellulase, protease variants as described herein;

[0880] - Water, sodium laureth sulfate, propylene glycol, C14-C15 pareth-7, sodium citrate, sodium palm kernel oleate, ethanol, sodium formate, quaternized sulfated ethoxylated hexamethylenediamine, sodium hydroxide, fragrance, sorbitol, calcium chloride, amylase, glycerin, glucosidase, glycosidase, sodium acetate, colorant, cellulase, protease variants as described herein;

[0881] - Water, sodium laureth sulfate, propylene glycol, C14-C15 pareth-7, sodium citrate, sodium palm kernel oleate, ethanol, sodium formate, quaternized sulfated ethoxylated hexamethylenediamine, sodium hydroxide, sorbitol, calcium chloride, amylase, glycerin, glycosidase, sodium acetate, cellulase, silica, protease variants as described herein;

[0882] -Water, sodium dodecylbenzenesulfonate, C 14 -C 15 Pareth-7, sodium citrate, propylene glycol, sodium palm kernel oleate, sodium laureth sulfate, MEA dodecylbenzene sulfonate, quaternized sulfated ethoxylated hexamethylene diamine, sodium cumene sulfonate, fragrance, PEG / vinyl acetate copolymer, sodium formate, C12-C14 pareth-7, hydrogenated castor oil, sodium diethylenetriamine pentamethylethylene phosphonate, PEG / PPG-10 / 2 propylheptyl ether, butylphenyl methylpropional, fluorescent brightener, sorbitol, glycerin, ethanolamine, sodium hydroxide, alpha-isomethyl ionone, amylase, calcium chloride, geraniol, linalool, citronellol, tripropylene glycol, sodium chloride, glycosidase, benzisothiazolinone, dimethicone, glycosidase, sodium acetate, cellulase, colorant, glyceryl stearate, hydroxyethylcellulose, silica, a protease variant as described herein;

[0883] -15%-30% anionic surfactants, nonionic surfactants, 5%-15% soap, <5% polycarboxylates, fragrances, phosphates, optical brighteners, protease variants as described herein;

[0884] -15%-30% anionic surfactant, 5%-15% nonionic surfactant, soap, benzisothiazolinone, methylisothiazolinone, fragrance, protease variant as described herein;

[0885] - 11% LAS, 2% AS / AEOS, 2% soap, 3% AEO, 15.15% sodium carbonate, 3% sodium silicate, 18.75% zeolite, 0.15% chelating agent, 2% sodium citrate, 1.65% AA / MA copolymer, 2.5% CMC and 0.5% SRP, a protease variant as described herein;

[0886] - 16.5% LAS, 15% zeolite, 12% sodium disilicate, 20% sodium carbonate, 1% sokalan, 35.5% sodium sulfate, a protease variant as described herein;

[0887] -15%-30% anionic surfactants, <5% nonionic surfactants, phosphonates, polycarboxylates, zeolites; enzymes, fragrances, hexylcinnamaldehyde, protease variants as described herein;

[0888] -15%-30% of the following: anionic surfactants, oxygen-based bleach and zeolite, less than 5% of the following: nonionic surfactants, phosphonates, polycarboxylates, soap, other ingredients: fragrance, hexyl cinnamaldehyde, benzyl salicylate, linalool, optical brighteners, enzymes and citronellol, protease variants as described herein;

[0889] - Water, Alcohol Ethoxysulfate, Diethylene Glycol, Alcohol Ethoxylate, Ethanolamine, Linear Alkylbenzene Sulfonate, Sodium Fatty Acid, Polyethyleneimine Ethoxylate, Citric Acid, Sodium Cumene Sulfonate, Propylene Glycol, DTPA, Disodium Diaminobenzene Disulfonate, Dipropyl Ethyltetramine, Sodium Hydroxide, Sodium Formate, Calcium Formate, Polydimethylsiloxane, Amylase, Liquitint TM , hydrogenated castor oil, fragrance, a protease variant as described herein;

[0890] -Linear Alkylbenzene Sulfonate, Propylene Glycol, Citric Acid, Sodium Hydroxide, Ethanolamine, Ethanol, Alcohol Sulfate, Polyethyleneimine Ethoxylate, Sodium Fatty Acid, Diquaternium Ethoxysulfate, Amylase, Diethylene Glycol, Laureth-9, Alkyl Dimethylamine Oxide, Fragrance, Disodium Diaminostilbene Disulfonate, DTPA, Sodium Formate, Calcium Formate, Polyethylene Glycol 4000, Mannanase, Liquitint TM Blue, polydimethylsiloxane, a protease variant as described herein;

[0891] - Water, sodium alcohol ethoxysulfate, propylene glycol, ethanol, linear alkylbenzenesulfonate sodium salt, polyethyleneimine ethoxylate, diethylene glycol, trans-sulfated and ethoxylated hexamethylenediamine, alcohol ethoxylate, linear alkylbenzenesulfonate MEA salt, sodium formate, sodium alkyl sulfate, DTPA, amine oxide, calcium formate, disodium diaminostilbene disulfonate, amylase, polydimethylsiloxane, benzisothiazolinone, protease variants as described herein;

[0892] - Water, Alcohol Ethoxysulfate, Linear Alkylbenzene Sulfonate, Diethylene Glycol, Propylene Glycol, Ethanolamine, Citric Acid, Alcohol Sulfate, Sodium Hydroxide, Polyethyleneimine Ethoxylate, Sodium Fatty Acid, Ethanol, Amylase, Laureth-9, Diquaternium Ethoxysulfate, Lauramine Oxide, Sodium Cumene Sulfonate, Fragrance, DTPA, Disodium Diaminostilbene Disulfonate, Sodium Formate, Disodium Distyryl Biphenyl Disulfonate, Calcium Formate, Polyethylene Glycol 4000, Mannanase, Pectinase, Liquitint TM Blue, polydimethylsiloxane, a protease variant as described herein;

[0893] - Water, Alcohol Ethoxysulfate, Propylene Glycol, Sodium Fatty Acid, Lauryl Trimethylammonium Chloride, Ethanol, Sodium Hydroxide, Sodium Cumene Sulfonate, Citric Acid, Ethanolamine, Diethylene Glycol, Silicone Polyether, Fragrance, Polyethyleneimine Ethoxylate, Amylase, Laureth-9, DTPA, Polyacrylamide Quaternary Ammonium Chloride, Disodium Diaminostilbene Disulfonate, Sodium Formate, Liquitint TM Orange, dipropylethyltetramine, polydimethylsiloxane, cellulase, a protease variant as described herein;

[0894] - Water, Sodium Alcohol Ethoxysulfate, Sodium Alkyl Sulfate, MEA Citrate, Linear Alkylbenzene Sulfonate MEA Salt, Propylene Glycol, Diethylene Glycol, Polyethyleneimine Ethoxylate, Ethanol, Sodium Fatty Acid, Ethanolamine, Lauramine Oxide, Laureth-9, DTPA, Sodium Cumene Sulfonate, Sodium Formate, Calcium Formate, Linear Alkylbenzene Sulfonate Sodium Salt, Alcohol Sulfate, Sodium Hydroxide, Diquaternium Ethoxysulfate, Fragrance, Amylase, Mannanase, Pectinase, Disodium Diaminostilbene Disulfonate, Benzisothiazolinone, Liquitint TM Blue, polydimethylsiloxane, dipropylethyltetramine, a protease variant as described herein;

[0895] - Water, Sodium Alcohol Ethoxysulfate, MEA Citrate, Sodium Alkyl Sulfate, Alcohol Ethoxylate, Linear Alkylbenzene Sulfonate MEA Salt, Sodium Fatty Acid, Polyethyleneimine Ethoxylate, Diethylene Glycol, Propylene Glycol, Diquaternium Ethoxysulfate, Polyethyleneimine, Ethoxylate Propoxylate, Ethanol, Sodium Cumene Sulfonate, Fragrance, DTPA, Disodium Diaminostilbene Disulfonate, Mannanase, Cellulase, Sodium Formate, Calcium Formate, Lauramine Oxide, Liquitint TM Blue, polydimethylsiloxane / polydimethyl silicone, protease variants as described herein;

[0896] - Water, Alcohol Ethoxysulfate, Linear Alkylbenzene Sulfonate, Alcohol Ethoxylate, Citric Acid, Ethanolamine, Sodium Fatty Acid, Diethylene Glycol, Propylene Glycol, Sodium Hydroxide, Polyethyleneimine Ethoxylate, Silicone Polyether, Alcohol, Amylase, Sodium Cumene Sulfonate, Diquaternium Ethoxysulfate, Laureth-9, Fragrance, DTPA, Disodium Diaminostilbene Disulfonate, Disodium Distyryl Biphenyl Disulfonate, Sodium Formate, Calcium Formate, Mannanase, Liquitint TM Orange, polydimethylsiloxane, polyacrylamide quaternary ammonium chloride, cellulase, dipropylethyltetramine, a protease variant as described herein;

[0897] - Water, Alcohol Ethoxysulfate, Diethylene Glycol, Monoethanolamine Citrate,

[0898] Sodium formate, propylene glycol, linear alkylbenzene sulfonate, ethanolamine, ethanol, polyethyleneimine ethoxylate, amylase, benzisothiazoline, calcium formate, citric acid, sodium diethylenetriamine pentaacetate, polydimethylsiloxane, diquaternium ethoxysulfate, disodium diaminostilbene disulfonate, laureth-9, mannanase, sodium cumene sulfonate, sodium fatty acid, a protease variant as described herein;

[0899] - Water, Alcohol Ethoxysulfate, MEA Citrate, Alcohol Sulfate, Alcohol Ethoxylate, Linear Alkylbenzene Sulfonic Acid MEA Salt, Sodium Fatty Acid, Polyethyleneimine Ethoxylate, Diethylene Glycol, Propylene Glycol, Diquaternium Ethoxysulfate, Polyethyleneimine Ethoxylate Propoxylate, Ethanol, Sodium Cumene Sulfonate, Fragrance, DTPA, Disodium Diaminostilbene Disulfonate, Mannanase, Cellulase, Amylase, Sodium Formate, Calcium Formate, Lauramine Oxide, Liquitint TM Blue, polydimethylsiloxane, a protease variant as described herein;

[0900] - Water, sodium alcohol ethoxysulfate, MEA citrate, linear alkylbenzene sulfonate: sodium salt, alcohol ethoxylate, linear alkylbenzene sulfonate: MEA salt, sodium fatty acid, polyethyleneimine ethoxylate, diethylene glycol, propylene glycol, diquaternary ammonium ethoxysulfate, amylase, polyethyleneimine ethoxylate propoxylate, ethanol, sodium cumene sulfonate, citric acid, DTPA, disodium diaminostilbene disulfonate, sodium formate, calcium formate, polydimethylsiloxane, a protease variant as described herein;

[0901] - Water, Alcohol Ethoxylate Sulfate, Sodium Linear Alkylbenzene Sulfonate / Mea Salt, Propylene Glycol, Diethylene Glycol, Sodium Formate, Ethanol, Sodium Fatty Acid, Fragrance, Laurylamine Oxide, DTPA, Polyvinylamine Ethoxylate, Calcium Formate, Disodium Diaminostilbene Disulfonate, Polydimethylsiloxane, Tetramine, Liquitint TM Blue, a protease variant as described herein;

[0902] -Linear alkylbenzene sulfonate, C 12-16 Pareth-9, propylene glycol, alcohol ethoxysulfate, water, polyethyleneimine ethoxylate, glycerin, soaps, PEG-136 polyvinyl acetate, ethylenediamine disuccinate, monoethanolamine citrate, sodium bisulfite, sodium diethylenetriamine pentaacetate, disodium distyrylbiphenyl disulfonate, calcium formate, mannanase, xyloglucanase, sodium formate, hydrogenated castor oil, natalase, dye, termamyl, subtilisin, benzisothiazoline, fragrance, protease variants as described herein;

[0903] - Deionized water, dipropylene glycol butyl ether, sodium alkyl sulfate, hydrogen peroxide, ethanol, magnesium sulfate, alkyl dimethylamine oxide, citric acid, sodium hydroxide, trimethoxybenzoic acid, fragrance, a protease variant as described herein;

[0904] - Water, alkyl ethoxylate, linear alkylbenzene sulfonate, hydrogen peroxide, diquaternium ethoxysulfate, ethanolamine, disodium distyrylbiphenyl disulfonate, tetrabutylethylenebisphenol, F&DC Yellow 3, fragrance, protease variants as described herein;

[0905] - sodium percarbonate, sodium sulfate, sodium carbonate, sodium aluminosilicate, nonanoyloxybenzenesulfonate, sodium polyacrylate, water, sodium alkylbenzenesulfonate, DTPA, polyethylene glycol, sodium palmitate, amylase, modified starch, FD&C Blue 1, flavor, protease variants as described herein;

[0906] - Water, Alkyl Ethoxylate, MEA Borate, Linear Alkylbenzene Sulfonate, Propylene Glycol, Diquaternium Ethoxysulfate, Calcium Chlorideenzyme, Ethanolamine, Benzisothiazolinone, Amylase, Sodium Citrate, Sodium Hydroxide, Fragrance, Protease Variants as Described herein;

[0907] - Water, alkylamine oxide, dipropylene glycol phenyl ether, hydrogen peroxide, citric acid, ethylenediamine disuccinate sodium salt, sodium alkyl sulfate, flavor, an amylase having at least 91% sequence identity to SEQ ID NO: 1, a protease variant as described herein;

[0908] - sodium bicarbonate, sodium carbonate, sodium percarbonate, alcohol ethoxylate, sodium chloride, maleic acid / acrylic acid copolymer, nonanoyloxybenzene sulfonate, sodium sulfate, colorant, diethylenetriamine pentaacetic acid sodium salt, hydrated aluminum silicate (zeolite), polyethylene glycol, sodium alkylbenzene sulfonate, sodium palmitate, starch, water, flavor, protease variants as described herein;

[0909] -Polyvinyl alcohol bag film, which contains liquid part and powder part: Liquid ingredients: dipropylene glycol, diquaternium ethoxysulfate, water, glycerin, Liquitint TM Orange, a protease variant as described herein;

[0910] - Powder ingredients: sodium percarbonate, nonanoyloxybenzenesulfonate, sodium carbonate, sodium sulfate, sodium aluminosilicate, sodium polyacrylate, sodium alkylbenzenesulfonate, maleic acid / acrylic acid copolymer, water, amylase, polyethylene glycol, sodium palmitate, modified starch, glycerin, DTPA, fragrance, protease variants as described herein;

[0911] - Water, Sodium Alcohol Ethoxysulfate, Sodium Linear Alkylbenzene Sulfonate / MEA Salt, MEA Citrate, Propylene Glycol, Polyethyleneimine Ethoxylate, Ethanol, Diethylene Glycol, Polyethyleneimine Propoxyethoxylate, Sodium Fatty Acid, Sodium Cumene Sulfonate, DTPA, Fragrance, Amylase, Disodium Diaminobenzene Disulfonate, Calcium Formate, Sodium Formate, Glucose, Polydimethylsiloxane, Liquitint TM Blue, mannanase, protease variants as described herein;

[0912] -Sodium carbonate, sodium aluminum silicate, sodium sulfate, linear alkylbenzene sulfonate, bentonite, water, sodium percarbonate, sodium polyacrylate, silicate, alkyl sulfate, nonanoyloxybenzene sulfonate, DTPA, polyethylene glycol 4000, silicone, ethoxylate, fragrance, polyethylene oxide, palmitic acid, disodium diaminobenzene disulfonate, amylase, liquitint TM Red, FD&C Blue 1, cellulase, protease variants as described herein;

[0913] - Water, Sodium Alcohol Ethoxysulfate, MEA Citrate, Linear Alkylbenzene Sulfonate: Sodium / MEA Salt, Propylene Glycol, Polyethyleneimine Ethoxylate, Ethanol, Diethylene Glycol, Polyethyleneimine, Propoxyethoxylate, Diquaternium Ethoxysulfate, Alcohol Sulfate, Polydimethylsiloxane, Fragrance, Sodium Fatty Acid, DTPA, Sodium Bisulfite, Disodium Diaminostilbene Disulfonate, Amylase, Gluconase, Castor Oil, Calcium Formate, MEA, Styrene Acrylic Acid Copolymer, Sodium Formate, Liquitint TM Blue, a protease variant as described herein;

[0914] - Water, Sodium Alcohol Ethoxysulfate, MEA Citrate, Linear Alkylbenzene Sulfonate: Sodium / MEA Salt, Propylene Glycol, Ethanol, Diethylene Glycol, Polyethylenimine Propoxyethoxylate, Polyethylenimine Ethoxylate, Alcohol Sulfate, Polydimethylsiloxane, Fragrance, Sodium Fatty Acid, DTPA, Amylase, Sodium Bisulfite, Disodium Diaminobenzyldisulfonate, Castor Oil, Calcium Formate, MEA, Styrene Acrylic Acid Copolymer, Propanol, Gluconolactone, Sodium Formate, Liquitint TM Blue, a protease variant as described herein;

[0915] - Water, Sodium Alcohol Ethoxysulfate, MEA Citrate, Linear Alkylbenzene Sulfonate: Sodium / MEA Salt, Propylene Glycol, Polyethyleneimine Ethoxylate, Ethanol, Diethylene Glycol, Polyethyleneimine Propoxyethoxylate, Diquaternium Ethoxysulfate, Alcohol Sulfate, Polydimethylsiloxane, Fragrance, Sodium Fatty Acid, DTPA, Sodium Bisulfite, Disodium Diaminobenzylbenzene Disulfonate, Amylase, Gluconolactone, Castor Oil, Calcium Formate, MEA, Styrene Acrylic Acid Copolymer, Propanol, Sodium Formate, Liquitint TM Blue, a protease variant as described herein;

[0916] - sodium carbonate, sodium aluminosilicate, alkyl sulfate, sodium sulfate, linear alkylbenzene sulfonate, water, sodium polyacrylate, silicate, ethoxylate, sodium percarbonate, polyethylene glycol 4000, amylase, disodium diaminostilbene disulfonate, silicone, cellulase, protease variants as described herein;

[0917] - sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, alkyl sulfate, sodium percarbonate, water, sodium polyacrylate, silicate, nonanoyloxybenzene sulfonate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, disodium diaminobenzene disulfonate, palmitic acid, amylase, silicone, cellulase, protease variants as described herein;

[0918] - sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, water, nonanoyloxybenzene sulfonate, alkyl sulfate, sodium polyacrylate, silicate, sodium percarbonate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, disodium diaminostilbene disulfonate, amylase, silicone, cellulase, protease variants as described herein;

[0919] - sodium carbonate, sodium aluminosilicate, sodium sulfate, sodium percarbonate, alkyl sulfates, linear alkylbenzene sulfonate, water, nonanoyloxybenzene sulfonate, sodium polyacrylate, silicate, ethoxylate, polyethylene glycol 4000, DTPA, fragrance, Natalase, palmitic acid, amylase, disodium diaminostilbene disulfonate, FD&C Blue 1, silicone, cellulase, alkyl ether sulfates, protease variants as described herein;

[0920] - sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, sodium percarbonate, nonanoyloxybenzene sulfonate, alkyl sulfate, water, silicate, sodium polyacrylate, ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, amylase, disodium diaminostilbene disulfonate, silicone, FD&C Blue 1, cellulase, alkyl ether sulfate, protease variants as described herein;

[0921] - sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, sodium percarbonate, alkyl sulfate, water, sodium polyacrylate, silicate, nonanoyloxybenzene sulfonate, ethoxylate, polyethylene glycol 4000, DTPA, fragrance, cellulase, amylase, disodium diaminostilbene disulfonate, silicone, FD&C Blue 1, a protease variant as described herein;

[0922] - Water, Sodium Alcohol Ethoxysulfate, MEA Citrate, Sodium Linear Alkylbenzene Sulfonate, Linear Alkylbenzene Sulfonate: MEA Salt, Alcohol Ethoxylate, Sodium Fatty Acid, Propylene Glycol, Diethylene Glycol, Polyethyleneimine Ethoxylate Propoxylate, Diquaternium Ethoxysulfate, Ethanol, Sodium Cumene Sulfonate, Fragrance, DTPA, Sodium Bisulfate, Disodium Diaminostilbene Disulfonate, Mannanase, Cellulase, Amylase, Sodium Formate, Calcium Formate, Lauramine Oxide, Liquitint TM Blue, dimethicone / polydimethylsiloxane, a protease variant as described herein;

[0923] - Water, Sodium Alcohol Ethoxysulfate, Linear Alkylbenzene Sulfonate: Sodium / MEA Salt, MEA Citrate, Propylene Glycol, Polyethyleneimine Ethoxylate, Fragrance, Ethanol, Diethylene Glycol, Polyethyleneimine Propoxyethoxylate, Amylase, Alcohol Sulfate, Sodium Fatty Acid, DTPA, Disodium Diaminobenzylbenzene Disulfonate, MEA, Mannanase, Glucose, Sodium Formate, Polydimethylsiloxane, Liquitint TM Blue, tetramine, a protease variant as described herein;

[0924] - Water, Sodium Alcohol Ethoxysulfate, MEA Citrate, Sodium Linear Alkylbenzene Sulfonate, Linear Alkylbenzene Sulfonate: MEA Salt, Alcohol Ethoxylate, Sodium Fatty Acid, Propylene Glycol, Diethylene Glycol, Polyethyleneimine Ethoxylate Propoxylate, Diquaternium Ethoxysulfate, Ethanol, Sodium Cumene Sulfonate, Fragrance, DTPA, Sodium Bisulfate, Disodium Diaminostilbene Disulfonate, Mannanase, Cellulase, Amylase, Sodium Formate, Calcium Formate, Lauramine Oxide, Liquitint TM Blue, dimethicone / polydimethylsiloxane, a protease variant as described herein;

[0925] - sodium carbonate, sodium aluminosilicate, sodium sulfate, linear alkylbenzene sulfonate, sodium percarbonate, nonanoyloxybenzene sulfonate, alkyl sulfate, water, silicate, sodium polyacrylate ethoxylate, polyethylene glycol 4000, fragrance, DTPA, palmitic acid, amylase, disodium diaminostilbene disulfonate, silicone, FD&C Blue 1, cellulase, alkyl ether sulfate, a protease variant as described herein; or

[0926] - Water, dodecylbenzenesulfonic acid, laureth-11, peg-75 lanolin, propylene glycol, alcohol denat. (alcohol holdenat.), potassium soyate, potassium hydroxide, disodium cocoamphodiacetate, ethylenediamine triacetate cocoalkylacetamide, fragrance, zinc ricinoleate, sodium chloride, benzisothiazolinone, methylisothiazolinone, ci 16255, benzyl alcohol, protease variants as described herein.

[0927] Preferably, the above formulations comprising a protease further comprise 4-FPBA and / or a peptide aldehyde protease inhibitor, most preferably Z-GAY or Z-VAL.

[0928] The protease variants described herein can be comprised in one of the following detergent compositions.

[0929]

[0930]

[0931]

[0932]

[0933]

[0934]

[0935]

[0936]

[0937] Compositions with antimicrobial agents

[0938] Antimicrobials are chemical compounds that kill microorganisms or inhibit their growth or reproduction. Microorganisms can be bacteria, yeasts, or molds. Preservatives are antimicrobial agents that can be added to water-based products and compositions to preserve the original properties, characteristics, and integrity of the products and compositions by killing contaminating microorganisms or inhibiting their growth.

[0939] The compositions / formulations comprising the protease of the present invention may contain one or more antimicrobial agents and / or preservatives as listed on pages 35 to 39 of WO 2021 / 115912 A1 (“Formulations comprising a hydrophobically modified polyethyleneimine and one or more enzymes”).

[0940] Of particular interest for cleaning compositions as well as fabric and home care products and particularly in laundry formulations are any of the following antimicrobial and / or preservatives:

[0941] 4,4'-Dichloro-2-hydroxydiphenyl ether (other names: 5-chloro-2-(4-chlorophenoxy)phenol, hydroxydichlorodiphenyl ether (Diclosan), DCPP), HP 100 (30 wt.% DCPP in 1,2-propylene glycol); 2-phenoxyethanol (alternative names: phenoxyethanol, methylphenyl glycol, phenoxyethanol, ethylene glycol phenyl ether, ethylene glycol monophenyl ether, 2-(phenoxy)ethanol, 2-phenoxy-1-ethanol); 2-bromo-2-nitropropane-1,3-diol (alternative name: 2-bromo-2-nitro-1,3-propanediol); Glutaraldehyde (alternative names: 1-5-glutaraldehyde, pentane-1,5-dialdehyde, glutaral, glutardialdehyde); Glyoxal (alternative name: ethandial) , oxylaldehyde, 1,2-ethandial); 5-bromo-5-nitro-1,3-dioxane (alternative name: 5-bromo-5-nitro-m-dioxane); phenoxypropanol (alternative names: propylene glycol phenyl ether, phenoxyisopropanol, 1-phenoxy-2-propanol, 2-phenoxy-1-propanol); glucoprotamine (chemical description: reaction products of glutamic acid and alkylpropylenediamines, alternative name: glucoprotamine 50); cyclohexylhydroxydiazenium-1-oxide, potassium salt (alternative name: N-cyclohexyl-diazenium dioxide, potassium HDO, Xyligene); formic acid (alternative name: methanoic acid acid) and its salts, such as sodium formate); tetrahydro-3,5-dimethyl-1,3,5-thiadiazine-2-thione (alternative names: 3,5-dimethyl-1,3-5-thiadiazinane-2-thione, Dazomet); 2,4-dichlorobenzyl alcohol (alternative names: dichlorobenzyl alcohol, 2,4-dichloro-benzyl alcohol, (2,4-dichloro-phenyl)-methanol, DCBA); 1-propanol (alternative names: n-propanol, propan-1-ol, n-propyl alcohol); 1,3,5-tris-(2-hydroxyethyl)-hexahydro-1,3,5-triazine (alternative names: hexahydrotriazine, tris(hydroxyethyl)-hexahydrotriazine, hexahydro-1,3-5 -tris(2-hydroxyethyl)-s-triazine, 2,2′,2″-(hexahydro-1,3,5-triazine-1,3,5-triyl)triethanol); 2-butyl-benzo[d]isothiazol-3-one (“BBIT”); 2-methyl-2H-isothiazol-3-one (“MIT”); 2-octyl-2H-isothiazol-3-one (“OIT”); 5-chloro-2-methyl-2H-isothiazol-3-one (“CIT” or “CMIT”); a mixture of 5-chloro-2-methyl-2H-isothiazol-3-one (“CMIT”) and 2-methyl-2H-isothiazol-3-one (“MIT”) (CMIT / MIT mixture);1,2-Benzisothiazol-3(2H)-one ("BIT"); hexa-2,4-dienoic acid (commonly known as "sorbic acid") and its salts, such as calcium sorbate and sodium sorbate; potassium (E,E)-hexa-2,4-dienoate (potassium sorbate); lactic acid and its salts; L-(+)-lactic acid; especially sodium lactate; benzoic acid and its salts, such as sodium benzoate, ammonium benzoate, calcium benzoate, and magnesium benzoate , MEA benzoate, potassium benzoate; salicylic acid and its salts, such as calcium salicylate, magnesium salicylate, MEA salicylate, sodium salicylate, potassium salicylate, TEA salicylate; benzalkonium chloride, benzalkonium bromide, benzalkonium saccharinate; didecyldimethylammonium chloride ("DDAC"); N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine ("diamine"); peracetic acid; hydrogen peroxide.

[0942] At least one antimicrobial agent or preservative may be added to the composition of the invention in a concentration ranging from 0.001% to 10% relative to the total weight of the composition.

[0943] Preferably, the composition contains 2-phenoxyethanol at a concentration of 0.1% to 2% or 4,4'-dichloro-2-hydroxydiphenyl ether (DCPP) at a concentration of 0.005% to 0.6%.

[0944] The present invention also encompasses a method for preserving an aqueous composition according to the invention against contamination or growth of microorganisms, which method comprises adding at least one antimicrobial agent or preservative, preferably 2-phenoxyethanol.

[0945] The present invention also encompasses methods of providing antimicrobial effects to textiles after treatment with solid laundry detergents (eg, powders, granules, capsules, tablets, bars, etc.), liquid laundry detergents, softeners, or post-rinse agents containing 4,4'-dichloro-2-hydroxydiphenyl ether (DCPP).

[0946] How to use

[0947] The protease variants as described herein can be used in a variety of applications.

[0948] The protease variants as described herein or compositions comprising the protease variants as described herein can be used in cleaning, food processing, animal feed, pulp and paper processing, baking, mining and oil well services, textile processing, leather processing, water treatment, brewing, ethanol production, circular economy, waste treatment or recycling.

[0949] The present invention further relates to the purposes of protease variants as described herein in cleaning process (such as laundry or hard surface cleaning), preferably for home care or I&I cleaning.Variant polypeptide of the present invention or the composition comprising said variant polypeptide can be used for various industrial and institutional cleaning applications, including commercial laundry (such as internal laundry or tunnel washing), mechanical appliance washing (such as in hood machine or tunnel washing machine), manual dishwashing cleaning, carpet cleaning, open plant cleaning (cleaning outside the pipeline), cleaning in situ (cleaning in the pipeline), film cleaning (such as in dairy products, food, beverage or water treatment), vehicle maintenance (such as pad cleaning), microbial removal, virus removal, insect removal or stench removal or veterinary cleaning.Variant polypeptide of the present invention or the composition comprising said variant polypeptide can also be used for textile processing, leather processing or water treatment.

[0950] The variant polypeptides of the present invention or compositions comprising the variant polypeptides can be used in the bioenergy industry, in particular bioethanol production, oil and gas recovery (in particular liquefaction for improved oil recovery) and food processing (in particular beverage production and / or processing).

[0951] The present invention also relates to the use of the protease variants described herein for providing detergent compositions with improved protease stability and / or for providing detergent compositions with improved cleaning performance, preferably on protease-sensitive stains.

[0952] The present invention therefore also relates to a method for cleaning, preferably laundry or hard surface cleaning, comprising the step of contacting a subject, preferably a textile or a hard surface, with a composition comprising a protease variant as described herein, preferably wherein the composition comprises at least one additional detergent component, preferably a surfactant and / or a builder.

[0953] Furthermore, the present invention relates to a method for improving the stability of a protease in a detergent composition and / or for improving the cleaning performance of a detergent composition (preferably against protease-sensitive stains), which method comprises the step of formulating a protease variant as described herein in a detergent composition.

[0954] The present invention also relates to a method of laundry laundering fabrics or cleaning hard surfaces, the method comprising treating the fabric or hard surface with a composition comprising a variant polypeptide of the present invention and further comprising 4,4'-dichloro-2-hydroxydiphenyl ether.

[0955] In one embodiment, the protease variants as described herein are used to improve the sustainability properties of a composition or method and / or for use in a circular economy.With regard to use in a circular economy, the protease variants as described herein can be used for waste treatment or recycling.

[0956] In one embodiment, the present invention relates to a method for waste treatment, preferably for dissolving waste, comprising the steps of contacting the waste with a protease variant as described herein and preferably with one or more enzymes selected from the group consisting of lipase, glucanase, amylase, pectate lyase and mannanase under conditions that support enzymatic dissolution of the waste. In one embodiment, the waste is municipal solid waste. In one embodiment, the liquefied waste can be used as a substrate for microbial fermentation.

[0957] Although the present invention has been shown and described in detail in the drawings and the foregoing description, such showing and description should be considered as illustrative or exemplary rather than restrictive. The present invention is not limited to the disclosed embodiments. By studying the drawings, the present disclosure and the appended claims, those skilled in the art can understand and realize other variations of the disclosed embodiments when practicing the claimed invention. The specific embodiments are merely exemplary in nature and are not intended to limit application and use. The following examples further illustrate the present invention, but do not limit the scope of the invention. Those skilled in the art can make various changes and modifications based on the description of the present invention, and such changes and modifications are also included in the present invention.

[0958] Preferred Embodiments

[0959] In particular, preferred herein are:

[0960] 1. A variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein:

[0961] (i) the polypeptide or fragment thereof has at least 65% but less than 100% identity to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9 ... an amino acid sequence having at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, but less than 100% sequence identity, and

[0962] (ii) the polypeptide or fragment thereof comprises an amino acid substitution at amino acid residue 183, compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and with reference to the numbering of SEQ ID NO: 2, and comprises at least one additional amino acid substitution at amino acid residues selected from the group consisting of 43, 78 and 204.

[0963] 2. The variant polypeptide of embodiment 1, wherein the fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0964] 3. The variant polypeptide of embodiment 1 or 2, wherein:

[0965] (a) the amino acid substitution at amino acid residue 43 is X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, preferably X43K;

[0966] (b) the amino acid substitution at amino acid residue 78 is X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, preferably X78N / D;

[0967] (c) the amino acid substitution at amino acid residue 183 is X183D / E / C / Q / A / M, preferably X183D / E, and / or

[0968] (d) the amino acid substitution at amino acid residue 204 is X204D / E / C / G, preferably X204D.

[0969] 4. The variant polypeptide of embodiments 1 to 3, wherein the polypeptide comprises one of the following combinations of amino acid substitutions:

[0970] (a) X183D / E and X43K / R;

[0971] (b) X183D / E and X78N / D;

[0972] (c) X183D / E and X204D;

[0973] (d) X183D / E, X43K / R and X78N / D;

[0974] (e) X183D / E, X43K / R and X204D;

[0975] (f) X183D / E, X78N / D and X204D; or

[0976] (g)X183D / E, X43K / R, X78N / D and X204D.

[0977] 5. The variant polypeptide of any one of the preceding embodiments, further comprising an amino acid substitution at amino acid residue 76, with reference to the numbering of SEQ ID NO: 2.

[0978] 6. The variant polypeptide of embodiment 5, wherein the amino acid substitution at amino acid residue 76 is X76D.

[0979] 7. The variant polypeptide of any preceding embodiment, wherein the polypeptide comprises amino acid substitutions X43K, X76D, X78N, X183D, and X204D.

[0980] 8. The variant polypeptide of any of the preceding embodiments, with reference to the numbering of SEQ ID NO: 2, further comprising at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 109, 144, 182, 237, 240, 248, 256, and 260.

[0981] 9. The variant polypeptide of embodiment 8, wherein:

[0982] (a) the amino acid substitution at amino acid residue 18 is X18A / D / C / E / Q;

[0983] (b) the amino acid substitution at amino acid residue 24 is X24K;

[0984] (c) the amino acid substitution at amino acid residue 56 is X56D;

[0985] (d) the amino acid substitution at amino acid residue 109 is X109K / A;

[0986] (e) the amino acid substitution at amino acid residue 144 is X144N / R;

[0987] (f) the amino acid substitution at amino acid residue 182 is X182K / R / E;

[0988] (g) the amino acid substitution at amino acid residue 237 is X237R / A;

[0989] (h) the amino acid substitution at amino acid residue 240 is X240E / N;

[0990] (i) the amino acid substitution at amino acid residue 248 is X248Q / R;

[0991] (j) the amino acid substitution at amino acid residue 256 is X256E / T / D / R / P; and / or

[0992] (k) The amino acid substitution at amino acid residue 260 is X260D / K.

[0993] 10. The variant polypeptide of any one of the preceding embodiments, wherein the polypeptide comprises a combination of substitutions selected from the group consisting of:

[0994] (a)X24K;

[0995] (b)X24K; X43K; X78N;

[0996] (c)X24K;

[0997] (d)X24K;

[0998] (e)X24K; X43K; X78N; X183D; X204D;

[0999] (f)X24K;

[1000] (g)X24K;

[1001] (h)X24K;

[1002] (i)X24K;

[1003] (j)X24K;

[1004] (k)X24K;

[1005] (l)X24K;X43K;X78N;X182E;X183D;X204D;X240E;X248R;X260K

[1006] (m)X24K;X43K;X78N;X183D;X204D;X237R;X240E;X248R X260K

[1007] (n)X24K;X43K;X56D;X78N;X109K;X144N;X182E;X183D;X204D;X248Q

[1008] (o)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X248Q;X260K

[1009] (p)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X237R;X248Q

[1010] (q)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q;X260D

[1011] (r)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q;X260K

[1012] (s)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X240E;X248Q

[1013] (t)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X248Q;X260D

[1014] (u)X24K;X43K;X56D;X144N;X182K;X183D;X204D;X237R;X248Q;X260D

[1015] (v)X24K;X43K;X56D;X78N;X109K;X144N;X182E;X183D;X204D;X248Q;X260D

[1016] (w)X24K;X43K;X56D;X78N;X109K;X144N;X182K;X183D;X204D;X248Q;X260D

[1017] (x)X24K;

[1018] (y)X24K;

[1019] (z)X24K; 8Q; X240E; X248Q; X260D(bb)X26I; X43K; 0G; X130G; 3S; X109A; X116E; X130G; X103S; X109A; X116E; X130G; X56D; X78D; X103S; X109A;

[1020] (gg)X26I;X43K;X56D;X78D;X103S;X109A;X116E;X130G;X144R;X183D;X204D;X240N;X248R, and

[1021] (hh)X26I;

[1022] 11. The variant polypeptide of any one of the preceding embodiments, wherein the polypeptide comprises a combination of substitutions selected from the group consisting of:

[1023] (a)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q

[1024] (b)S24K;N43K;S78N;N183D;N204D;N248R;T260K

[1025] (c)S24K;N43K;S78N;Q182E;N183D;N204D;N248R;T260K

[1026] (d)S24K;N43K;S78N;N183D;N204D;K237R;N248R;T260K

[1027] (e)S24K;N43K;S78N;N183D;N204D;S240E;N248R;T260K

[1028] (f)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;N248Q

[1029] (g)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q

[1030] (h)S24K;N43K;S56D;S144N;Q182K;N183D;N204D;S240E;N248Q

[1031] (i)S24K;N43K;S56D;S144N;Q182K;N183D;N204D;N248Q;T260D

[1032] (j)S24K;N43K;S78N;S156D;N183D;N204D;S240E;N248R;T260K

[1033] (k)S24K;N43K;S78N;Q182E;N183D;N204D;K237R;N248R;T260K

[1034] (l)S24K;N43K;S78N;Q182E;N183D;N204D;S240E;N248R;T260K

[1035] (m)S24K;N43K;S78N;N183D;N204D;K237R;S240E;N248R;T260K

[1036] (n)S24K;N43K;S56D;S78N;Q109K;S144N;Q182E;N183D;N204D;N248Q

[1037] (o)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;N248Q;T260K

[1038] (p)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;K237R;N248Q

[1039] S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q;T260D

[1040] (q)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q;T260K

[1041] (r)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;S240E;N248Q

[1042] S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;N248Q;T260D

[1043] (s)S24K;N43K;S56D;S144N;Q182K;N183D;N204D;K237R;N248Q;T260D

[1044] (t)S24K;N43K;S56D;S78N;Q109K;S144N;Q182E;N183D;N204D;N248Q;T260D

[1045] (u)S24K;N43K;S56D;S78N;Q109K;S144N;Q182K;N183D;N204D;N248Q;T260D

[1046] (v)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;K237R;S240E;N248Q

[1047] (w)S24K;N43K;S56D;S78N;Q109K;S144N;Q182K;N183D;N204D;N248Q;T260D

[1048] (x)S24K; N43K; S56D; S78N; Q109K; S144N; Q182K; N183D; N204D; S240E; N248Q; T260K

[1049] (y)S24K; N43K; S56D; S78N; S144N; Q182K; N183D; N204D; K237R; S240E; N248Q; T260D

[1050] (z)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R (aa) V26I; S56D; S78D; A103S; Q109A; N116E; S1 30G; S144R; N183D; N204D; S240N; N248R(bb)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R(cc)V 26I; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237R; S240N; N248R(dd)V26I; S56D; S78D; A103S; Q109A; N116E; S130G ;S144R;N183D;N204D;S240N;N248R(ee)V26I;N43K;S56D;S78D;A103S;Q109A;N116E;S130G;S144R;N183D;N204D;S240N;N248R, and

[1051] (ff)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237A; S240E; N248R.

[1052] 12. The variant polypeptide of any one of the preceding embodiments, wherein the polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101, with reference to the numbering of SEQ ID NO: 2.

[1053] 13. The variant polypeptide of any preceding embodiment, wherein the polypeptide does not comprise the amino acid substitutions S3T, V4I, and V199I.

[1054] 14. The variant polypeptide of any preceding embodiment, wherein the polypeptide exhibits one or more improved properties compared to the protease of SEQ ID NO: 3.

[1055] 15. The variant polypeptide of claim 14, wherein the improved properties are selected from the group consisting of:

[1056] (i) increased stability,

[1057] (ii) increased storage stability, and

[1058] (iii) Increased storage stability in detergent compositions.

[1059] 16. A polynucleotide encoding the variant polypeptide of any preceding embodiment.

[1060] 17. A formulation comprising the variant polypeptide of any one of embodiments 1 to 15 and at least one additional component.

[1061] 18. The formulation of embodiment 17, wherein the formulation comprises an enzyme stabilization system, wherein the enzyme stabilization system preferably comprises at least one compound selected from the group consisting of a polyol (preferably 1,3-propylene glycol, ethylene glycol, glycerol, 1,2-propylene glycol, or sorbitol), an inorganic salt (preferably CaCl2, MgCl2, or NaCl), a short chain (preferably C1-C3) carboxylic acid or a salt thereof (preferably formic acid, a formate salt (preferably sodium formate), acetic acid, an acetate salt, or a lactate salt), a borate salt, boric acid, a boronic acid (preferably 4-formylphenylboronic acid (4-FPBA)), a peptide aldehyde (preferably Z-VAL-H or Z-GAY-H), a peptide acetal, and a peptide aldehyde bisulfite adduct, preferably a peptide aldehyde (preferably Z-VAL-H or Z-GAY-H).

[1062] 19. The formulation of embodiment 17 or 18, wherein the formulation comprises one or more second enzymes different from the variant polypeptides mentioned in any of the preceding embodiments, preferably one or more second enzymes selected from the group consisting of an amylase, a second protease, a lipase, a cellulase, a hemicellulase, a mannanase, a xylanase, a DNase, a dispersin, a pectinase, an oxidoreductase, and a cutinase, preferably selected from an amylase, a mannanase, and a lipase, most preferably an amylase.

[1063] 20. A detergent composition comprising the variant polypeptide of any one of embodiments 1 to 15, preferably a laundry detergent composition or a hard surface cleaning detergent composition.

[1064] 21. The detergent composition of embodiment 20, wherein the composition comprises one or more surfactants and / or one or more builders, preferably strong chelating builders.

[1065] 22. The detergent composition of embodiment 21, wherein the composition comprises a builder, wherein the builder is selected from the group consisting of MDGA, GLDA, DTPMP, HEDP and EDDS, preferably MDGA or EDDS.

[1066] 23. The detergent composition of embodiment 21, wherein the composition comprises a surfactant, wherein the surfactant is selected from the group consisting of nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, and combinations thereof.

[1067] 24. The detergent composition of any one of embodiments 20 to 23, wherein the detergent composition is free of anionic surfactants.

[1068] 25. The detergent composition of any one of embodiments 21 to 24, wherein the surfactant and / or the builder is biodegradable and / or bio-based.

[1069] 26. The detergent composition of any one of embodiments 20 to 25, wherein the detergent composition is a liquid or a solid.

[1070] 27. The detergent composition of any one of embodiments 20 to 26, wherein the detergent composition is in the form of a sachet.

[1071] 28. The detergent composition of any one of embodiments 20 to 27, wherein the detergent composition is a liquid laundry detergent composition.

[1072] 29. The detergent composition of any one of embodiments 20 to 28, wherein the detergent composition is free of boron.

[1073] 30. The detergent composition of any one of embodiments 20 to 29, wherein the detergent composition does not contain a preservative.

[1074] 31. A detergent composition as described in any of embodiments 20 to 29, wherein the composition further comprises 2-phenoxyethanol, preferably comprising phenoxyethanol in an amount ranging from 2 ppm to 5% by weight of the composition; more preferably comprising 0.1% to 2% phenoxyethanol.

[1075] 32. The detergent composition of any one of embodiments 20 to 31, further comprising 4,4'-dichloro-2-hydroxydiphenyl ether at a concentration of 0.001% to 3%, preferably 0.002% to 1%, more preferably 0.01% to 0.6%, each by weight of the composition.

[1076] Examples

[1077] Materials and Methods

[1078] 1. Library generation

[1079] The gene encoding the protease is cloned into a Gram-positive expression vector comprising a promoter sequence, a sequence encoding a secretory signal peptide, and a ribosome binding site by a standard protocol based on restriction endonuclease digestion-ligation. After the reaction, the plasmid assembly mixture is transformed into Bacillus subtilis PY79 by the natural competence transformation method established. Successful transformation is selected by plating on LB agar plates supplemented with 20 μg / ml kanamycin sulfate and incubating overnight at 37 degrees Celsius. After overnight selection, individual colonies are grown overnight at 1000 rpm shaking in TB culture medium with 20 μg / ml kanamycin sulfate. The cells are then precipitated by centrifugation, and the QIAprep rotary microprep kit from Qiagen is used to separate the plasmid DNA by alkaline lysis method. The isolated DNA is transformed into electrocompetent Bacillus licheniformis cells. For example, by making the Bacillus licheniformis strain be grown in the culture medium rich in concentrated osmotic agent (for example, LB broth with 0.5M D-sorbitol) and harvesting cells in the early exponential growth period to prepare cells.By cooling on ice and harvesting cells by centrifugation.After harvesting, wash cells to remove salt by 3 suspension-precipitation (by centrifugation) cycles with a wash buffer rich in osmotic agent (for example, 10% glycerol with 0.5M D-sorbitol and 0.5M D-mannitol).Finally, concentrate cells by being resuspended in wash buffer with 1% to 10% of the original culture volume.Once prepared, plasmid DNA is added to the Bacillus licheniformis electrocompetent cells in the electroporation cuvette of 0.2cm Bio-Rad.According to the manufacturer's instructions, cells are electroporated using the Gene Pulser Xcell of Bio-Rad.After impact, rescue cells immediately by adding 1ml of culture medium rich in concentrated osmotic agent. After two hours of recovery at 37 degrees Celsius, successful transformants were then selected by plating on LB agar plates supplemented with 20 μg / ml kanamycin sulfate and incubated overnight at 37 degrees Celsius.

[1080] 2. Preparation and expression of variants

[1081] The single bacterium colony of expression strain is picked into 600 μ L rich culture medium (such as LB nutrient solution) supplemented with 20 μ g / mL kanamycin sulfate in 96-well plates. Make culture grow at 30 degrees Celsius with 1000rpm shaking for 16 hours, then use 6 μ L culture to inoculate 600 μ L defined type glucose-mineral medium and 20 μ g / mL kanamycin sulfate in 96-well plates. Make culture grow at 30 degrees Celsius with 1000rpm shaking for 48 hours, then make cell precipitation and remove residual culture solution and gather in the crops supernatant by using centrifugal.

[1082] 3. Protease activity assay

[1083] Proteolytic activity was determined using the Suc-AAPF-AMC substrate (Bachem, product number 4012873). Suc-AAPF-AMC, short for N-succinyl-Ala-Ala-Pro-Phe-7-amino-4-methylcoumarin, is a blocking peptide that can be cleaved by endoproteases. After proteolytic cleavage, free AMC molecules are released and measured by spectrofluorometry at an excitation wavelength of 360 nm and an emission wavelength of 448 nm. Under a given set of conditions, the slope of the time-dependent fluorescence signal increase (V max ) is proportional to the amount of protease in solution and the specific activity (activity per mg of enzyme) of the protease in question. Prior to activity assessment, the protease sample was diluted in assay buffer (100 mM Tris-HCl, 0.1% Brij-35, pH 8.6) or detergent challenge solution, for example to a pre-assay dilution of between 400-800 times. The assay was performed by transferring 10 μL of the diluted enzyme sample to a 384-well microtiter plate containing 40 μL of substrate working solution or by transferring 20 uL of the diluted enzyme sample to a 96-well microtiter plate containing 80 μL of substrate working solution. The solution was mixed at room temperature and the plate was read using a standard plate reader with fluorescence capability (Synergy BioTek). or ) The fluorescence signal was measured every minute at an excitation wavelength of 360 nm and an emission wavelength of 448 nm within 15 minutes. The substrate conversion rate (V max ), and the residual activity is calculated by dividing the activity after the storage time by the activity of the sample at time point 0. In some cases, the residual activity is normalized to the residual activity of a specific reference sample, which is expressed as the normalized residual activity.

[1084] Example 1: Conditions for accelerated stability studies in standard detergents

[1085] To assess stability in detergent, the expressed protease supernatant was first diluted 10-15 fold into a stability buffer (20 mM HEPES, pH 8.0) (Table 3) containing a defined amount of calcium and added to a standard liquid detergent (standard A or B, Table 4), allowed to equilibrate for 1-2 hours, and then assessed for activity at time zero. The samples were then stored at 37 or 45 degrees Celsius in liquid detergent (standard A or B) and residual activity was assessed after 1, 2, 3, and / or 7 days. Prior to activity assessment using the Suc-AAPF-AMC assay performed as described in the Materials and Methods section, the protease samples stimulated in detergent were pre-diluted 5-40 fold in an activity buffer (100 mM Tris-HCl, 0.1% Brij-35, pH 8.6).

[1086] Table 3. Stimulation conditions for accelerated stability studies

[1087]

[1088] Table 4. Standard detergents for accelerated stability studies

[1089]

[1090] Example 2: Accelerated Storage Stability Evaluation of Mutants from the Combinatorial Library under Condition 1

[1091] Mutants from SEQ ID NO: 3 were generated in a combinatorial approach and a portion of the library was expressed in 96DWP as described in Materials and Methods. The supernatant containing the protease was stimulated under Condition 1 (see Example 1), subjected to thorough mixing, and equilibrated for one hour. The activity of the non-stressed samples was measured using the Suc-AAPF-AMC assay. The detergent plates were sealed and placed at an elevated temperature (37 degrees Celsius) for a duration of 18-160 hours. At designated time points during the incubation period, the protease activity of the stressed samples was measured and compared to the non-stressed control to calculate the residual activity. SEQ ID NO: 3 was included as a reference control.

[1092] Combinatorial libraries based on SEQ ID NO: 3 generated variants containing 2-19 mutations at different selected positions compared to SEQ ID NO: 3. Progressive and synergistic stabilization of the variants was observed, with increasing numbers of mutations from the selected stabilizing core group consisting of N183D, N43K, S78N / D, and N204D (Table 5).

[1093] Table 5

[1094]

[1095] *Residual activity is defined as the activity after 160 h of storage at 37°C divided by the activity at time zero

[1096] Example 3: Accelerated Storage Stability Assay of Mutants from a Single Site Mutagenesis Library under Condition 2

[1097] A single site mutagenesis library containing four stabilizing core group positions (N43K, S78N, N183D, and N204D) was constructed from SEQ ID NO:34. Each position of the core group was reversed to the amino acid residues in SEQ ID NO:3. Mutants were expressed according to the protocol listed in Materials and Methods, and the supernatant containing the protease was stimulated under Condition 2 (see Example 1), subjected to thorough mixing, and equilibrated for 2 h. The activity of non-stressed samples was measured using the Suc-AAPF-AMC assay. The detergent plates were sealed and placed at an elevated temperature (45 degrees Celsius) for a duration of 44 hours. The protease activity of the stressed samples was measured and compared to the non-stressed control to calculate the residual activity. SEQ ID NO:3 was included as a reference control. The stabilizing effect (increased residual activity compared to SEQ ID NO:3) was significantly reduced in variants with reversal mutations that were reversed to the residue identity present in SEQ ID NO:3 (Table 6), which supports the role of the stabilizing core group.

[1098] Table 6

[1099]

[1100] *Residue identity to SEQ ID NO: 3 at the indicated positions

[1101] **Normalized value relative to the residual activity of SEQ ID NO: 34

[1102] Example 4: Accelerated Storage Stability Evaluation of Mutants from the Combinatorial Library under Condition 3

[1103] Mutants from SEQ ID NO:3 were generated using a combinatorial approach and a portion of the library was expressed in 96DWP as described in Materials and Methods. The supernatant containing protease was stimulated under Condition 3 (see Example 1), fully mixed, and balanced for 1 h. The activity of non-stressed samples was measured using Suc-AAPF-AMC. The detergent plates were sealed and placed in an elevated temperature (45 degrees Celsius) for a duration of 18-160 hours. At designated time points during incubation, the protease activity of the stressed samples was measured and compared with the non-stressed control to calculate the residual activity. SEQ ID NO:3 was included as a reference control. The residual activity of the selected variants and reference controls is reported in Table 7.

[1104] Table 7

[1105]

[1106]

[1107] *Residual activity is defined as the activity after 160 h of storage under the conditions divided by the activity at time zero

[1108] Example 5: Stable detergent formulation

[1109] Liquid laundry detergent formulations were prepared with or without (comparative) 0.5% by weight of a variant polypeptide of the invention and 0.2% by weight of a biocide HP 100 (from BASF SE) or 1% 2-phenoxyethanol ( PE, BASF AG). These formulations were prepared by first preparing a premix containing the AEO and AES surfactants, the solvents 1,2-propylene glycol and ethanol, and (where relevant) HP 100 or 2-phenoxyethanol. This premix was stirred at room temperature to form a homogeneous mixture. LAS, fatty acids, and citric acid, as shown in Table 8, were then added, along with water until the mixture reached 90%. The pH was then adjusted to 8.5 using NaOH. The final formulation was then prepared by stirring at room temperature: 90% of this resulting mixture, 0.5% of the polypeptide of the present invention, and water until the mixture reached 100%.

[1110] The compositions and results are shown in Table 8.

[1111] Table 8

[1112]

[1113]

[1114] AEO:C 13 / C 15 Oxo-alcohol (7EO) AO7 (BASF SE) (CAS 68002-97-1)

[1115] AES:C 12 / C 14 - Fatty alcohol ether sulfate (2EO), sodium salt: N 70 (BASF SE) (CAS 68891-38-3)

[1116] LAS: linear alkylbenzene sulfonic acid DBS / LC (BASF SE) (CAS 85536-14-7)

[1117] Coconut fatty acids: K12-18 (Emery Oleochemicals) (CAS90990-15-1)

[1118] 1,2-Propanediol: racemic mixture (CAS 57-55-6)

[1119] HP 100 is a commercial product from BASF AG which contains 30% of antimicrobially active 4,4'-dichloro-2-hydroxydiphenyl ether (CAS 3380-30-1) in 1,2-propylene glycol (CAS 122-99-6).

[1120] 2-Phenoxyethanol can be used as PE acquired from BASF AG

[1121] The concentrations of commercial surfactants are given in the table above.

[1122] As is clear from the table above, it is possible to combine the polypeptides of the present invention with Tinosan HP 100 or 2-phenoxyethanol in liquid laundry formulations.

Claims

1. A variant polypeptide having protease activity or a fragment of said polypeptide having protease activity, wherein: (i) the polypeptide or fragment thereof has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, and (ii) the polypeptide or fragment thereof comprises an amino acid substitution at amino acid residue 183, compared to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and with reference to the numbering of SEQ ID NO: 2, and comprises at least one additional amino acid substitution at amino acid residues selected from the group consisting of 43, 78 and 204.

2. The variant polypeptide of claim 1, wherein: (a) the amino acid substitution at amino acid residue 43 is X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, preferably X43K; (b) the amino acid substitution at amino acid residue 78 is X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, preferably X78N / D; (c) the amino acid substitution at amino acid residue 183 is X183D / E / C / Q / A / M, preferably X183D / E, and / or (d) the amino acid substitution at amino acid residue 204 is X204D / E / C / G, preferably X204D.

3. The variant polypeptide of claim 1 or 2, wherein the polypeptide comprises one of the following combinations of amino acid substitutions: (a) X183D / E and X43K / R; (b) X183D / E and X78N / D; (c) X183D / E and X204D; (d) X183D / E, X43K / R and X78N / D; (e) X183D / E, X43K / R and X204D; (f) X183D / E, X78N / D and X204D; or (g)X183D / E, X43K / R, X78N / D and X204D.

4. The variant polypeptide of any preceding claim, with reference to the numbering of SEQ ID NO: 2, further comprising an amino acid substitution at amino acid residue 76, preferably wherein the amino acid substitution at amino acid residue 76 is X76D.

5. The variant polypeptide of any one of the preceding claims, wherein the polypeptide comprises amino acid substitutions X43K, X76D, X78N, X183D, and X204D.

6. The variant polypeptide of any of the preceding claims, with reference to the numbering of SEQ ID NO: 2, further comprising at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 109, 144, 182, 237, 240, 248, 256 and 260, preferably wherein: (a) the amino acid substitution at amino acid residue 18 is X18A / D / C / E / Q; (b) the amino acid substitution at amino acid residue 24 is X24K; (c) the amino acid substitution at amino acid residue 56 is X56D; (d) the amino acid substitution at amino acid residue 109 is X109K / A; (e) the amino acid substitution at amino acid residue 144 is X144N / R; (f) the amino acid substitution at amino acid residue 182 is X182K / R / E; (g) the amino acid substitution at amino acid residue 237 is X237R / A; (h) the amino acid substitution at amino acid residue 240 is X240E / N; (i) the amino acid substitution at amino acid residue 248 is X248Q / R; (j) the amino acid substitution at amino acid residue 256 is X256E / T / D / R / P; and / or (k) The amino acid substitution at amino acid residue 260 is X260D / K.

7. The variant polypeptide of any one of the preceding claims, wherein the polypeptide comprises a combination of substitutions selected from the group consisting of: (a)X24K; (b)X24K; X43K; X78N; (c)X24K; (d)X24K; (e)X24K; X43K; X78N; X183D; X204D; (f)X24K; (g)X24K; (h)X24K; (i)X24K; (j)X24K; (k)X24K; (l)X24K; (m)X24K; (n)X24K;X43K;X56D;X78N;X109K;X144N;X182E;X183D;X204D;X248Q (o)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X248Q;X260K (p)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X237R;X248Q (q)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q;X260D (r)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X248Q;X260K (s)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X240E;X248Q (t)X24K;X43K;X56D;X78N;X144N;X182E;X183D;X204D;X248Q;X260D (u)X24K;X43K;X56D;X144N;X182K;X183D;X204D;X237R;X248Q;X260D (v)X24K;X43K;X56D;X78N;X109K;X144N;X182E;X183D;X204D;X248Q;X260D (w)X24K;X43K;X56D;X78N;X109K;X144N;X182K;X183D;X204D;X248Q;X260D (x)X24K;X43K;X56D;X78N;X144N;X182K;X183D;X204D;X237R;X240E;X248Q (y)X24K;X43K;X56D;X78N;X109K;X144N;X182K;X183D;X204D;X248Q;X260D (z)X24K; a) X24K; X43K; X56D; X78N; )X26I; X26I; X56D; X78D; X103S; X109A; 26I; X43K; X56D; X78D; X103S; 6I; X56D; X78D; X103S; I;X56D;X78D;X103S;X109A;X116E;X130G;X144R;X183D;X204D;X240N;X248R(gg)X26I;X43K;X56D;X78D;X103S;X109A;X116E;X130G;X144R;X183D;X204D;X240N;X248R, and (hh)X26I; 8. The variant polypeptide of any one of the preceding claims, wherein the polypeptide comprises a combination of substitutions selected from the group consisting of: (a)S24K; N43K; S56D; S78N; S144N; Q182K; N183D; N204D; N248Q (b)S24K; N43K; S78N; N183D; N204D; N248R; T260K (c)S24K; N43K; S78N; Q182E; N183D; N204D; N248R; T260K (d)S24K; N43K; S78N; N183D; N204D; K237R; N248R; T260K (e)S24K; N43K; S78N; N183D; N204D; S240E; N248R; T260K (f)S24K; N43K; S56D; S78N; S144N; Q182E; N183D; N204D; N248Q (g)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q (h)S24K;N43K;S56D;S144N;Q182K;N183D;N204D;S240E;N248Q (i)S24K;N43K;S56D;S144N;Q182K;N183D;N204D;N248Q;T260D (j)S24K;N43K;S78N;S156D;N183D;N204D;S240E;N248R;T260K (k)S24K;N43K;S78N;Q182E;N183D;N204D;K237R;N248R;T260K (l)S24K;N43K;S78N;Q182E;N183D;N204D;S240E;N248R;T260K (m)S24K;N43K;S78N;N183D;N204D;K237R;S240E;N248R;T260K (n)S24K;N43K;S56D;S78N;Q109K;S144N;Q182E;N183D;N204D;N248Q (o)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;N248Q;T260K (p)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;K237R;N248Q (q)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q;T260D (r)S24K;N43K;S56D;S78N;S144N;Q182K;N183D;N204D;N248Q;T260K (s)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;S240E;N248Q (t)S24K;N43K;S56D;S78N;S144N;Q182E;N183D;N204D;N248Q;T260D (u)S24K;N43K;S56D;S144N;Q182K;N183D;N204D;K237R;N248Q;T260D (v)S24K;N43K;S56D;S78N;Q109K;S144N;Q182E;N183D;N204D;N248Q;T260D (w)S24K;N43K;S56D;S78N;Q109K;S144N;Q182K;N183D;N204D;N248Q;T260D (x)S24K; N43K; S56D; S78N; S144N; Q182K; N183D; N204D; K237R; S240E; N248Q (y)S24K; N43K; S56D; S78N; Q109K; S144N; Q182K; N183D; N204D; N248Q; T260D (z)S24K; N43K; S56D; S78N; Q109K; S144N; Q182K; N183D; N204D; S240E; N248Q; T260K (a a)S24K; N43K; S56D; S78N; S144N; Q182K; N183D; N204D; K237R; S240E; N248Q; T260D (bb )V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R(cc) V26I; S56D; S78D; A103S; Q109A; N116E; S130G; S144R; N183D; N204D; S240N; N248R(dd)V 26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; S240N; N248R(ee)V2 6I; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237R; S240N; N248R(ff)V26 I;S56D;S78D;A103S;Q109A;N116E;S130G;S144R;N183D;N204D;S240N;N248R(gg)V26I;N43K;S56D;S78D;A103S;Q109A;N116E;S130G;S144R;N183D;N204D;S240N;N248R, and (hh)V26I; N43K; S56D; S78D; A103S; Q109A; N116E; S130G; N183D; N204D; K237A; S240E; N248R.

9. The variant polypeptide of any one of the preceding claims, wherein the polypeptide comprises amino acid residue D or E at position 101, preferably E at position 101, with reference to the numbering of SEQ ID NO:

2.

10. The variant polypeptide of any one of the preceding claims, wherein the polypeptide exhibits one or more improved properties compared to the protease SEQ ID NO: 3, preferably wherein the improved properties are selected from: (i) increased stability, (ii) increased storage stability, and (iii) Increased storage stability in detergent compositions.

11. A polynucleotide encoding a variant polypeptide according to any one of the preceding claims.

12. A composition comprising the variant polypeptide of any one of claims 1 to 10 and at least one additional component, preferably wherein the composition comprises an enzyme stabilization system, wherein the enzyme stabilization system preferably comprises at least one compound selected from the group consisting of a polyol (preferably 1,3-propylene glycol, ethylene glycol, glycerol, 1,2-propylene glycol or sorbitol), an inorganic salt (preferably CaCl2, MgCl2 or NaCl), a short chain (preferably C1-C3) carboxylic acid or a salt thereof (preferably formic acid, a formate (preferably sodium formate), acetic acid, acetate, or lactate), a borate, boric acid, boronic acid (preferably 4-formylphenylboronic acid (4-FPBA)), a peptide aldehyde (preferably Z-VAL-H or Z-GAY-H), a peptide acetal and a peptide aldehyde bisulfite adduct, preferably a peptide aldehyde (preferably Z-VAL-H or Z-GAY-H).

13. The composition of claim 12, wherein the composition comprises one or more second enzymes different from the variant polypeptide as mentioned in any of the preceding claims, preferably one or more second enzymes selected from the group consisting of an amylase, a second protease, a lipase, a cellulase, a hemicellulase, a mannanase, a xylanase, a DNase, a dispersin, a pectinase, an oxidoreductase and a cutinase, preferably selected from an amylase, a mannanase and a lipase, most preferably an amylase.

14. A composition according to any one of claims 12 or 13, wherein the composition is a detergent composition, preferably a laundry detergent composition or a hard surface cleaning detergent composition, preferably wherein the composition comprises one or more surfactants and / or one or more builders, preferably a strong chelating builder.

15. The composition of any one of claims 12 to 14, wherein the composition further comprises 2-phenoxyethanol and / or 4,4'-dichloro-2-hydroxydiphenyl ether, preferably comprising phenoxyethanol in an amount ranging from 2 ppm to 5% by weight of the composition; more preferably comprising 0.1% to 2% by weight of the composition of phenoxyethanol and / or preferably comprising 4,4'-dichloro-2-hydroxydiphenyl ether in a concentration of 0.001% to 3%, preferably 0.002% to 1%, more preferably 0.01% to 0.6%, each by weight of the composition.

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