Polypeptides having protease activity for use in cleaning compositions

Abstract

In the present invention, novel proteases are provided. More particularly, 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] In this invention, a method for using a novel protease and a composition containing the novel protease is provided. Background Technology

[0002] Enzymes are increasingly being used in a variety of applications as a sustainable alternative to petrochemicals. Enzymes are biodegradable and have been able to exert their catalytic activity at lower temperatures, thereby reducing energy consumption. Specifically, in the detergent industry, enzymes are used in detergent formulations to improve cleaning efficiency and / or reduce energy consumption in the washing process.

[0003] Proteases are enzymes capable of hydrolyzing 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 under elevated temperatures and / or denaturing conditions in detergents and washing solutions.

[0004] WO 2016 / 096711 and WO 2016 / 096714 describe subtilisin enzyme variants and detergents containing these variants, which exhibit improved stability and / or improved detergency in liquid detergents compared to the parental subtilisin enzyme. WO 2016 / 001450 and WO 2020 / 002255 disclose subtilisin enzyme variants with increased stability. WO 2010 / 056640 also describes subtilisin protease variants. US 6,376,450 discloses multi-substituted protease variants that provide improved and enhanced cleaning power. US 2020 / 172890 A1 discloses protease variants with enhanced performance and storage stability. WO 99 / 20770 A2, WO 03 / 062381 A2, WO 2011 / 140364 A1, WO 2011 / 072117 A1, WO2011 / 072099 A2, WO 2012 / 151534 A1, WO 2015 / 144932 A1, WO Protease variants are also disclosed in 2017 / 207762 A1, WO 2017 / 192692A1, WO 2018 / 069158 A1, WO 2022 / 225696 A2, WO 2024 / 094732, WO 2024 / 094735 and WO 2024 / 094733.

[0005] However, new proteases are still needed that are stable in harsh cleaning compositions and exhibit good washing performance. Summary of the Invention

[0006] This invention relates to a method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a polypeptide having protease activity or a fragment of said protease-active polypeptide.

[0007] The polypeptide with protease activity mentioned above:

[0008] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0009] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0010] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261 and 271; and

[0011] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260.

[0012] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0013] The present invention further relates to a composition comprising:

[0014] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0015] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0016] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0017] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261 and 271; and

[0018] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260.

[0019] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0020] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol. Detailed Implementation

[0021] The invention can be more readily understood by referring to the following detailed description of embodiments of the invention and the examples included herein.

[0022] Although the invention will be described with reference to specific embodiments, such description should not be construed as limiting.

[0023] definition

[0024] Unless otherwise stated, the terminology used herein should be understood in accordance with the usual usage of those skilled in the art.

[0025] Before describing exemplary embodiments of the invention in detail, definitions important for understanding the invention are given. Unless otherwise stated or obvious from the nature of the definitions, the definitions apply to all compounds, methods, and uses described herein.

[0026] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular form “a / an” also includes the corresponding plural form.

[0027] In the context of this invention, the terms "about" and "approximately" refer to a range of precision that, as will be understood by those skilled in the art, still ensures the technical effect of the features in question. This term typically indicates a deviation from the indicated value of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5%.

[0028] Furthermore, the terms “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” etc., used in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequential or chronological order. It should be understood that such terms are interchangeable where appropriate, and the embodiments of the invention described herein can be operated in orders other than those described or illustrated herein. Where the terms “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” “i,” “ii,” etc., relate to steps of a method or use or measurement, unless otherwise indicated in this application set forth above or below, there is no temporal or time interval continuity between these steps; that is, these steps may be performed simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months, or even years between such steps.

[0029] This application references numerous publications throughout. The disclosures of all such publications, along with the references cited within them, are hereby incorporated in their full text by reference in order to provide a more comprehensive description of the prior art to which this invention pertains.

[0030] It should be understood that the term "comprising" is not limiting. For the purposes of this invention, the term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as containing at least a certain number of members, this means that groups consisting solely of those members are also included.

[0031] "Amino acid substitution" is described by providing the original amino acid, followed by its position number within the amino acid sequence, and then the substituted amino acid. For example, histidine at position 120 being substituted with alanine is specified as "His120Ala" or "H120A". Substitution can also be described by naming only the amino acid obtained in the variant without specifying the parent amino acid at that position, for example by using "X120A" or "120A" or "Xaa120Ala" or "120Ala".

[0032] Variations containing multiple substitutions are separated by "+", for example, "Arg170Tyr+Gly195Glu", "R170Y+G195E", or "X170Y+X195E" represent arginine at position 170 and glycine at position 195 being 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 position, the different substitutions are separated by commas, for example, "Arg170Tyr,Glu" and "R170T,E" represent arginine at position 170 being replaced by tyrosine or glutamic acid, respectively. Alternative substitutions at a specific position can also be expressed as "X120A,G,H", "120A,G,H", "X120A / G / H", or "120A / G / H". Alternatively, different substitutions can be indicated in parentheses, such as "Arg170[Tyr,Gly]" or "Arg170{Tyr,Gly}" or abbreviated as "R170 [Y,G]" or "R170 {Y,G}".

[0033] The amino acid residues of the protease described herein are numbered as is commonly used in the art for proteases (see PN Bryan, Biochimica et Biophysica Acta [Chinese Journal of Biochemistry and Biophysics] 1543(2000), 203-222, see page 204, left column, paragraph 3), according to the numbering of BPN' subtilis protease from Bacillus amyloliquefaciens (whose sequence is shown in SEQ ID NO: 2) (i.e., according to the numbering of SEQ ID NO: 2 or according to the "BPN' numbering").

[0034] Alternatively, the amino acid position can be described by referring to the numbering of SEQ ID NO: 1 or SEQ ID NO: 3 (i.e., according to the numbering of SEQ ID NO: 1 or according to the numbering of SEQ ID NO: 3). Table 1 below shows the amino acid numbers according to SEQ ID NO: 2 and the corresponding amino acid numbers in the sequence according to SEQ ID NO: 1 or 3:

[0035] Table 1

[0036]

[0037] The term “natural” (or naturally, wild-type, or endogenous) cell or organism or polynucleotide or polypeptide refers to a cell or organism or polynucleotide or polypeptide found in nature (i.e., without any human intervention).

[0038] The term “heterogeneous” (or exogenous, foreign, recombinant, non-native, or non-natural) polypeptide is defined herein as follows: a polypeptide that is not natural to the host cell; a polypeptide that is natural to the host cell but has been structurally modified (e.g., deletion, substitution, and / or insertion) by recombinant DNA technology to alter the natural polypeptide; or a polypeptide that is natural to the host cell but whose expression has been quantitatively altered as a result of manipulation of the host cell’s DNA by recombinant DNA technology (e.g., a stronger promoter) or whose expression is directed by a genomic location different from that of the natural host cell. Similarly, the term "heterologous" (or exogenous, foreign, recombinant, non-native, or unnatural) polynucleotide refers to a polynucleotide that is not native to the host cell; a polynucleotide native to the host cell but whose natural expression has been altered by recombinant DNA technology (e.g., deletion, substitution, and / or insertion); a polynucleotide native to the host cell but whose expression has been quantitatively altered as a result of manipulation of the regulatory elements of the polynucleotide by recombinant DNA technology (e.g., a stronger promoter); or a polynucleotide native to the host cell but which has not been integrated into its natural genetic environment as a result of genetic manipulation by recombinant DNA technology. Regarding the relationship between two or more polynucleotide sequences or two or more amino acid sequences, the term "heterologous" is used to characterize two or more polynucleotide sequences or two or more amino acid sequences that do not naturally exist in a specific combination of each other.

[0039] For the purposes of this invention, "recombination" (or transgenic) in relation to cells or organisms means cells or organisms containing heterologous polynucleotides introduced artificially using genetic technology. Regarding polynucleotides, "recombination" includes all constructs produced using genetic technology / recombinant DNA technology, wherein...

[0040] (a) A sequence of a polynucleotide or a portion thereof, or

[0041] (b) One or more genetic control sequences operatively linked to the polynucleotide, including but not limited to promoters, or

[0042] (c) both a) and b)

[0043] It is not located in its wild-type genetic environment or has been artificially modified.

[0044] "Synthetic" compounds are obtained through in vitro chemical and / or enzymatic synthesis.

[0045] Variant polynucleotide and variant polypeptide sequences can be defined by their sequence identity when compared to parental sequences. Sequence identity is typically provided as "sequence identity %" or "identity %". To calculate sequence identity, sequence alignments are generated in the first step. According to the invention, paired global alignments are generated, which means that the two sequences are aligned along their full length, typically generated using mathematical methods known as alignment algorithms.

[0046] According to the present invention, alignments are generated using the algorithm of Needleman and Wunsch (J. Mol. Biol. [Journal of Molecular Biology] (1970) 48, pp. 443-453). Preferably, for the purposes of the present invention, the program "NEEDLE" (European Molecular Biology Open Software Suite (EMBOSS)) is used, with the program default parameters (polynucleotides: vacancy open = 10.0, vacancy extension = 0.5, and matrix = EDNAFULL; peptides: vacancy open = 10.0, vacancy extension = 0.5, and matrix = EBLOSUM62). After aligning the two sequences, in a second step, an identity value is determined based on the generated alignment. For this purpose, the identity % is calculated by dividing the number of identical residues by the length of the aligned region that displays the corresponding sequence of the present invention over its full length, and then multiplying by 100: Identity % = (identical residues / length of the aligned region that displays the corresponding sequence of the present invention over its full length). 100.

[0047] A special aspect of amino acid substitution is conserved mutations, which generally appear to have minimal impact on protein folding, allowing the enzyme properties of the corresponding variant to be largely maintained compared to the parent enzyme. A conserved mutation is a mutation in which an amino acid is exchanged for a similar amino acid. Such an exchange is unlikely to alter the enzyme properties. This paper considers the following conserved exchanges: 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; and amino acid Y is similar to amino acids F, H, and W.

[0048] Conserved amino acid substitutions can occur along the full-length sequence of a functional protein, such as an enzyme. Preferably, such mutations do not involve the functional domains of the enzyme, and more preferably, conserved mutations do not involve the catalytic center of the enzyme.

[0049] As used herein, a “fragment” or “subsequence” refers to a portion of an amino acid sequence. A polypeptide containing one or more amino acid deletions at its N-terminus and / or C-terminus while substantially retaining protease activity is designated herein as a “functional fragment.” Preferably, the functional fragment represents 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 with the original full-length amino acid sequence, 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 functional fragment comprises 100 to 259, 120 to 259, 140 to 259, 160 to 259, 180 to 259, 200 to 259, or 220 to 259 consecutive amino acids of the full-length variant polypeptide. More preferably, the functional fragment retains at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80% identity with the original full-length amino acid sequence, 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 enzyme activity of the original full-length amino acid sequence. The functional fragment comprises amino acids consecutively compared to the original full-length amino acid sequence. In the context of this invention, "original full-length amino acid sequence" is an amino acid sequence that is at least 80% identical to the amino acid sequence according to SEQ ID NO: 1 and has the same length as the amino acid sequence according to SEQ ID NO: 1.

[0050] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention does not contain any internal deletions compared to the amino acid sequence according to SEQ ID NO: 1. However, as discussed above, the variant polypeptide used in the methods and compositions of the present invention may contain the deletion of one or more amino acids at the N-terminus and / or C-terminus of the polypeptide.

[0051] As used herein, a “gene construct” or “expression cassette” is a nucleic acid molecule consisting of at least one target sequence to be expressed, which is operatively linked to one or more control sequences (at least to a promoter) as described herein.

[0052] As used herein, the term "vector" encompasses any kind of construct suitable for carrying a foreign polynucleotide sequence for transfer to another cell or for stable or transient expression within a given cell. As used herein, the term "vector" covers any kind of cloning vector, such as, but not limited to, plasmids, phage particles, viral vectors (e.g., phages), bacteriophages, baculoviruses, entomopathogenic plasmids, artificial chromosomes, and any other vector specific to a particular target host. The foreign polynucleotide sequence typically contains the coding sequence of what may be referred to herein as the "target gene." The target gene may contain introns and exons, depending on the origin or destination of the host cell.

[0053] As used herein, the terms “introduction of polynucleotides” or “conversion of polynucleotides” encompass the transfer of exogenous polynucleotides into host cells, regardless of the method used for transfer. That is, as used herein, the term “conversion of polynucleotides” is independent of the vector, shuttle system, or host cell, and it not only refers to conversion in polynucleotide transfer methods known in the art (see, for example, Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), but also covers any other kind of polynucleotide transfer method, such as, but not limited to, transduction or transfection.

[0054] Polynucleotides encoding polypeptides can be "expressed." The term "expression" or "gene expression" refers to the transcription of one or more specific genes or specific nucleic acid constructs. The term "expression" or "gene expression" also refers to the transcription of one or more genes or gene constructs into structural RNA (e.g., rRNA, tRNA) or mRNA, followed by translation of the latter into a protein or no translation. This process includes DNA transcription and processing of the resulting mRNA product.

[0055] The term "purification" refers to the process of separating and transferring at least one component (e.g., the target protein) from at least another component (e.g., particulate matter from the fermentation broth) into different compartments or phases, wherein these 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 filtrate and osmate; examples of such different phases are precipitate and supernatant or filter cake and filtrate, respectively. The solution obtained after purifying the target enzyme from the fermentation broth is referred to herein as a "purified enzyme solution".

[0056] "Protein formulation" (or "enzyme preparation" or "peptide formulation"), such as "protease formulation," means any non-complex formulation containing a small number of ingredients (preferably 2-8 components) for the purpose of stabilizing the proteins / peptides contained in the protein / peptide formulation and / or stabilizing the protein / peptide formulation itself. Preferably, non-complex protein / peptide formulations contain a higher concentration of proteins / peptides than complex formulations (e.g., than detergent formulations or cleaning compositions). Thus, preferably, non-complex protein formulations are concentrated protein / peptide formulations. Preferably, non-complex protein / peptide formulations contain 20 to 120 mg / g of active enzyme, while complex formulations such as detergent compositions contain 0.002 to 10 mg / g of active enzyme. Compared to non-complex formulations, complex formulations herein mean formulations containing a higher number of ingredients (preferably 9-30 components) for the purpose of stabilizing the proteins contained in the protein formulation and / or stabilizing the protein formulation itself, but additionally, complex formulations contain components for the purposes of complex formulations (e.g., detergent compositions). An example of a non-complex protein formulation is a concentrated enzyme composition used as a stock solution to prepare a complex formulation, such as a detergent composition, wherein the detergent composition contains other compounds (e.g., surfactants and / or chelating agents) that serve the cleaning purpose of the detergent composition.

[0057] "Enzyme properties" include, but are not limited to, catalytic activity, substrate / cofactor specificity, product specificity, stability over time, thermal stability, pH stability, and chemical stability. "Enzyme activity" or "catalytic activity" refers to the catalytic effect exerted by an enzyme, expressed in units per milligram of enzyme (specific activity) or substrate molecules converted per minute per enzyme molecule (molecular activity). Enzyme activity can be specified by the enzyme's actual function; for example, proteases exert proteolytic activity by catalyzing the hydrolysis of peptide bonds, lipases exert lipolytic activity by catalyzing the hydrolysis of ester bonds, and amylase activity involves the hydrolysis of glycosidic bonds in polysaccharides. 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, DelMar et al. (1979), Analytical Biochem 99, 316-320) or Suc-AAPF-AMC (7-amido-4-methylcoumarin) as a substrate. Cleavage of pNA or AMC from the substrate molecule via proteolytic hydrolysis results in the release of free yellow pNA or a change in fluorescence properties, which can be quantified by measuring emission at 460 nm after excitation at 380 nm. Other methods using, for example, casein as a protein substrate are known to those skilled in the art.

[0058] The term "enzyme stability" according to the 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 referred to as "storage stability" and is preferred in the context of the invention.

[0059] To determine and quantify the change in catalytic activity of an enzyme stored or used under certain conditions over time, “enzyme activity” is measured under defined conditions at time zero (100%) and at a later time point (x%). By comparing the measured values, the degree of residual enzyme activity can be determined. The degree of residual enzyme activity determines whether the enzyme is stable or unstable.

[0060] As used in this article, an "enzyme inhibitor" is a compound that slows down or stops enzyme activity. Enzyme inhibitors often also stabilize the three-dimensional structure of enzymes. Therefore, enzyme inhibitors often also act as "enzyme stabilizers."

[0061] "pH stability" refers to the ability of an enzyme to maintain its activity after being exposed to a certain pH value.

[0062] The term "detergent stability" (also referred to herein as "residual activity in detergent" or "storage stability in cleaning composition") refers to the ability of an enzyme to exhibit catalytic activity or cleaning performance after storage in a cleaning composition (preferably at temperatures of 37°C, 45°C, or 50°C for up to 14 days, as described herein in ASEAN2 detergents). Most preferably, "detergent stability" is determined by measuring the residual activity of the protease after storage in a cleaning composition (preferably in ASEAN2 detergents as described herein) at 40°C for 14 days.

[0063] 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 increased cleaning performance added to the cleaning composition by the enzyme’s properties. The term “washing performance” is used similarly herein for both laundry and hard surface cleaning. Washing performance is compared under relevant washing conditions. The term “relevant washing conditions” is used herein to refer to conditions of actual household use in a detergent segment, particularly washing temperature, time, washing machinery, foam concentration, detergent type, and water hardness. The term “improved washing performance” is used to indicate a better end result in stain removal under relevant washing conditions, or, relative to the corresponding control conditions, by weight, a reduction in the amount of enzyme required to achieve the same end result. The term “comparable washing performance” is used to indicate a substantially equal end result in stain removal under relevant washing conditions, or, relative to the corresponding control conditions, by weight, a substantially equal amount of enzyme required to achieve the same end result. As used herein, “substantially equal end result” means that the end result obtained using a specific enzyme variant differs from the end result obtained using the corresponding control conditions (e.g., the corresponding wild-type enzyme) by 30% or less. In this invention, the washing performance is preferably determined for egg stains.

[0064] As used herein, the term "specific performance" refers to the cleaning and removal of a specific stain or dirt per unit of active enzyme. In some embodiments, specific performance is determined using stains or dirt such as egg, egg yolk, milk, grass, minced meat, blood, chocolate sauce, baby food, sebum, etc.

[0065] The term "detergent composition" or "detergent" means a composition specified for cleaning soiled materials. Within this application, the terms "detergent composition" and "cleaning composition" are used interchangeably. A detergent composition is a compound formulation as further defined herein. Detergent compositions include detergent compositions for various applications such as laundry and hard surface cleaning. Detergent compositions also include biodegradable detergent compositions. The term "detergent component" is defined herein to mean a class of chemicals that can be used in a detergent composition. A typical detergent component is a surfactant. "Surfactant" (used herein synonymously with "surface active agent") means an organic chemical that, when added to a liquid, alters the properties of that liquid at the interface. Depending on their ionic charge, surfactants are referred to as nonionic surfactants, anionic surfactants, cationic surfactants, or amphoteric surfactants. The term "effective amount of 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 power), the amount of certain components that effectively provide optical benefits (e.g., optical whitening, dye transfer inhibition, color care), and the amount of certain components that effectively aid processing (maintaining physical characteristics during processing, storage, and use; e.g., rheology modifiers, water-soluble additives, desiccants). Detergent compositions typically have a protease concentration of 0.002 to 10 mg / g of active enzyme.

[0066] The term "laundry" or "laundering" encompasses both household and industrial laundry, and refers to the process of treating textiles and / or fabrics with a solution containing a detergent composition. Laundry can be performed using technical equipment such as household or industrial washing machines. Alternatively, laundry can be done manually.

[0067] The term “textiles” means any textile material, including yarn (threads made of natural or synthetic fibers for knitting or weaving), yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and fabrics (such as garments, cloths, and other articles) made from these materials. As used herein, the terms “fabric” (textiles made from woven, knitted, or felted fibers) or “garment” (any clothing made of textiles) are also intended to include the broader term “textiles.”

[0068] The term "fiber" includes natural fibers, synthetic fibers, and blends thereof. Examples of natural fibers are plant-derived fibers (such as flax, jute, and cotton) or animal-derived fibers containing proteins like collagen, keratin, and fibroin (such as silk, sheep's wool, angora wool, mohair, and cashmere). Examples of synthetic fibers are polyurethane fibers (such as Spandex).® or Lycra ® ), polyester fibers, polyolefins (such as elastofin), or polyamide fibers (such as nylon). The fiber can be a monofilament or part of a textile (such as knitted, woven, or nonwoven fabric).

[0069] The term "hard surface cleaning" encompasses both household and industrial hard surface cleaning, and refers to the process of treating hard surfaces with a solution containing a detergent composition. Hard surfaces can include any hard surface in a household or industrial setting, such as floors, furniture, walls, sanitary ceramics, glass, metal surfaces (including knives or cutlery), and medical devices such as diagnostic instruments, trays, discs, clamps, shelves, tweezers, scissors, shearing machines, saws (e.g., bone saws and their blades), hemostats, knives, chisels, bone forceps, files, pliers, drills, drill bits, coarse files, dental drills, lateral spreaders, lithotripters, lifters, clamps, needle holders, support frames, clips, hooks, chisels, scrapers, retractors, straighteners, punches, extractors, spoons, corneal knives, tongue depressors, squeezers, cannulas, dilators, interbody fusion devices, glassware, tubing, catheters, cannulas, plugs, stents, endoscopes, arthroscopes, and related equipment. One specific form of hard surface cleaning is dishwashing, particularly automatic dishwashing (ADW).

[0070] The term "dishwashing" refers to all forms of dishwashing, such as manual or automatic dishwashing. Dishwashing includes, but is not limited to, cleaning all forms of earthenware, such as plates, cups, glasses, and bowls; all forms of cutlery, such as spoons, knives, forks, and communal cutlery; as well as ceramic, plastic (such as melamine), metal, porcelain, glass, and acrylic products.

[0071] Cleaning performance is evaluated under relevant cleaning conditions. In this paper, the term "relevant cleaning conditions" refers to the conditions actually used in a washing machine, automatic dishwashing machine, or manual cleaning process, particularly cleaning temperature, time, cleaning machinery, foam concentration, detergent type, and water hardness.

[0072] The term "medical device cleaning" refers to the cleaning steps involved in reprocessing reusable medical devices. Medical device cleaning methods can be categorized into two types: manual cleaning methods and mechanical / automatic cleaning methods. Manual cleaning is used when no mechanical device is available or when the medical device to be cleaned is too fragile or difficult to clean mechanically. Mechanical / automatic cleaning methods remove dirt and microorganisms through automated cleaning and rinsing processes, including ultrasonic cleaning and washing.

[0073] In the field of stain removal, the term "stain" is generally used when referring to the cleaning of clothing, such as textiles, fabrics, or fibers, while the term "dirt" is generally used when referring to the cleaning of hard surfaces, such as tableware and knives. However, the terms "stain" and "dirt" should be used interchangeably in this article.

[0074] As used herein, the difference between a “chelating builder” and a precipitating builder is that when the builder is used in an amount sufficient to bind with all calcium ions in an aqueous solution initially having a hardness of 7 °dH (German hardness) at neutral pH, no significant amount of precipitation is formed. “Strong builder” is classified as a highly effective chelating agent, capable of strongly binding divalent cations (such as Ca2+), and the logarithmic stability constant (Log K) of the cation / chelating agent complex is [missing value]. Ca The stability constant is greater than 4, particularly greater than 5, 6, or 7. The stability constant is determined at an ionic strength of 0.1 M and a temperature of 25°C. "Strong chelating builders" combine the above two properties. Strong chelating builders include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetra(methylenephosphonic acid) (EDTMP), triazinetrimethylenephosphonic acid (NTMP), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), methylglycine diacetic acid (MGDA), triazinetrimethyleneacetic acid (NTA), 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP), sodium tripolyphosphate (STPP), iminodisuccinic acid (IDS), tetrasodium N,N-diacetic acid (GLDA), pyrophosphate, and ethylenediaminedisuccinic acid (EDDS), preferably MGDA and / or EDDS.

[0075] "Antimicrobial agents" are compounds that kill or inhibit the growth or reproduction of microorganisms. Microorganisms can be bacteria, yeasts, or molds. "Preservatives" are antimicrobial agents that can be added to aqueous products and compositions to maintain the original properties, characteristics, and integrity of the product and composition by killing or inhibiting the growth of contaminating microorganisms.

[0076] A composition that is “substantially free” of a compound shall be meant herein to mean that the corresponding compound is not intentionally added to the composition, which means that there is at most an ineffective amount, and most preferably the composition contains 0% of the compound.

[0077] A “composition” is a mixture of multiple components (also called ingredients) prepared according to a specific formulation. The composition can be liquid or solid. The liquid compositions of the present invention include solutions, emulsions and dispersions, gels, etc., as long as the liquid is fluid and pourable. The dynamic viscosity of the liquid compositions according to the present invention is preferably from about 500 to about 20,000 mPa. Within the range of s, it is determined according to the Brookfield method, for example, using a Brookfield viscometer LVT-II with rotor No. 3 at 20 rpm at 25°C.

[0078] As used in this article, "gel" refers to a shear-thinned lamellar gel with a pour viscosity of 100 to 5,000 mPa. Within the range of millipascal-seconds (s), more preferably less than 3,000 mPa s, most preferably less than 1,500 mPa The composition includes an enzyme composition (e.g., a liquid enzyme composition as described herein) and a cleaning composition (e.g., a liquid cleaning composition as described herein).

[0079] The components of a composition can be separated by formulating them in different compartments (such as the different compartments of a multi-compartment pouch or bottle with different chambers), and the liquid is poured out simultaneously from these different compartments in predetermined amounts to ensure that the correct amount of each component from each compartment is applied at each individual point of use. Such multi-compartment pouches and bottles are also known to those skilled in the art.

[0080] The compositions of the present invention can be delivered in dual- or multi-compartment containers, in single- or multi-phase unit doses, in spray or foam detergents, as pre-wetted wipes (i.e., cleaning compositions combined with nonwoven materials, as discussed in US 6,121,165, Mackey et al.), and in other homogeneous, non-homogeneous, or single- or multi-phase cleaning product forms.

[0081] Detailed description

[0082] In this invention, methods and compositions comprising novel proteases are provided.

[0083] Protease variants

[0084] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0085] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0086] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0087] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261 and 271; and

[0088] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260.

[0089] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0090] The variant peptides used in the methods and compositions of the present invention are variant peptides with protease activity, wherein:

[0091] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0092] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0093] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261 and 271; and

[0094] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260.

[0095] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0096] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0097] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0098] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X21I / V / F / W / Y, X25D, X38R / K / H / W, X160H / S / D / E / P, X210I / V, X212G / M / R / K / P / A / F / H / W / N / Y / D, X228S, X261L / M / F / E / W / Y / C and X271D;

[0099] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q / E / D, X24K, X56D, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X103S, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X218S / T / D, X248Q / R and X260D / E / K.

[0100] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0101] In one embodiment, the variant polypeptide or fragment thereof, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions X21I / V / F / W / Y, X25D, X38R / K / H / W, X160H / S / D / E / P, X210I / V, X212G / M / R / K / P / A / F / H / W / N / Y / D, X228S, X261L / M / F / E / W / Y / C, and X271D. In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all of the amino acid substitutions X18Q / E / D, X24K, X56D, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X103S, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X218S / T / D, X248Q / R, and X260D / E / K.

[0102] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof comprises amino acid substitutions X21I / V / F / W / Y, X25D, X38R / K / H / W, X160H / S / D / E / P, X210I / V, X212G / M / R / K / P / A / F / H / W / N / Y / D, X228S, X261L / M / F / E / W / Y / C, and X271D, as well as amino acid substitution X18Q / E / D, X24K, X56D, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X103S, / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X218S / T / D, X248Q / R, and

[0103] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0104] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0105] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0106] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21V, X25D, X38K, X160H, X210I, X212P, X228S, X261F and X271D;

[0107] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q, X24K, X56D, X78N, X103S, X109K, X144N, X182K, X183D, X194E, X204D, X218S, X248Q and X260D.

[0108] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0109] In one embodiment, the variant polypeptide or fragment thereof comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions X21V, X25D, X38K, X160H, X210I, X212P, X228S, X261F, and X271D, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2. In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all of the amino acid substitutions X18Q, X24K, X56D, X78N, X103S, X109K, X144N, X182K, X183D, X194E, X204D, X218S, X248Q, and X260D.

[0110] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof comprises amino acid substitutions of X21V, X25D, X38K, X160H, X210I, X212P, X228S, X261F and X271D, and amino acid substitutions of X18Q, X24K, X56D, X78N, X103S, X109K, X144N, X182K, X183D, X194E, X204D, X218S, X248Q and X260D.

[0111] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0112] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0113] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0114] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of L21I / V / F / W / Y, G25D, T38R / K / H / W, S160H / S / D / E / P, P210I / V, S212G / M / R / K / P / A / F / H / W / N / Y / D, A228S, N261L / M / F / E / W / Y / C and E271D;

[0115] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of N18Q / E / D, S24K, S56D, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, A103S, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, N218S / T / D, N248Q / R and T260D / E / K.

[0116] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0117] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions L21I / V / F / W / Y, G25D, T38R / K / H / W, S160H / S / D / E / P, P210I / V, S212G / M / R / K / P / A / F / H / W / N / Y / D, A228S, N261L / M / F / E / W / Y / C, and E271D. In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all of the amino acid substitutions N18Q / E / D, S24K, S56D, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, A103S, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, N218S / T / D, N248Q / R, and T260D / E / K.

[0118] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof comprises amino acid substitutions L21I / V / F / W / Y, G25D, T38R / K / H / W, S160H / S / D / E / P, P210I / V, S212G / M / R / K / P / A / F / H / W / N / Y / D, A228S, N261L / M / F / E / W / Y / C and E271D, and amino acid substitution N18Q / E / D, S24K, S56D, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, A103S, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, N218S / T / D, N248Q / R and T260D / E / K.

[0119] In a preferred embodiment, referring to SEQ ID NO: 2, the variant polypeptide or fragment thereof having protease activity used in the methods and compositions of the present invention contains amino acid residue Q at position 59.

[0120] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0121] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0122] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0123] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261 and 271; and

[0124] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260.

[0125] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0126] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0127] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0128] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0129] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven or at least eight amino acid substitutions X21I / V / F / W / Y, X25D, X38R / K / H / W, X160H / S / D / E / P, X210I / V, X212G / M / R / K / P / A / F / H / W / N / Y / D, X228S, X261L / M / F / E / W / Y / C and X271D;

[0130] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen amino acid substitutions of X18Q / E / D, X24K, X56D, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X103S, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X218S / T / D, X248Q / R, and X260D / E / K.

[0131] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0132] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0133] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0134] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0135] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions X21I / V / F / W / Y, X25D, X38R / K / H / W, X160H / S / D / E / P, X210I / V, X212G / M / R / K / P / A / F / H / W / N / Y / D, X228S, X261L / M / F / E / W / Y / C and X271D;

[0136] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions X18Q / E / D, X24K, X56D, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X103S, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X218S / T / D, X248Q / R, and X260D / E / K.

[0137] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0138] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0139] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0140] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0141] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven or at least eight amino acid substitutions of X21V, X25D, X38K, X160H, X210I, X212P, X228S, X261F and X271D;

[0142] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen amino acid substitutions of X18Q, X24K, X56D, X78N, X103S, X109K, X144N, X182K, X183D, X194E, X204D, X218S, X248Q, and X260D.

[0143] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0144] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0145] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0146] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0147] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions X21V, X25D, X38K, X160H, X210I, X212P, X228S, X261F and X271D;

[0148] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions of X18Q, X24K, X56D, X78N, X103S, X109K, X144N, X182K, X183D, X194E, X204D, X218S, X248Q, and X260D.

[0149] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0150] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0151] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0152] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0153] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven or at least eight amino acid substitutions of L21I / V / F / W / Y, G25D, T38R / K / H / W, S160H / S / D / E / P, P210I / V, S212G / M / R / K / P / A / F / H / W / N / Y / D, A228S, N261L / M / F / E / W / Y / C and E271D;

[0154] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen amino acid substitutions of N18Q / E / D, S24K, S56D, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, A103S, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, N218S / T / D, N248Q / R, and T260D / E / K.

[0155] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0156] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0157] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0158] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0159] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions L21I / V / F / W / Y, G25D, T38R / K / H / W, S160H / S / D / E / P, P210I / V, S212G / M / R / K / P / A / F / H / W / N / Y / D, A228S, N261L / M / F / E / W / Y / C and E271D;

[0160] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions N18Q / E / D, S24K, S56D, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, A103S, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, A194E / D / C, N204D / E / C / G, N218S / T / D, N248Q / R, and T260D / E / K.

[0161] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0162] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0163] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0164] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0165] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 38, 212, 261 and 271; and

[0166] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 18 and 218.

[0167] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0168] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0169] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0170] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0171] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X38R / K / H / W, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C and X271D;

[0172] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q / E / D and X218S / T / D.

[0173] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0174] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof contains at least two, at least three, or all of the amino acid substitutions X38R / K / H / W, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, and X271D. In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof contains all of the amino acid substitutions X18Q / E / D and X218S / T / D.

[0175] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof contains amino acid substitutions X38R / K / H / W, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C and X271D, and amino acid substitutions X18Q / E / D and X218S / T / D.

[0176] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0177] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0178] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0179] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X38K, X212P, X261F and X271D;

[0180] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q and X218S.

[0181] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0182] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof contains at least two, at least three, or all of the amino acid substitutions X38K, X212P, X261F, and X271D. In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof contains all of the amino acid substitutions X18Q and X218S.

[0183] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof contains amino acid substitutions X38K, X212P, X261F and X271D and amino acid substitutions X18Q and X218S.

[0184] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0185] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0186] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0187] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of T38R / K / H / W, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C and E271D;

[0188] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of N18Q / E / D and N218S / T / D.

[0189] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0190] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof contains amino acid substitutions for at least two, at least three, or all of T38R / K / H / W, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, and E271D. In one embodiment, the variant polypeptide or fragment thereof contains amino acid substitutions for all of N18Q / E / D and N218S / T / D.

[0191] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof contains amino acid substitutions T38R / K / H / W, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C and E271D, and amino acid substitutions N18Q / E / D and N218S / T / D.

[0192] In a preferred embodiment, referring to SEQ ID NO: 2, the variant polypeptide or fragment thereof having protease activity used in the methods and compositions of the present invention contains amino acid residue Q at position 59.

[0193] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0194] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0195] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0196] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 38, 212, 261 and 271; and

[0197] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 18 and 218.

[0198] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0199] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0200] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0201] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0202] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one, at least two, or at least three amino acid substitutions selected from the group consisting of X38R / K / H / W, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, and X271D;

[0203] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q / E / D and X218S / T / D.

[0204] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0205] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0206] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0207] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0208] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions X38R / K / H / W, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C and X271D;

[0209] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, containing amino acid substitutions X18Q / E / D and X218S / T / D,

[0210] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0211] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0212] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0213] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0214] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one, at least two, or at least three amino acid substitutions selected from the group consisting of X38K, X212P, X261F, and X271D;

[0215] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q and X218S.

[0216] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0217] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0218] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0219] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0220] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions X38K, X212P, X261F and X271D;

[0221] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions X18Q and X218S.

[0222] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0223] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0224] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0225] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0226] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one, at least two, or at least three amino acid substitutions selected from the group consisting of T38R / K / H / W, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, and E271D;

[0227] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of N18Q / E / D and N218S / T / D.

[0228] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0229] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0230] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0231] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0232] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions T38R / K / H / W, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C and E271D;

[0233] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions N18Q / E / D and N218S / T / D.

[0234] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0235] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0236] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0237] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0238] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at the amino acid residue selected from the group consisting of 21, 38, 160, 210 and 228; and

[0239] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 109, 144, 182, 183, 204, 218 and 248.

[0240] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0241] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0242] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0243] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0244] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21I / V / F / W / Y, X38R / K / H / W, X160H / S / D / E / P, X210I / V and X228S;

[0245] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q / E / D, X24K, X56D, 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, X218S / T / D and X248Q / R.

[0246] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0247] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof contains at least two, at least three, at least four, or all of the amino acid substitutions X21I / V / F / W / Y, X38R / K / H / W, X160H / S / D / E / P, X210I / V, and X228S. In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the amino acid substitutions X18Q / E / D, X24K, X56D, 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, X218S / T / D, and X248Q / R.

[0248] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof comprises amino acid substitutions X21I / V / F / W / Y, X38R / K / H / W, X160H / S / D / E / P, X210I / V and X228S, and amino acid substitutions X18Q / E / D, X24K, X56D, 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, X218S / T / D and X248Q / R.

[0249] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0250] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0251] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0252] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21V, X38K, X160H, X210I and X228S;

[0253] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q, X24K, X56D, X78N, X109K, X144N, X182K, X183D, X204D, X218S and X248Q.

[0254] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0255] In one embodiment, the variant polypeptide or fragment thereof, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions X21V, X38K, X160H, X210I, and X228S. In another embodiment, the variant polypeptide or fragment thereof, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all of the amino acid substitutions X18Q, X24K, X56D, X78N, X109K, X144N, X182K, X183D, X204D, X218S, and X248Q.

[0256] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof comprises amino acid substitutions X21V, X38K, X160H, X210I and X228S, and amino acid substitutions X18Q, X24K, X56D, X78N, X109K, X144N, X182K, X183D, X204D, X218S and X248Q.

[0257] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0258] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0259] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0260] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of L21I / V / F / W / Y, T38R / K / H / W, S160H / S / D / E / P, P210I / V and A228S;

[0261] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of N18Q / E / D, S24K, S56D, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, N204D / E / C / G, N218S / T / D and N248Q / R.

[0262] The fragment of this variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0263] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof contains at least two, at least three, at least four, or all of the amino acid substitutions L21I / V / F / W / Y, T38R / K / H / W, S160H / S / D / E / P, P210I / V, and A228S. In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the amino acid substitutions N18Q / E / D, S24K, S56D, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, N204D / E / C / G, N218S / T / D, and N248Q / R.

[0264] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof comprises amino acid substitutions L21I / V / F / W / Y, T38R / K / H / W, S160H / S / D / E / P, P210I / V and A228S, and amino acid substitutions N18Q / E / D, S24K, S56D, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, N204D / E / C / G, N218S / T / D and N248Q / R.

[0265] In a preferred embodiment, referring to SEQ ID NO: 2, the variant polypeptide or fragment thereof having protease activity used in the methods and compositions of the present invention contains amino acid residue Q at position 59.

[0266] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0267] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0268] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0269] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at the amino acid residue selected from the group consisting of 21, 38, 160, 210 and 228; and

[0270] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 109, 144, 182, 183, 204, 218 and 248.

[0271] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0272] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0273] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0274] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0275] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one, at least two, at least three or at least four amino acid substitutions selected from the group consisting of X21I / V / F / W / Y, X38R / K / H / W, X160H / S / D / E / P, X210I / V and X228S;

[0276] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid substitutions selected from the group consisting of X18Q / E / D, X24K, X56D, 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, X218S / T / D, and X248Q / R.

[0277] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0278] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0279] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0280] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0281] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions X21I / V / F / W / Y, X38R / K / H / W, X160H / S / D / E / P, X210I / V and X228S;

[0282] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions X18Q / E / D, X24K, X56D, 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, X218S / T / D, and X248Q / R.

[0283] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0284] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0285] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0286] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0287] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one, at least two, at least three or at least four amino acid substitutions selected from the group consisting of X21V, X38K, X160H, X210I and X228S;

[0288] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid substitutions selected from the group consisting of X18Q, X24K, X56D, X78N, X109K, X144N, X182K, X183D, X204D, X218S, and X248Q.

[0289] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0290] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0291] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0292] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0293] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions X21V, X38K, X160H, X210I and X228S;

[0294] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions of X18Q, X24K, X56D, X78N, X109K, X144N, X182K, X183D, X204D, X218S, and X248Q.

[0295] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0296] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0297] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0298] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0299] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one, at least two, at least three or at least four amino acid substitutions selected from the group consisting of L21I / V / F / W / Y, T38R / K / H / W, S160H / S / D / E / P, P210I / V and A228S;

[0300] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid substitutions selected from the group consisting of N18Q / E / D, S24K, S56D, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, N204D / E / C / G, N218S / T / D, and N248Q / R.

[0301] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0302] The variant polypeptide used in the methods and compositions of the present invention is a variant polypeptide with protease activity or a fragment of said variant polypeptide with protease activity, wherein the variant polypeptide:

[0303] (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1;

[0304] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0305] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions L21I / V / F / W / Y, T38R / K / H / W, S160H / S / D / E / P, P210I / V and A228S;

[0306] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions N18Q / E / D, S24K, S56D, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, Q109K / A, S144N / R, Q182K / R / E, N183D / E / C / Q / A / M, N204D / E / C / G, N218S / T / D, and N248Q / R.

[0307] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0308] The protease-active variant polypeptides used in the methods and compositions of the present invention are non-naturally occurring proteases. Preferably, the protease-active variant polypeptides used in the methods and compositions of the present invention are purified, isolated, synthetic, and / or recombinant proteases. Preferably, the protease-active variant polypeptides used in the methods and compositions of the present invention are purified and recombinant proteases.

[0309] The protease according to the invention has “proteolytic activity” or “protease activity.” “Proteolytic activity” or “protease activity” describes the ability to hydrolyze peptide bonds in a polypeptide. 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 a substrate. Cleavage of pNA or AMC from substrate molecules via proteolytic hydrolysis results in the release of free yellow pNA or a change in fluorescence properties, which can be quantified by measuring emission at 460 nm after excitation at 380 nm.

[0310] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, and at least 95%. 4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5% sequence identity, and

[0311] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0312] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X21I / V / F / W / Y, X25D, X38R / K / H / W, X160H / S / D / E / P, X210I / V, X212G / M / R / K / P / A / F / H / W / N / Y / D, X228S, X261L / M / F / E / W / Y / C and X271D, and

[0313] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q / E / D, X24K, X56D, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X103S, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X218S / T / D, X248Q / R and X260D / E / K.

[0314] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, and at least 95%. 4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5% sequence identity, and

[0315] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0316] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21V, X25D, X38K, X160H, X210I, X212P, X228S, X261F and X271D, and

[0317] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q, X24K, X56D, X78N, X103S, X109K, X144N, X182K, X183D, X194E, X204D, X218S, X248Q and X260D.

[0318] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, and at least 95%. 4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5% sequence identity, and

[0319] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0320] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X21I / V / F / W / Y, X25D, X38R / K / H / W, X160H / S / D / E / P, X210I / V, X212G / M / R / K / P / A / F / H / W / N / Y / D, X228S, X261L / M / F / E / W / Y / C and X271D, and

[0321] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q / E / D, X24K, X56D, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X103S, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X218S / T / D, X248Q / R, and X260D / E / K.

[0322] Referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0323] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, and at least 95%. 4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5% sequence identity, and

[0324] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0325] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21V, X25D, X38K, X160H, X210I, X212P, X228S, X261F and X271D, and

[0326] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q, X24K, X56D, X78N, X103S, X109K, X144N, X182K, X183D, X194E, X204D, X218S, X248Q and X260D.

[0327] Referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0328] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with a content of 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 80% to 98%, 90.3% to 98%, 90.5% to 98%, or 90.7%. The sequence identity was 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, and 95.5% to 98%.

[0329] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0330] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X21I / V / F / W / Y, X25D, X38R / K / H / W, X160H / S / D / E / P, X210I / V, X212G / M / R / K / P / A / F / H / W / N / Y / D, X228S, X261L / M / F / E / W / Y / C and X271D, and

[0331] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q / E / D, X24K, X56D, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X103S, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X218S / T / D, X248Q / R and X260D / E / K.

[0332] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with a content of 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 80% to 98%, 90.3% to 98%, 90.5% to 98%, or 90.7%. The sequence identity was 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, and 95.5% to 98%.

[0333] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0334] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21V, X25D, X38K, X160H, X210I, X212P, X228S, X261F and X271D, and

[0335] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q, X24K, X56D, X78N, X103S, X109K, X144N, X182K, X183D, X194E, X204D, X218S, X248Q and X260D.

[0336] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with a content of 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 80% to 98%, 90.3% to 98%, 90.5% to 98%, or 90.7%. The sequence identity was 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, and 95.5% to 98%.

[0337] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0338] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X21I / V / F / W / Y, X25D, X38R / K / H / W, X160H / S / D / E / P, X210I / V, X212G / M / R / K / P / A / F / H / W / N / Y / D, X228S, X261L / M / F / E / W / Y / C and X271D; and

[0339] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q / E / D, X24K, X56D, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X103S, X109K / A, X144N / R, X182K / R / E, X183D / E / C / Q / A / M, X194E / D / C, X204D / E / C / G, X218S / T / D, X248Q / R, and X260D / E / K.

[0340] Referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0341] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with a content of 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 80% to 98%, 90.3% to 98%, 90.5% to 98%, or 90.7%. The sequence identity was 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, and 95.5% to 98%.

[0342] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0343] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X21V, X25D, X38K, X160H, X210I, X212P, X228S, X261F and X271D; and

[0344] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q, X24K, X56D, X78N, X103S, X109K, X144N, X182K, X183D, X194E, X204D, X218S, X248Q and X260D.

[0345] Referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0346] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, and at least 95%. 4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5% sequence identity, and

[0347] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0348] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X38R / K / H / W, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C and X271D, and

[0349] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q / E / D and X218S / T / D.

[0350] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, and at least 95%. 4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5% sequence identity, and

[0351] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0352] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X38K, X212P, X261F and X271D, and

[0353] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q and X218S.

[0354] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, and at least 95%. 4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5% sequence identity, and

[0355] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0356] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X38R / K / H / W, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C and X271D, and

[0357] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q / E / D and X218S / T / D.

[0358] Referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0359] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, and at least 95%. 4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5% sequence identity, and

[0360] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0361] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X38K, X212P, X261F and X271D, and

[0362] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q and X218S.

[0363] Referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0364] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with a content of 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 80% to 98%, 90.3% to 98%, 90.5% to 98%, or 90.7%. The sequence identity was 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, and 95.5% to 98%.

[0365] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0366] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X38R / K / H / W, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C and X271D, and

[0367] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q / E / D and X218S / T / D.

[0368] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with a content of 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 80% to 98%, 90.3% to 98%, 90.5% to 98%, or 90.7%. The sequence identity was 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, and 95.5% to 98%.

[0369] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0370] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X38K, X212P, X261F and X271D, and

[0371] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q and X218S.

[0372] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with a content of 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 80% to 98%, 90.3% to 98%, 90.5% to 98%, or 90.7%. The sequence identity was 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, and 95.5% to 98%.

[0373] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0374] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X38R / K / H / W, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C and X271D, and

[0375] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q / E / D and X218S / T / D.

[0376] Referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0377] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with a content of 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 80% to 98%, 90.3% to 98%, 90.5% to 98%, or 90.7%. The sequence identity was 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, and 95.5% to 98%.

[0378] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0379] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X38K, X212P, X261F and X271D, and

[0380] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X18Q and X218S.

[0381] Referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0382] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, and at least 95%. 4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5% sequence identity, and

[0383] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0384] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21I / V / F / W / Y, X38R / K / H / W, X160H / S / D / E / P, X210I / V and X228S, and

[0385] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q / E / D, X24K, X56D, 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, X218S / T / D and X248Q / R.

[0386] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, and at least 95%. 4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5% sequence identity, and

[0387] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0388] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21V, X38K, X160H, X210I and X228S, and

[0389] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q, X24K, X56D, X78N, X109K, X144N, X182K, X183D, X204D, X218S and X248Q.

[0390] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, and at least 95%. 4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5% sequence identity, and

[0391] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0392] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21I / V / F / W / Y, X38R / K / H / W, X160H / S / D / E / P, X210I / V and X228S, and

[0393] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q / E / D, X24K, X56D, 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, X218S / T / D and X248Q / R.

[0394] Referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0395] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, and at least 95%. 4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5% sequence identity, and

[0396] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0397] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21V, X38K, X160H, X210I and X228S, and

[0398] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q, X24K, X56D, X78N, X109K, X144N, X182K, X183D, X204D, X218S and X248Q.

[0399] Referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0400] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with a content of 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 80% to 98%, 90.3% to 98%, 90.5% to 98%, or 90.7%. The sequence identity was 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, and 95.5% to 98%.

[0401] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0402] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21I / V / F / W / Y, X38R / K / H / W, X160H / S / D / E / P, X210I / V and X228S, and

[0403] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q / E / D, X24K, X56D, 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, X218S / T / D and X248Q / R.

[0404] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with a content of 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 80% to 98%, 90.3% to 98%, 90.5% to 98%, or 90.7%. The sequence identity was 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, and 95.5% to 98%.

[0405] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0406] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21V, X38K, X160H, X210I and X228S, and

[0407] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q, X24K, X56D, X78N, X109K, X144N, X182K, X183D, X204D, X218S and X248Q.

[0408] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with a content of 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 80% to 98%, 90.3% to 98%, 90.5% to 98%, or 90.7%. The sequence identity was 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, and 95.5% to 98%.

[0409] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0410] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21I / V / F / W / Y, X38R / K / H / W, X160H / S / D / E / P, X210I / V and X228S, and

[0411] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q / E / D, X24K, X56D, 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, X218S / T / D and X248Q / R.

[0412] Referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0413] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with a content of 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 80% to 98%, 90.3% to 98%, 90.5% to 98%, or 90.7%. The sequence identity was 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, and 95.5% to 98%.

[0414] (a) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0415] (b) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution selected from the group consisting of X21V, X38K, X160H, X210I and X228S, and

[0416] (c) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it comprises at least one amino acid substitution selected from the group consisting of X18Q, X24K, X56D, X78N, X109K, X144N, X182K, X183D, X204D, X218S and X248Q.

[0417] Referring to SEQ ID NO: 2, the variant polypeptide contains glutamine at position 59.

[0418] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, or at least 95.4%. The sequence identity is at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5%, with reference to SEQ ID NO: 2, containing a serine residue at position 9, and compared with the amino acid sequence shown in SEQ ID NO: 3 and with reference to SEQ ID NO: 2, containing one of the combinations of amino acid substitutions in Table 2.

[0419] Table 2

[0420]

[0421]

[0422] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 of at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, at least 95%... 0.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, 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% but less than 97.5% sequence identity, referring to SEQ ID NO: 2, containing a serine residue at position 9, referring to SEQ ID NO: 2, containing glutamine at position 59, and compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to SEQ ID NO: 2, containing one of the combinations of amino acid substitutions in Table 2.

[0423] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with a content of 79% to 98%, 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7%. Sequence identity of 95% to 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, or 95.5% to 98%, refer to SEQ ID. Number NO: 2 contains a serine residue at position 9, and compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to number SEQ ID NO: 2, contains one of the combinations of amino acid substitutions in Table 2.

[0424] In one embodiment, the variant polypeptide used in the methods and compositions of the present invention preferably has an amino acid sequence according to SEQ ID NO: 1 with 79% to 98%, 79.5% to 98%, 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, and 90.5% to 98% of the amino acid sequence. Sequence identity of 90.7% to 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, or 95.5% to 98%, refer to SEQ. The sequence number SEQ ID NO: 2 contains a serine residue at position 9, and glutamine at position 59, compared with the amino acid sequence shown in SEQ ID NO: 3 and with reference to the sequence number SEQ ID NO: 2, contains one of the combinations of amino acid substitutions in Table 2.

[0425] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide or fragment thereof contains a combination of substitutions selected from the group consisting of:

[0426] (a) X18Q; X21V; X24K; X25D; X38K;

[0427] X183D;

[0428] (b)X18Q;X21V;X24K;X38W;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;

[0429] X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X260D;X261F;X271D;

[0430] (c)X18Q;X21V;X24K;X38K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;

[0431] X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X260D;X261F;X271D;

[0432] (d)X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;

[0433] X183D;X194E;X204D;X210I;X218S;X228S;X248Q;X260D;X261F;X271D;

[0434] (e)X18Q;X21V;X24K;X25D;X38K;X43R;X56D;X78N;X103S;X109K;X144N;X160H;X182K;

[0435] X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X261F;

[0436] (f)X18Q;X21V;X24K;X25D;X38K;X43R;X56D;X78N;X109K;X144N;X160H;X182K;X183D;

[0437] X194E;X204D;X210I;X212P;X218S;X228S;X248Q;

[0438] (g)X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X109K;X144N;X160H;X182K;X183D;

[0439] X194E; X204D; X210I; X212P; X218S;

[0440] (h)X18Q;

[0441] X194E; X204D; X210I; X212P; X218S;

[0442] (i)X18Q; X21V; X24K; X38K; X56D;

[0443] X183D; X194E; X204D; X210I; X212P; X218S; X228S; X248Q; X260D; X271D; and

[0444] (j)X18Q;

[0445] X183D; X194E; X204D; X210I;

[0446] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the variant polypeptide comprises a combination of substitutions selected from the group consisting of:

[0447] (a)N18Q; L21V; S24K; G25D; T38K; S56D; S78N; A103S; Q109K; S144N; S160H; Q182K;

[0448] N183D; A194E; N204D; P210I; S212P; N218S; A228S; N248Q; T260D; N261F; E271D;

[0449] (b)N18Q; L21V; S24K; T38W; S56D; S78N; A103S; Q109K; S144N; S160H; Q182K; N183D;

[0450] A194E;N204D;P210I;S212P;N218S;A228S;N248Q;T260D;N261F;E271D;

[0451] (c)N18Q;L21V;S24K;T38K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;

[0452] N204D;P210I;S212P;N218S;A228S;N248Q;S259D;T260D;N261F;E271D;

[0453] (d)N18Q;L21V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;

[0454] N183D;A194E;N204D;P210I;N218S;A228S;N248Q;T260D;N261F;E271D;

[0455] (e)N18Q;L21V;S24K;G25D;T38K;N43R;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;

[0456] N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;N261F;

[0457] (f)N18Q;L21V;S24K;G25D;T38K;N43R;S56D;S78N;Q109K;S144N;S160H;Q182K;N183D;

[0458] A194E;N204D;P210I;S212P;N218S;A228S;N248Q;

[0459] (g)N18Q;L21V;S24K;G25D;T38K;N43K;S56D;S78N;Q109K;S144N;S160H;Q182K;N183D;

[0460] A194E;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;N261F;

[0461] (h)N18Q; L21V; S24K; T38W; S56D; Q59K; S78N; A103S; Q109K; S144N; S160H; Q182K; N183D;

[0462] A194E; N204D; P210I; S212P; N218S; A228S; N248Q; T260D; N261F; E271D;

[0463] (i)N18Q; L21V; S24K; T38K; S56D; Q59K; S78N; A103S; Q109K; S144N; S160H; Q182K;

[0464] N183D; A194E; N204D; P210I; S212P; N218S; A228S; N248Q; T260D; E271D; and

[0465] (j)N18Q; L21V; S24K; T38K; S56D; Q59K; S78N; A103S; Q109K; S144N; S160H; Q182K;

[0466] N183D; A194E; N204D; P210I; S212P; N218S; A228S; N248Q; T260D; N261F; E271D.

[0467] In one embodiment, the variant protease has an amino acid sequence that is at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or 100% sequence identical to the amino acid sequence of any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0468] In one embodiment, the protease variant of the present invention exhibits one or more improved properties compared to the parental protease, and preferably compared to the protease according to SEQ ID NO: 3.

[0469] Preferably, the protease-active variant polypeptides of the present invention exhibit increased stability, particularly storage stability, compared to the parent protease according to SEQ ID NO: 3. Increased stability is preferably expressed as residual activity after a stability challenge. Preferably, storage stability is expressed as residual activity after storage under appropriate storage conditions, preferably in a cleaning 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 protease according to SEQ ID NO: 3. This is preferably determined by measuring the residual activity of the protease after storage at 40°C for 14 days in a cleaning composition (preferably, in an ASEAN2 detergent as described herein).

[0470] Preferably, the improved property is improved stability in a detergent composition, preferably a laundry detergent composition, preferably in a cleaning composition containing 1%-20%, more preferably 5%-10% anionic surfactant (preferably LAS) and / or greater than 50% water, preferably in a type B detergent as described herein. In one embodiment, the improved property is improved stability in a detergent composition, preferably a laundry detergent composition, preferably in a cleaning composition containing 0.1%-5%, preferably 0.3%-3% a strong chelating builder. In this embodiment, the cleaning composition preferably contains 1%-20%, more preferably 5%-10% anionic surfactant (preferably LAS) and / or greater than 50% water.

[0471] Preferably, the variant polypeptides with protease activity used in the methods and compositions of the present invention exhibit comparable washing performance compared to the protease according to SEQ ID NO: 3.

[0472] Within the scope of this invention, "comparable washing performance" means that the variant polypeptide with protease activity used in the methods and compositions of this invention exhibits washing performance that is less than 30% higher or less than 28% higher or less than 26% higher or less than 24% higher or less than 22% higher or less than 20% higher or less than 18% higher or less than 16% higher or less than 14% higher or less than 12% higher or less than 10% higher or less than 10% higher than the washing performance of the protease according to SEQ ID NO: 3.

[0473] Preferably, the washing performance is determined at 30°C in the formulations shown in Table 3a or at 40°C in the formulations shown in Table 3b. Also preferably, the protease-active variant peptides used in the methods and compositions of the present invention exhibit washing performance comparable to the protease according to SEQ ID NO: 3 against stains selected from the group consisting of eggs, blood / milk / ink, milk / chocolate, and grass / spinach; more preferably, the protease-active variant peptides used in the methods and compositions of the present invention exhibit washing performance comparable to the protease according to SEQ ID NO: 3 against egg stains.

[0474] Methods for cleaning objects

[0475] The present invention relates to a method for cleaning an object, the method comprising contacting the object with a cleaning composition comprising a variant polypeptide as defined herein at a temperature of 15°C to 65°C.

[0476] In one embodiment, the object is a textile or hard surface, preferably tableware. In another embodiment, the object is a textile or hard surface, preferably tableware, with protease-sensitive stains, preferably egg stains.

[0477] To clean the object, any cleaning composition as described herein may be used.

[0478] Cleaning objects using a cleaning composition containing a variant peptide as defined herein at temperatures of 15°C to 65°C, 25°C to 60°C, 25°C to 55°C, 25°C to 50°C, 30°C to 60°C, 30°C to 55°C, 30°C to 50°C, 30°C to 45°C, or 30°C to 40°C.

[0479] Protease composition

[0480] This document also claims a composition comprising a variant polypeptide with protease activity as described herein and at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerolactone, 1,6-hexanediol, and diethylene glycol. In one embodiment, the composition comprising a variant polypeptide with protease activity as described herein and at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerolactone, 1,6-hexanediol, and diethylene glycol further comprises glycerol and / or monopropylene glycol (MPG).

[0481] Therefore, the present invention also provides a composition comprising a variant polypeptide with protease activity as described herein, neopentyl glycol, and glycerol. The present invention also provides a composition comprising a variant polypeptide with protease activity as described herein, neopentyl glycol, and MPG. The present invention also provides a composition comprising a variant polypeptide with protease activity as described herein, neopentyl glycol, glycerol, and MPG. The present invention also provides a composition comprising a variant polypeptide with protease activity as described herein, 1,6-hexanediol, and glycerol. The present invention also provides a composition comprising a variant polypeptide with protease activity as described herein, 1,6-hexanediol, and MPG. The present invention also provides a composition comprising a variant polypeptide with protease activity as described herein, 1,6-hexanediol, glycerol, and MPG. The present invention also provides a composition comprising a variant polypeptide with protease activity as described herein, 1,6-hexanediol, neopentyl glycol, glycerol, and MPG. The present invention also provides a composition comprising a variant polypeptide with protease activity as described herein, glycerol, and MPG.

[0482] Therefore, the present invention also relates to a method for preparing a composition, the method comprising the step of mixing the following:

[0483] a) variant peptides with protease activity as described herein; and

[0484] b) At least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0485] Furthermore, the present invention also relates to a method for improving the stability of a protease in a composition, the method comprising the step of mixing the following:

[0486] a) variant peptides with protease activity as described herein; and

[0487] b) At least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0488] In one embodiment, the composition comprises neopentyl glycol (NPG; IUPAC name: 2,2-dimethylpropane-1,3-diol, sometimes also referred to as 2,2-dimethyl-1,3-propanediol) having the following formula (II):

[0489] (II).

[0490] In one embodiment, the liquid composition of the present invention comprises a neopentyl glycol in an amount ranging from about 0.01% to 25%.

[0491] In one embodiment, the liquid composition of the present invention is a liquid enzyme composition comprising, by weight, a portion of neopentyl glycol ranging from about 5% to 40%, preferably from about 10% to 25% by weight. For example, the total amount of neopentyl glycol in the liquid enzyme composition of the present invention may be at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% by weight. The total amount of neopentyl glycol in the liquid enzyme composition may be no more than 25%, no more than 24%, no more than 23%, no more than 22%, no more than 21%, no more than 20%, no more than 19%, no more than 18%, no more than 17%, no more than 16%, no more than 15%, no more than 14%, no more than 13%, or no more than 12% by weight.

[0492] In another embodiment, the liquid composition of the present invention is a liquid cleaning composition comprising a neopentyl glycol in an amount ranging from about 0.01% to 1%, preferably from about 0.05% to 0.2% by weight. For example, the total amount of neopentyl glycol in the liquid cleaning composition of the present invention may be at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, or at least 0.05% by weight. The total amount of neopentyl glycol in the liquid cleaning composition may be no more than 1%, no more than 0.8%, no more than 0.6%, no more than 0.4%, no more than 0.2%, or no more than 0.15% by weight.

[0493] In another embodiment, the composition comprises γ-valerolactone (GVL; IUPAC name: 5-methyloxacyclopentan-2-one, sometimes also called γ-valerolactone, 5-methyldihydrofuran-2(3H)-one, 4-pentanolide, 4-valerolactone, 4-pentalactone, 4-hydroxyvalerolactone) having the following formula (III):

[0494] (III).

[0495] In one embodiment, the liquid composition of the present invention comprises an amount ranging from about 0.005% to 10% of γ-valerolactone.

[0496] In one embodiment, the liquid composition of the present invention is a liquid enzyme composition comprising an amount of γ-valerolactone ranging from about 1% to 10% by weight, preferably about 2% to 8% by weight, and more preferably about 2% to 5% by weight. For example, the total amount of γ-valerolactone in the liquid enzyme composition of the present invention may be at least 1%, at least 2%, at least 3%, at least 4%, or at least 4.5% by weight. The total amount of γ-valerolactone in the liquid enzyme composition of the present invention may be no more than 10%, no more than 9%, no more than 8%, no more than 7%, no more than 6%, no more than 5.5%, or no more than 5% by weight, all relative to the total weight of the liquid composition.

[0497] In another embodiment, the liquid composition of the present invention is a liquid cleaning composition comprising an amount ranging from about 0.005% to 0.5%, preferably from about 0.01% to 0.1% by weight, of γ-valerolactone. For example, the total amount of γ-valerolactone in the liquid cleaning composition of the present invention may be at least 0.005%, at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, or at least 0.05% by weight. The total amount of γ-valerolactone in the liquid cleaning composition may be no more than 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2%, no more than 0.15%, or no more than 0.1% by weight.

[0498] In another embodiment, the composition comprises 1,6-hexanediol having the following formula (IV) (IUPAC name: hex-1,2-diol, CAS number 6920-22-5):

[0499] .

[0500] In one embodiment, the liquid composition of the present invention comprises 1,6-hexanediol in an amount ranging from about 10% to 40% by weight. In one embodiment, the liquid composition of the present invention comprises 1,2-propanediol (MPG, monopropylene glycol) and 1,6-hexanediol. In one embodiment, the liquid composition of the present invention comprises a total amount of 1,2-propanediol and 1,6-hexanediol ranging from about 40% to 70%, preferably about 45% to 65% by weight. For example, the total amount of 1,2-propanediol and 1,6-hexanediol in the liquid composition of the present invention may be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, or about 70% by weight, all relative to the total weight of the liquid composition. Liquid compositions containing 1,6-hexanediol are disclosed in WO 2022 / 063689 A1.

[0501] In another embodiment, the composition comprises diethylene glycol (DEG; IUPAC name: 2,2′-oxydiethanol, sometimes also called 2,2′-oxybis(ethylene-1-ol), 2-(2-hydroxyethoxy)ethylene-1-ol, ethylene diglycol, diglycol, 2,2′-oxydiethanol, 2,2′-oxydiethanol, 3-oxa-1,5-pentanediol, dihydroxydiethyl ether, diethylene glycol, diigol) having the following formula (V):

[0502] (V).

[0503] In one embodiment, the liquid composition of the present invention comprises an amount ranging from about 0.01% to 15% of diethylene glycol.

[0504] In one embodiment, the liquid composition of the present invention is a liquid enzyme composition comprising diethylene glycol in an amount ranging from about 5% to about 15% by weight, preferably from about 8% to about 12% by weight. For example, the total amount of diethylene glycol in the liquid enzyme composition of the present invention may be at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 9.5%, or at least 10% by weight, all relative to the total weight of the liquid composition.

[0505] The total amount of diethylene glycol in the liquid enzyme composition of the present invention may be no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, or no more than 10.5% by weight, all relative to the total weight of the liquid composition.

[0506] In another embodiment, the liquid composition of the present invention is a liquid cleaning composition comprising diethylene glycol in an amount ranging from about 0.01% to 1%, preferably from about 0.05% to 0.2% by weight. For example, the total amount of diethylene glycol in the liquid cleaning composition of the present invention may be at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, or at least 0.05% by weight. The total amount of neopentyl glycol in the liquid cleaning composition may be no more than 1%, no more than 0.8%, no more than 0.6%, no more than 0.4%, no more than 0.2%, or no more than 0.15% by weight.

[0507] The total amount of organic solvent in the protease composition of the present invention is 20% to 80%, preferably 30% to 75%, and more preferably 40% to 70%. Therefore, the total amount of neopentyl glycol, 1,6-hexanediol, MPG, and glycerol in the protease composition of the present invention is 20% to 80%, preferably 30% to 75%, and more preferably 40% to 70%.

[0508] The composition may be a non-complex formulation (e.g., a protease formulation) as described herein, or a complex formulation (e.g., a detergent composition).

[0509] In one embodiment of the invention, a variant polypeptide having protease activity is formulated as a protease formulation, preferably a concentrated protease formulation. The protease formulation may be solid or liquid. The protease 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 / 19444 A1 and WO 97 / 43482A1.

[0510] Liquid protease formulations may contain 2% to 40%, 2% to 30%, 2% to 25%, 2% to 12%, or preferably 2% to 10% of protease by weight, all relative to the total weight of the enzyme formulation. These amounts refer to the content of active protease within the liquid protease formulation.

[0511] In one embodiment, the protease formulation, particularly a liquid protease 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 formulation is substantially surfactant-free, i.e., the protease formulation contains less than 1% surfactant, preferably less than 0.5% surfactant. The solvent may be water and / or an organic solvent. The aqueous protease formulation of the present invention may contain water in an amount of more than about 30%, more than about 40%, more than about 50%, or more than about 60% by weight, all relative to the total weight of the protease formulation. The protease-containing formulation of the present invention may contain an organic solvent in an amount of more than 30%, more than 40%, more than about 50%, more than about 60%, more than about 70%, or more than about 80% by weight, all relative to the total weight of the protease formulation. The organic solvent may be a water-miscible solvent. The organic solvent may be one or more selected from the group consisting of glycerol, propylene glycol, polypropylene glycol and polyethylene glycol.

[0512] In one embodiment, the protease formulation contains at least one preservative. Preferably, a preservative means a substance added to the liquid composition for preservation purposes; more preferably, compounds known to have preservative properties contained in the liquid composition formed during the production process are excluded from the term preservative. In one embodiment, the preservative is selected from the group consisting of 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol, formic acid (in acid form or as a salt thereof), and 4,4'-dichloro-2-hydroxydiphenyl ether. Typically, the liquid composition of the present invention contains at least one preservative in an amount less than 10 ppm, such as in an amount ranging from 2 ppm to 5% by weight relative to the total weight of the liquid composition. Preferably, the protease formulation is preservative-free, meaning the content of the preservative is less than 1 ppm, preferably 0 ppm.

[0513] In one embodiment, the protease formulation comprises an enzyme stabilizing system. Preferably, the enzyme stabilizing system comprises at least one compound selected from the group consisting of: polyols (preferably 1,3-propanediol, 1,2-hexanediol, 1,6-hexanediol, ethylene glycol, glycerol, 1,2-propanediol, neopentyl glycol, or sorbitol), inorganic salts (preferably CaCl2, MgCl2, or NaCl), short-chain (preferably C1-C3) carboxylic acids or their salts (preferably formic acid, formate (preferably sodium formate), acetic acid, acetate, or lactate), borates, boric acid, boronic acids (preferably 4-formylphenylboronic acid (4-FPBA)), peptide aldehydes (preferably benzyloxycarbonyl-VAL-H (Z-VAL-H or Cbz-VAL-H) ​​or benzyloxycarbonyl-GAY-H (Z-GAY-H or Cbz-GAY-H)), peptide acetals, and peptide aldehyde bisulfite adducts. Preferably, the enzyme stabilizing 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, organoboronic acids (preferably 4-formylphenylboronic acid (4-FPBA)), peptide aldehydes, peptide acetals, and peptide aldehyde bisulfite adducts. In a particularly preferred embodiment, the stabilizing system comprises a protease inhibitor, preferably selected from the group consisting of borates, boric acid, organoboronic acids (preferably 4-FPBA), peptide aldehydes (preferably peptide aldehydes like Z-VAL-H or Z-GAY-H), peptide acetals, and peptide aldehyde bisulfite adducts, preferably peptide aldehydes, preferably Z-VAL-H or Z-GAY-H. In one embodiment, the stabilizing system does not contain a protease inhibitor. Preferably, the composition is boron-free. Preferably, the protease formulation comprises a calcium salt, preferably calcium chloride.

[0514] Preferably, the molar ratio of protease inhibitor to protease in the composition does not exceed 3:1. In one embodiment, the molar ratio of protease inhibitor to protease in the composition is less than 3:1. More preferably, the molar ratio of protease inhibitor to protease in the composition does not exceed 2:1. Even more preferably, the molar ratio of protease inhibitor to protease in the composition is less than 2:1. Most preferably, the molar ratio of protease inhibitor to protease in the composition does not exceed 1:1, and more preferably, the molar ratio of protease inhibitor to protease in the composition is less than 1:1.

[0515] In one embodiment, the protease formulation comprises a protease inhibitor, preferably a peptidaldehyde, the amount of which is in the range of about 0.02% to 0.6%, 0.02% to 0.5%, 0.02% to 0.4%, or 0.02% to 0.3% by weight relative to the total weight of the liquid protease variant formulation. Preferably, the peptidaldehyde content is in the range of about 0.03% to 0.25%, about 0.03% to 0.225%, or about 0.04% to 0.20% by weight, all relative to the total weight of the liquid protease formulation.

[0516] Preferably, the liquid protease formulation comprises or consists of the following: a variant polypeptide with protease activity as described herein, a solvent, an enzyme stabilizing system, and optionally a preservative, and optionally an additional enzyme different from the protease variant. Preferably, the formulation of the variant polypeptide with protease activity as described herein is substantially free of surfactant, i.e., the protease formulation contains less than 1% surfactant, preferably less than 0.5% surfactant.

[0517] Therefore, the present invention also relates to a method for preparing a protease formulation, preferably a concentrated protease formulation, the method comprising the step of mixing the following:

[0518] a) variant peptides with protease activity as described herein; and

[0519] b) One or more components selected from the group consisting of: solvents, enzyme stabilizing systems, preservatives, and other enzymes different from proteases.

[0520] Furthermore, the present invention relates to a method for improving the stability of a protease in a composition, the method comprising the step of mixing the following:

[0521] a) variant peptides with protease activity as described herein; and

[0522] b) One or more components selected from the group consisting of: solvents, enzyme stabilizing systems, preservatives, and other enzymes different from proteases.

[0523] In one embodiment, the liquid protease formulation does not contain the enzyme stabilization system described above. Preferably, the liquid protease formulation does not contain peptide aldehydes. More preferably, the liquid protease formulation does not contain Z-VAL or Z-GAY.

[0524] In one embodiment, the variant polypeptides of the present invention having protease activity are part of a microorganism (live, attenuated, or inactivated), probiotic, or prebiotic.

[0525] Other enzymes

[0526] In another embodiment, the composition comprising the variant polypeptide of the present invention having protease activity further comprises one or more additional enzymes different from proteases. Preferably, the additional enzymes are selected from the group consisting of: amylase, lipase, proteases other than the protease-active polypeptides of the present invention, cellulase, mannanase, hemicellulase, phospholipase, esterase, pectinase, lactase, peroxidase, xylanase, keratinase, pectate lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, amylopectinase, tanninase, pentosanase, malanases, β-glucanase, arabinoside arabinase, hyaluronidase, and chondroitinase. Laccase, nuclease, ribonuclease (RNase), deoxyribonuclease (DNase), phosphodiesterase, phytase, carbohydrate enzyme, galactanase, xanthan gumase, xyloglucanase, oxidoreductase, hydrolase, aminopeptidase, asparaginase, carbohydrate enzyme, carboxypeptidase, catalase, chitinase, cyclodextrin glycosyltransferase, α-galactosidase, β-galactosidase, glucosylamylase, α-glucosidase, β-glucosidase, invertase, transglutaminase, and dispersin, and combinations of at least two of the foregoing types. More preferably, the additional enzymes are selected from the group consisting of: amylase, lipase, proteases other than the polypeptides with protease activity of the present invention, cellulase, mannanase, xylanase, DNase, dispersin, pectinase, oxidoreductase, and keratinase, and combinations of at least two of the foregoing types. More preferably, the additional enzyme is selected from the group consisting of: amylase, lipase, proteases other than the polypeptides with protease activity of the present invention, cellulase, mannanase, and combinations of at least two of the foregoing types. Most preferably, the additional enzyme is amylase, preferably α-amylase.

[0527] The compositions of the present invention may contain more than one different type of enzyme (e.g., amylase and protease), or more than one of the same type of enzyme (e.g., two or more different proteases), or mixtures thereof (e.g., amylase and two different proteases, one of which is a variant polypeptide with protease activity as described herein).

[0528] Cleaning Composition

[0529] In one embodiment, the present invention relates to the use of a variant polypeptide with protease activity as described herein in a cleaning composition. Therefore, the present invention also relates to a cleaning composition comprising the variant polypeptide with protease activity of the present invention and one or more auxiliary cleaning additives.

[0530] Publication IPCOM000274907D, published on www.IP.com, is considered reference RF1 and is incorporated herein by reference in its entirety. Prior art disclosures in publications (Issue 684; paragraphs

[3000] to

[3061] ; ISSN: 2198-4786; date of publication: February 12, 2024) are considered reference RF2 and are incorporated herein by reference in their entirety.

[0531] As used herein, the phrase “cleaning composition” includes compositions and formulations designed for cleaning soiled materials. Such compositions and formulations include those designed for cleaning any kind of soiled material or surface, more preferably compositions for fabrics and home care. The term “cleaning composition” is defined in more detail in paragraphs

[0001] ,

[0002] ,

[0004] , and

[0007] of reference RF1.

[0532] "Compositions for fabric and home care" include cleaning compositions and formulations, including but not limited to laundry cleaning compositions and detergents, and hard surface cleaning compositions, including dishwashing compositions, more preferably liquid laundry detergent formulations, solid laundry detergent compositions, liquid manual dishwashing formulations, automatic dishwashing (ADW) gels, and automatic dishwashing (ADW) solid compositions. "Compositions for fabric and home care" are defined in more detail in paragraph

[0003] of reference RF1.

[0533] The cleaning compositions of the present invention, which contain one or more of the enzymes of the present invention, may—and preferably do—contain auxiliary cleaning additives (also referred to herein as “auxiliaries”), such auxiliary agents preferably being other than surfactant systems as previously defined.

[0534] Suitable auxiliary cleaning additives include polymers, surfactants or surfactant systems, builders, co-builders, additional enzymes, enzyme stabilizers, structural agents or thickeners, clay stain removers / anti-redeposition agents, solubilizers, chelating agents, bleaching compounds, bleaching agents, bleaching activators, bleaching catalysts, brighteners, odor control agents, pigments, dyes, opacifiers, toners, dye transfer inhibitors, chelating agents, foaming agents, defoamers, color smear removers, silver care products, anti-tarnish agents and / or corrosion inhibitors, alkalinity sources, pH adjusters, pH buffers, water-soluble agents, scrubbing particles, antibacterial agents, antioxidants, softeners, carriers, processing aids, fragrance precursors, dye fixatives, and fragrances.

[0535] Therefore, the present invention also relates to a method for preparing a cleaning composition, the method comprising the step of mixing the following:

[0536] a) variant peptides with protease activity as described herein; and

[0537] b) One or more auxiliary cleaning additives described herein.

[0538] The present invention also relates to a cleaning composition for preparing a cleaning composition having improved protease stability and a method for providing a cleaning composition having improved washing performance, the method comprising the step of mixing the following:

[0539] a) variant peptides with protease activity as described herein; and

[0540] b) One or more auxiliary cleaning additives described herein.

[0541] The liquid protease formulation is added to the detergent composition, preferably the liquid detergent composition, typically at a weight ratio of about 1:1000, 1:500, 1:100, 1:50, 1:30, 1:25, 1:20 or 1:10 of the liquid protease formulation to the cleaning composition.

[0542] In a preferred embodiment, the cleaning composition comprises one or more of the enzymes and polymers of the present invention, preferably cleaning polymers and / or dirt-releasing polymers. “Cleaning polymers and dirt-releasing polymers” are defined in more detail in paragraphs

[0032] through

[0034] of reference RF1. These polymers include polycarboxylates, alkoxylated polyalkyleneamines, alkoxylated polyalkyleneimides, polyether-based polymers, rheology-modified polymers, dye-inhibiting polymers, and dirt-releasing polymers (as defined in more detail in paragraphs

[3035] through

[3044] of reference RF2).

[0543] Polymers may include, but are not limited to, "polyfunctional alkoxylated polyethyleneimine," "polyfunctional alkoxylated diamine," and terephthalic acid-based polyesters, such as Clariant's TexCare®, including TexCare® SRN 170, TexCare® SRN 172, TexCare® SRN 260, TexCare® SRN 260 SG Terra, and TexCare® SRA 300, as well as various combinations of all the aforementioned polymers. Grafted polymers are also included, which comprise a polyepoxide-based backbone having grafted side chains of vinyl ester monomers and optionally N-vinylpyrrolidone monomers.

[0544] In a preferred embodiment, the cleaning composition comprises one or more of the enzymes of the present invention and a surfactant or surfactant system. “Surfactant” is defined in more detail in paragraphs

[3008] through

[3034] of reference RF2 as anionic surfactant, nonionic surfactant, cationic surfactant, amphoteric surfactant, and zwitterionic surfactant. Furthermore, these surfactants are described in more detail in paragraphs

[0008] through

[0013] of reference RF1.

[0545] The anionic surfactants used in the cleaning compositions of the present invention include linear alkylbenzene sulfonates (LAS), alkyl sulfates (AS), alkylalkoxy sulfates (AES), alkylalkoxycarboxylates, modified alkylbenzene sulfonates (MLAS), methyl ester sulfonates (MES), alkyl sulfosuccinates, α-olefin sulfonates (AOS), alkyl polysaccharides (APG), and biosurfactants such as rhamnolipids and sophorolipids. The nonionic surfactants used in the cleaning compositions of the present invention include alkoxylates, alkoxylated alcohols, alkoxylated fatty acids, and alkoxylated (poly)saccharides. The cationic surfactants used in the cleaning compositions of the present invention include surfactants containing quaternary ammonium compounds. The amphoteric surfactants used in the cleaning compositions of the present invention include amine oxides. The amphoteric surfactants used in the cleaning compositions of the present invention include betaine.

[0546] In a preferred embodiment, the cleaning composition comprises one or more of the enzymes and cleaning aids of the present invention. “Cleansing aids” are defined in more detail in paragraphs

[0014] through

[0018] of reference RF1. These cleaning aids include nonphosphate-based cleaning aids (NPB) and phosphonates (CoP), which are described in more detail in paragraphs

[3001] through

[3005] of reference RF2.

[0547] Builders may include, but are not limited to, methylglycine diacetic acid (MGDA), ethylenediamine disuccinic acid (EDDS), glutamic acid diacetate (GLDA), citric acid and its salts.

[0548] In a preferred embodiment, the cleaning composition comprises one or more of the enzymes of the present invention and additional enzymes. The term “(additional) enzymes” is defined in more detail in paragraphs

[0020] through

[0027] of reference RF1.

[0549] These additional enzymes may include hydrolases such as (other) proteases, amylases, lipases, DNases, cellulases, hemicellulases, phospholipases, esterases, mannanases, xylanases, dispersins, oxidoreductases, keratins, pectinases, pectinases, lactases, and peroxidases. In a more preferred embodiment, in addition to one or more of the compounds of the present invention, the cleaning composition further comprises an enzyme stabilization system as described in the “Enzyme Stabilization System” section herein or in reference RF1, which preferably comprises a protease inhibitor, preferably selected from borates, boric acid, organoboronic acids (preferably, 4-FPBA), peptidaldehydes (preferably, peptidaldehydes like Z-VAL-H or Z-GAY-H), peptidaldehydes, and peptidaldehyde bisulfite adducts, preferably peptidaldehydes, preferably Z-VAL-H, and / or Z-GAY-H. Preferably, the protease inhibitor is Z-VAL-H and / or Z-GAY-H.

[0550] In a preferred embodiment, the cleaning composition comprises one or more of the enzymes and biocides of the present invention. “Biocides” are defined in more detail in paragraphs

[0035] and

[0036] of reference RF1. These biocides also include compounds as defined in more detail in paragraphs

[3006] and

[3007] of reference RF2.

[0551] Biocides may include, but are not limited to, 2-phenoxyethanol and 4,4'-dichloro-2-hydroxydiphenyl ether.

[0552] Paragraphs

[0005] ,

[0006] ,

[0019] ,

[0028] to

[0031] and

[0037] to

[0039] of reference RF1 include and describe in more detail additional auxiliary cleaning additives.

[0553] Liquid laundry detergent formulations, solid laundry detergent compositions, liquid manual dishwashing formulations, automatic dishwashing (ADW) gels, and automatic dishwashing (ADW) solid compositions comprising one or more enzymes of the present invention are defined in more detail in paragraph

[0044] of reference RF1.

[0554] How to use

[0555] Variant peptides with protease activity, as described herein, can be used in a variety of applications.

[0556] This invention relates to the use of protease-active variant peptides as described herein in cleaning processes (such as laundry or hard surface cleaning), preferably for use in home care or I&I cleaning. Protease-active variant peptides as described herein, or compositions containing said peptides, can be used in a variety of industrial and institutional cleaning applications, including commercial laundry (such as on-premises laundry or tunnel washing), machine cleaning (such as in hooded or tunnel washing machines), manual dishwashing, carpet cleaning, open-plan factory cleaning (external duct cleaning), in-place cleaning (internal duct cleaning), membrane cleaning (such as in dairy, food, beverage, or water treatment), vehicle maintenance (such as mat cleaning), microbial removal, virus removal, insect removal, or odor removal, or veterinary cleaning.

[0557] In one embodiment, the protease variant described herein can be combined with a metalloproteinase for the removal and / or prevention of redeposition of bloodstains or other stains containing metal ions.

[0558] The present invention also relates to the use of variant peptides having protease activity as described herein for providing cleaning compositions with improved protease stability and for providing cleaning compositions with improved washing performance (preferably for protease-sensitive stains).

[0559] Therefore, the present invention also relates to a method for cleaning, preferably laundry or hard surface cleaning, comprising the step of contacting a test subject, preferably textile or hard surface, with a composition comprising a variant polypeptide having protease activity as described herein, preferably wherein the composition comprises at least one additional auxiliary cleaning additive, preferably a surfactant and / or a detergent builder.

[0560] Furthermore, the present invention relates to a method for improving the stability of proteases in cleaning compositions and for improving the washing performance of detergent compositions (preferably for protease-sensitive stains), the method comprising the step of formulating a variant polypeptide having protease activity as described herein into the cleaning composition.

[0561] The present invention also relates to a method for washing fabrics or cleaning hard surfaces, the method comprising treating the fabric or hard surface with a composition comprising a variant polypeptide having protease activity as described herein and further comprising 4,4'-dichloro-2-hydroxydiphenyl ether.

[0562] While the invention has been detailed and described in the accompanying drawings and foregoing description, such description and illustration should be considered illustrative or exemplary rather than restrictive. The invention is not limited to the disclosed embodiments. By studying the drawings, this disclosure, and the appended claims, those skilled in the art can understand and implement other variations of the disclosed embodiments in practicing the claimed invention. The detailed description is merely exemplary in nature and is not intended to limit application or use. The following examples further illustrate the invention but do not limit its scope. Various changes and modifications can be made by those skilled in the art based on the description of the invention, and such changes and modifications are also included in the invention. Preferred embodiment

[0563] Specifically, this paper prefers:

[0564] 1. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0565] The variant polypeptide having protease activity described above:

[0566] (i) having an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 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 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%, and at least 97.5% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0567] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0568] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261 and 271; and

[0569] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260.

[0570] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0571] 2. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0572] The variant polypeptide having protease activity described above:

[0573] (i) having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0574] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0575] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid residues selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261, and 271 contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions; and

[0576] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid residues selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248, and 260 contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all of the amino acid substitutions.

[0577] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0578] 3. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0579] The variant polypeptide having protease activity described above:

[0580] (i) having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0581] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0582] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions at amino acid residues 21, 25, 38, 160, 210, 212, 228, 261 and 271; and

[0583] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, amino acid substitutions are contained at amino acid residues 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260.

[0584] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0585] 4. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0586] The variant polypeptide having protease activity described above:

[0587] (i) having an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 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 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%, and at least 97.5% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0588] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0589] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 38, 212, 261 and 271; and

[0590] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 18 and 218.

[0591] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0592] 5. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0593] The variant polypeptide having protease activity described above:

[0594] (i) having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0595] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0596] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least two, at least three, or all of the amino acid residues selected from the group consisting of 38, 212, 261, and 271 are substituted with amino acid substitutions; and

[0597] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, all amino acid substitutions are included at amino acid residues 18 and 218.

[0598] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0599] 6. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0600] The variant polypeptide having protease activity described above:

[0601] (i) having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0602] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0603] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, amino acid substitutions are contained at amino acid residues 38, 212, 261, and 271; and

[0604] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, amino acid substitutions are included at amino acid residues 18 and 218.

[0605] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0606] 7. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0607] The variant polypeptide having protease activity described above:

[0608] (i) having an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 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 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%, and at least 97.5% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0609] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0610] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at the amino acid residue selected from the group consisting of 21, 38, 160, 210 and 228; and

[0611] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 109, 144, 182, 183, 204, 218 and 248.

[0612] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0613] 8. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0614] The variant polypeptide having protease activity described above:

[0615] (i) having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0616] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0617] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least two, at least three, at least four, or all amino acid substitutions at amino acid residues selected from the group consisting of 21, 38, 160, 210, and 228; and

[0618] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid residues selected from the group consisting of 18, 24, 56, 78, 109, 144, 182, 183, 204, 218, and 248 contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the amino acid substitutions.

[0619] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0620] 9. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0621] The variant polypeptide having protease activity described above:

[0622] (i) having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0623] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0624] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions at amino acid residues 21, 38, 160, 210, and 228; and

[0625] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, amino acid substitutions are contained at amino acid residues 18, 24, 56, 78, 109, 144, 182, 183, 204, 218 and 248.

[0626] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0627] 10. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0628] The variant polypeptide having protease activity described above:

[0629] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0630] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0631] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261 and 271; and

[0632] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260.

[0633] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0634] 11. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0635] The variant polypeptide having protease activity described above:

[0636] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0637] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0638] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid residues selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261, and 271 contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions; and

[0639] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid residues selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248, and 260 contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all of the amino acid substitutions.

[0640] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0641] 12. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0642] The variant polypeptide having protease activity described above:

[0643] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0644] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0645] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions at amino acid residues 21, 25, 38, 160, 210, 212, 228, 261 and 271; and

[0646] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, amino acid substitutions are contained at amino acid residues 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260.

[0647] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0648] 13. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0649] The variant polypeptide having protease activity described above:

[0650] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0651] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0652] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 38, 212, 261 and 271; and

[0653] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 18 and 218.

[0654] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0655] 14. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0656] The variant polypeptide having protease activity described above:

[0657] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0658] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0659] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least two, at least three, or all of the amino acid residues selected from the group consisting of 38, 212, 261, and 271 are substituted with amino acid substitutions; and

[0660] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, all amino acid substitutions are included at amino acid residues 18 and 218.

[0661] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0662] 15. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0663] The variant polypeptide having protease activity described above:

[0664] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0665] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0666] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, amino acid substitutions are contained at amino acid residues 38, 212, 261, and 271; and

[0667] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, amino acid substitutions are included at amino acid residues 18 and 218.

[0668] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0669] 16. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0670] The variant polypeptide having protease activity described above:

[0671] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0672] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0673] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at the amino acid residue selected from the group consisting of 21, 38, 160, 210 and 228; and

[0674] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 109, 144, 182, 183, 204, 218 and 248.

[0675] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0676] 17. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0677] The variant polypeptide having protease activity described above:

[0678] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0679] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0680] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least two, at least three, at least four, or all amino acid substitutions at amino acid residues selected from the group consisting of 21, 38, 160, 210, and 228; and

[0681] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid residues selected from the group consisting of 18, 24, 56, 78, 109, 144, 182, 183, 204, 218, and 248 contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the amino acid substitutions.

[0682] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0683] 18. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity.

[0684] The variant polypeptide having protease activity described above:

[0685] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0686] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0687] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions at amino acid residues 21, 38, 160, 210, and 228; and

[0688] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, amino acid substitutions are contained at amino acid residues 18, 24, 56, 78, 109, 144, 182, 183, 204, 218 and 248.

[0689] The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0690] 19. The method according to any one of the foregoing embodiments, wherein, with reference to SEQ ID NO: 2, the variant polypeptide or a fragment thereof contains glutamine at position 59.

[0691] 20. The method according to any one of the foregoing embodiments, wherein:

[0692] (a) The amino acid substitution at amino acid residue 18 is X18Q / E / D; and / or

[0693] (b) The amino acid substitution at amino acid residue 21 is X21I / V / F / W / Y, preferably X21V / I; and / or

[0694] (c) The amino acid substitution at amino acid residue 24 is X24K; and / or

[0695] (d) The amino acid substitution at amino acid residue 25 is X25D; and / or

[0696] (e) The amino acid substitution at amino acid residue 38 is X38R / K / H / W; and / or

[0697] (f) The amino acid substitution at amino acid residue 56 is X56D; and / or

[0698] (g) 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; and / or

[0699] (h) The amino acid substitution at amino acid residue 103 is X103S; and / or

[0700] The substitution of this amino acid at amino acid residue 109 is X109K / A; and / or

[0701] (i) The amino acid substitution at amino acid residue 144 is X144N / R; and / or

[0702] (j) The amino acid substitution at amino acid residue 160 is X160H / S / D / E / P, preferably X160H;

[0703] (k) The amino acid substitution at amino acid residue 182 is X182K / R / E; and / or

[0704] (l) The amino acid substitution at amino acid residue 183 is X183D / E / C / Q / A / M, preferably X183D / E; and / or

[0705] (m) The amino acid substitution at amino acid residue 194 is X194E / C / D, preferably X194E / D; and / or

[0706] (n) The amino acid substitution at amino acid residue 204 is X204D / E / C / G, preferably X204D / E; and / or

[0707] (o) The amino acid substitution at amino acid residue 210 is X210I / V, preferably X210I; and / or

[0708] (p) The amino acid substitution at amino acid residue 212 is X212G / M / R / K / P / A / F / H / W / N / Y / D, preferably X212P / A / G / RK / F / Y / M; and / or

[0709] (q) The amino acid substitution at amino acid residue 218 is X218S / T / D, preferably X218S / T; and / or

[0710] (r) The amino acid substitution at amino acid residue 228 is X228S; and / or

[0711] (s) The amino acid substitution at amino acid residue 248 is X248Q / R; and / or

[0712] (t) The amino acid substitution at amino acid residue 260 is X260D / E / K, preferably X260D / E; and / or

[0713] (u) The amino acid substitution at amino acid residue 261 is X261L / M / F / E / W / Y / C, preferably X261F / W / Y / L; and / or

[0714] (v) The amino acid substitution at amino acid residue 271 is X271D.

[0715] 21. The method according to any one of the foregoing embodiments, wherein the variant polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

[0716] 22. The method according to any one of the foregoing embodiments, wherein the variant polypeptide exhibits one or more improved properties compared to the protease according to SEQ ID NO: 3.

[0717] 23. The method according to Example 22, wherein these improved properties are selected from:

[0718] (i) Increased stability

[0719] (ii) Increased storage stability, and

[0720] (iii) Increased storage stability in detergent compositions.

[0721] 24. The method according to any one of the foregoing embodiments, wherein the object is a textile, tableware, or a hard surface.

[0722] 25. The method according to any one of the foregoing embodiments, wherein the object has protease-sensitive stains.

[0723] 26. The method according to any one of the foregoing embodiments, wherein the object has egg stains.

[0724] 27. The method according to any one of the foregoing embodiments, wherein the polypeptide has washing properties comparable to those of the protease according to SEQ ID NO:3 and increased storage stability compared to the protease according to SEQ ID NO:3.

[0725] 28. The method according to Example 27, wherein the polypeptide has washing properties comparable to the protease according to SEQ ID NO: 3 for stains selected from the group consisting of eggs, blood / milk / ink, milk / chocolate and grass / spinach, preferably for egg stains.

[0726] 29. The method according to any one of the foregoing embodiments, wherein the cleaning composition is a liquid cleaning composition.

[0727] 30. The method according to any one of the foregoing embodiments, wherein the cleaning composition comprises one or more additional enzymes different from the variant polypeptides described in any one of the foregoing embodiments, preferably one or more additional enzymes selected from the group consisting of: amylase, additional protease, lipase, cellulase, hemicellulase, mannanase, xylanase, DNase, dispersin, pectinase, oxidoreductase and keratinase, preferably selected from amylase, mannanase, cellulase and lipase, most preferably amylase.

[0728] 31. The method according to any one of the foregoing embodiments, wherein the cleaning composition comprises one or more surfactants and / or one or more builder agents, preferably strongly chelating builder agents.

[0729] 32. The method according to Example 31, wherein the composition comprises a detergent builder selected from MDGA, GLDA, DTPMP, HEDP and EDDS, preferably MDGA or EDDS.

[0730] 33. The method according to Example 31, wherein the composition comprises a surfactant selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, and combinations thereof.

[0731] 34. The method according to Examples 29 to 32, wherein the cleaning composition is free of anionic surfactants.

[0732] 35. The method according to any one of Examples 31 to 34, wherein the surfactant and / or the detergent builder is biodegradable and / or bio-based.

[0733] 36. The method according to any one of Examples 29 to 35, wherein the cleaning composition is a liquid laundry detergent composition.

[0734] 37. The method according to any one of Examples 29 to 36, wherein the cleaning composition is boron-free.

[0735] 38. The method according to any one of Examples 29 to 37, wherein the cleaning composition does not contain a preservative.

[0736] 39. The method according to any one of Examples 29 to 38, wherein the composition further comprises 2-phenoxyethanol, preferably comprising an amount ranging from 2 ppm to 5% by weight of the composition; more preferably comprising 0.1% to 2% phenoxyethanol.

[0737] 40. The method according to any one of Examples 29 to 39, wherein the composition further comprises 0.001% to 3%, preferably 0.002% to 1%, more preferably 0.01% to 0.6% of 4,4'-dichloro-2-hydroxydiphenyl ether, each by weight of the composition.

[0738] 41. The method according to any one of Examples 29 to 40, wherein the cleaning composition comprises 5% to 10% LAS and / or more than 50% water.

[0739] 42. The method according to any one of Examples 29 to 41, wherein the cleaning composition does not contain Z-VAL or Z-GAY, and preferably does not contain any peptide aldehyde stabilizer.

[0740] 43. A composition comprising:

[0741] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0742] (i) having an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 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 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%, and at least 97.5% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0743] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0744] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261 and 271; and

[0745] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260.

[0746] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0747] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0748] 44. A composition comprising:

[0749] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0750] (i) having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0751] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0752] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid residues selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261, and 271 contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions; and

[0753] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid residues selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248, and 260 contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all of the amino acid substitutions.

[0754] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0755] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0756] 45. A composition comprising:

[0757] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0758] (i) having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0759] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0760] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions at amino acid residues 21, 25, 38, 160, 210, 212, 228, 261 and 271; and

[0761] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, amino acid substitutions are contained at amino acid residues 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260.

[0762] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0763] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0764] 46. ​​A composition comprising:

[0765] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0766] (i) having an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 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 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%, and at least 97.5% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0767] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0768] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 38, 212, 261 and 271; and

[0769] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 18 and 218.

[0770] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0771] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0772] 47. A composition comprising:

[0773] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0774] (i) having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0775] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0776] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least two, at least three, or all of the amino acid residues selected from the group consisting of 38, 212, 261, and 271 are substituted with amino acid substitutions; and

[0777] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, all amino acid substitutions are included at amino acid residues 18 and 218.

[0778] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0779] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0780] 48. A composition comprising:

[0781] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0782] (i) having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0783] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0784] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, amino acid substitutions are contained at amino acid residues 38, 212, 261, and 271; and

[0785] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, amino acid substitutions are included at amino acid residues 18 and 218.

[0786] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0787] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0788] 49. A composition comprising:

[0789] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0790] (i) having an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 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 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%, and at least 97.5% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0791] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0792] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at the amino acid residue selected from the group consisting of 21, 38, 160, 210 and 228; and

[0793] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 109, 144, 182, 183, 204, 218 and 248.

[0794] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0795] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0796] 50. A composition comprising:

[0797] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0798] (i) having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0799] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0800] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least two, at least three, at least four, or all amino acid substitutions at amino acid residues selected from the group consisting of 21, 38, 160, 210, and 228; and

[0801] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid residues selected from the group consisting of 18, 24, 56, 78, 109, 144, 182, 183, 204, 218, and 248 contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the amino acid substitutions.

[0802] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0803] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0804] 51. A composition comprising:

[0805] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0806] (i) having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0807] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0808] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions at amino acid residues 21, 38, 160, 210, and 228; and

[0809] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, amino acid substitutions are contained at amino acid residues 18, 24, 56, 78, 109, 144, 182, 183, 204, 218 and 248.

[0810] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0811] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0812] 52. A composition comprising:

[0813] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0814] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0815] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0816] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261 and 271; and

[0817] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260.

[0818] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0819] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0820] 53. A composition comprising:

[0821] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0822] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0823] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0824] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid residues selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261, and 271 contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acid substitutions; and

[0825] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid residues selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248, and 260 contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or all of the amino acid substitutions.

[0826] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0827] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0828] 54. A composition comprising:

[0829] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0830] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0831] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0832] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions at amino acid residues 21, 25, 38, 160, 210, 212, 228, 261 and 271; and

[0833] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, amino acid substitutions are contained at amino acid residues 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260.

[0834] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0835] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0836] 55. A composition comprising:

[0837] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0838] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0839] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0840] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 38, 212, 261 and 271; and

[0841] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 18 and 218.

[0842] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0843] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0844] 56. A composition comprising:

[0845] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0846] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0847] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0848] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least two, at least three, or all of the amino acid residues selected from the group consisting of 38, 212, 261, and 271 are substituted with amino acid substitutions; and

[0849] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, all amino acid substitutions are included at amino acid residues 18 and 218.

[0850] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0851] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0852] 57. A composition comprising:

[0853] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0854] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0855] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0856] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, amino acid substitutions are contained at amino acid residues 38, 212, 261, and 271; and

[0857] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, amino acid substitutions are included at amino acid residues 18 and 218.

[0858] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0859] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0860] 58. A composition comprising:

[0861] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0862] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0863] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0864] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at the amino acid residue selected from the group consisting of 21, 38, 160, 210 and 228; and

[0865] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 109, 144, 182, 183, 204, 218 and 248.

[0866] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0867] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0868] 59. A composition comprising:

[0869] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0870] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0871] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0872] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least two, at least three, at least four, or all amino acid substitutions at amino acid residues selected from the group consisting of 21, 38, 160, 210, and 228; and

[0873] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid residues selected from the group consisting of 18, 24, 56, 78, 109, 144, 182, 183, 204, 218, and 248 contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the amino acid substitutions.

[0874] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0875] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0876] 60. A composition comprising:

[0877] (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is:

[0878] (i) having an amino acid sequence that is 80% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0879] (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9;

[0880] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains amino acid substitutions at amino acid residues 21, 38, 160, 210, and 228; and

[0881] (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the numbering of SEQ ID NO: 2, amino acid substitutions are contained at amino acid residues 18, 24, 56, 78, 109, 144, 182, 183, 204, 218 and 248.

[0882] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and

[0883] (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

[0884] 61. The composition according to any one of Examples 43 to 60, wherein, with reference to SEQ ID NO: 2, the variant polypeptide or a fragment thereof contains glutamine at position 59.

[0885] 62. The composition according to any one of Examples 43 to 61, wherein:

[0886] (a) The amino acid substitution at amino acid residue 18 is X18Q / E / D; and / or

[0887] (b) The amino acid substitution at amino acid residue 21 is X21I / V / F / W / Y, preferably X21V / I; and / or

[0888] (c) The amino acid substitution at amino acid residue 24 is X24K; and / or

[0889] (d) The amino acid substitution at amino acid residue 25 is X25D; and / or

[0890] (e) The amino acid substitution at amino acid residue 38 is X38R / K / H / W; and / or

[0891] (f) The amino acid substitution at amino acid residue 56 is X56D; and / or

[0892] (g) 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; and / or

[0893] (h) The amino acid substitution at amino acid residue 103 is X103S; and / or

[0894] The substitution of this amino acid at amino acid residue 109 is X109K / A; and / or

[0895] (i) The amino acid substitution at amino acid residue 144 is X144N / R; and / or

[0896] (j) The amino acid substitution at amino acid residue 160 is X160H / S / D / E / P, preferably X160H;

[0897] (k) The amino acid substitution at amino acid residue 182 is X182K / R / E; and / or

[0898] (l) The amino acid substitution at amino acid residue 183 is X183D / E / C / Q / A / M, preferably X183D / E; and / or

[0899] (m) The amino acid substitution at amino acid residue 194 is X194E / C / D, preferably X194E / D; and / or

[0900] (n) The amino acid substitution at amino acid residue 204 is X204D / E / C / G, preferably X204D / E; and / or

[0901] (o) The amino acid substitution at amino acid residue 210 is X210I / V, preferably X210I; and / or

[0902] (p) The amino acid substitution at amino acid residue 212 is X212G / M / R / K / P / A / F / H / W / N / Y / D, preferably X212P / A / G / RK / F / Y / M; and / or

[0903] (q) The amino acid substitution at amino acid residue 218 is X218S / T / D, preferably X218S / T; and / or

[0904] (r) The amino acid substitution at amino acid residue 228 is X228S; and / or

[0905] (s) The amino acid substitution at amino acid residue 248 is X248Q / R; and / or

[0906] (t) The amino acid substitution at amino acid residue 260 is X260D / E / K, preferably X260D / E; and / or

[0907] (u) The amino acid substitution at amino acid residue 261 is X261L / M / F / E / W / Y / C, preferably X261F / W / Y / L; and / or

[0908] (v) The amino acid substitution at amino acid residue 271 is X271D.

[0909] 63. The composition according to any one of Examples 43 to 62, wherein the variant polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.

[0910] 64. The composition according to any one of Examples 43 to 63, wherein the composition contains a protease inhibitor and the molar ratio of the protease inhibitor to the protease in the composition is not more than 3:1, preferably not more than 1:1, or wherein the composition does not contain a protease inhibitor, preferably wherein the composition does not contain a peptidaldehyde protease inhibitor, or a salt or bisulfite adduct thereof.

[0911] 65. The composition according to any one of Examples 43 to 64, further comprising sodium formate.

[0912] 66. The composition according to any one of Examples 43 to 65, further comprising at least one additional enzyme.

[0913] 67. The composition according to Example 66, wherein the at least one additional enzyme is selected from amylase, mannanase, cellulase, esterase, lipase, pectinase, pectic acid lyase, xylanase, DNase, nuclease, dispersin, oxidoreductase, peroxidase, oxygenase, glycosidase, glucosidase, xanthan gum lyase and other proteases, preferably amylase, mannanase and cellulase.

[0914] 68. The composition according to any one of Examples 43 to 67, wherein the composition further comprises glycerol and / or further comprises monopropylene glycol (MPG).

[0915] 69. The composition according to Example 68, wherein the composition comprises one of the following:

[0916] (i) Neopentyl glycol and MPG;

[0917] (ii) Neopentyl glycol and glycerol;

[0918] (iii) Neopentyl glycol, MPG, and glycerol;

[0919] (iv) 1,6-hexanediol and MPG;

[0920] (v) 1,6-hexanediol and glycerol;

[0921] (vi) 1,6-hexanediol, MPG and glycerol;

[0922] (vii) Neopentyl glycol, 1,6-hexanediol, MPG and glycerol.

[0923] 70. The composition according to any one of Examples 43 to 69, wherein the composition is a liquid enzyme composition, wherein the concentration of the protease variant is 0.05%-10%, preferably 2%-10%, and the concentration of the solubilizer is:

[0924] (i) Neopentyl glycol: 2.5%-30%,

[0925] (ii) γ-valerolactone: 1%-6%, preferably 4.5%-5.5%,

[0926] (iii) 1,6-Hexanediol: 2.5%-30%, and / or

[0927] (iv) Diethylene glycol: 8%-12%, preferably 9.5%-10.5%.

[0928] 71. The liquid enzyme composition according to Example 70, wherein the liquid enzyme composition is substantially free of anionic surfactants and detergent builders.

[0929] 72. The composition according to any one of Examples 43 to 71, wherein the composition is a liquid cleaning composition further comprising at least one detergent component.

[0930] 73. The liquid cleaning composition according to Example 72, wherein the detergent component is a compound selected from anionic surfactants and builder agents, wherein the anionic surfactant is preferably selected from linear alkylbenzene sulfonates (LAS) or alcohol ethoxysulfates (AES), and wherein the builder agent is preferably a non-phosphate-based builder agent, preferably selected from citrates, phosphonates and aminocarboxylates.

[0931] 74. The liquid cleaning composition according to Example 72 or 73, wherein the concentration of the protease is 0.0005%-0.1%, preferably 0.02%-0.06%, and the concentration of the solubilizer is:

[0932] (i) Neopentyl glycol: 0.01%-1%

[0933] (ii) γ-valerolactone: 0.005%-0.5%, preferably 0.01%-0.1%,

[0934] (iii) 1,6-Hexanediol: 0.01%-1%, and / or

[0935] (iv) Diethylene glycol: 0.01%-1%, preferably 0.05%-0.2%.

[0936] 75. The composition according to any one of Examples 43 to 74, further comprising 2-phenoxyethanol, preferably in an amount ranging from 2 ppm to 5% by weight of the composition, more preferably from 0.1% to 2% by weight.

[0937] 76. The method or composition according to any one of the foregoing embodiments, wherein the polypeptide having protease activity has 80% to 98%, 80.5% to 98%, 81% to 98%, 81.5% to 98%, 82% to 98%, 82.5% to 98%, 83% to 98%, 83.5% to 98%, 84% to 98%, 84.5% to 98%, 85% to 98%, 85.5% to 98%, 86% to 98%, 86.5% to 98%, 87% to 98%, 87.5% to 98%, 88% to 98%, 88.5% to 98%, 89% to 98%, 89.5% to 98%, 90% to 98%, 90.3% to 98%, 90.5% to 98%, 90.7% of the amino acid sequence shown in SEQ ID NO: 1. The same amino acid sequence up to 98%, 91% to 98%, 91.3% to 98%, 91.5% to 98%, 91.7% to 98%, 92% to 98%, 92.3% to 98%, 92.5% to 98%, 92.7% to 98%, 93% to 98%, 93.3% to 98%, 93.5% to 98%, 93.7% to 98%, 94% to 98%, 94.3% to 98%, 94.5% to 98%, 94.7% to 98%, 95% to 98%, 95.1% to 98%, 95.2% to 98%, 95.3% to 98%, 95.4% to 98%, or 95.5% to 98%.

[0938] Example

[0939] Cleaning Composition

[0940] The cleaning compositions ASEAN2 and ES1-C as described in Tables 3a and 3b were used for testing.

[0941] Table 3a: ASEAN2 formulations:

[0942]

[0943] Table 3b: ES1-C formulations:

[0944]

[0945] Protease activity measurement

[0946] The protease activity was measured by monitoring the release of free amines after hydrolyzing N,N-dimethylcasein at 50°C and pH 9.5. Therefore, a 0.3% solution of N,N-dimethylcasein (DMC, Sigma-Aldrich C9801) in 100 mM CHES (Sigma-Aldrich C2885) at pH 9.5, 0.4% (w / w) Na2SO3 (Sigma-Aldrich 31454), and 0.02% Brij-35 (Sigma-Aldrich 16005) was prepared as the DMC solution. Prior to the assay, 400 µL of 60 mM 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma-Aldrich 92822) was added to 18 mL of the DMC solution. The solution containing the protease was diluted with 100 mM CHES pH 9.5 and 0.15% CaCl2x2H2O and transferred to test tubes. The reaction was initiated by adding DMC / TNBS starting solution, and the kinetics of the reaction were measured at 405 nm for 10 min. Protease standards with defined activity were used as references and for calibration.

[0947] The slope of the time-dependent absorption curve is proportional to the specific activity (activity / mg enzyme) of the protease under a given set of conditions.

[0948] Storage stability in ASEAN2 detergent at 40°C for 14 days.

[0949] To assess stability in detergents, a protease was formulated in ASEAN2 detergent (see Table 3a). To determine changes in proteolytic activity over time, the “initial enzyme activity” of the protease in the detergent was measured at time zero under defined conditions, and the enzyme activity was measured after storage at 40°C for 14 days. The residual activity (x%) can be calculated by dividing the latter activity by the activity at time zero.

[0950] After 14 days, the residual activity of the corresponding proteases was classified as - (<15%), + (15%-35%), ++ (>35%-60%), and +++ (>60%).

[0951] Table 4: Storage stability of proteases in ASEAN2 detergent at 40°C. Residual activity after 14 days of challenge:

[0952]

[0953] As can be seen from Table 4 above, the protease variant of the present invention (SEQ ID NO: 4) is more stable than the reference protease according to SEQ ID NO: 3.

[0954] Determining washing performance in full-size washing machines:

[0955] Multi-stain test items were washed in a full-size washing machine at 30°C or 40°C in the presence of cotton ballast fabrics with liquid laundry detergents containing different proteases. After washing, the stain test items were air-dried. The washing performance against egg stains was determined by measuring the CIE-Lab values ​​of the soiled fabrics before and after washing using a MACH5 multi-zone colorimeter. For data evaluation, the average value was determined, and ΔE between unwashed and washed stains was calculated.

[0956] Detergent performance was evaluated in ASEAN2 (Tables 5 and 6) and ES1-C (Tables 7 and 8) detergent formulations.

[0957] Table 5: Washing Conditions

[0958]

[0959] 1) Obtained from the Center for Testmaterials (BV), NL-3130 AC Vlaardingen.

[0960] Table 6: Full-size washing performance of ASEAN2 preparations under the washing conditions given in Table 5. The data shows the summary performance for all six types of egg stains, expressed as ΔE:

[0961]

[0962] As can be seen from Table 6 above, in ASEAN2 detergent formulations, the protease variant of the present invention (SEQ ID NO: 4) provides the best cleaning results for egg stains compared with other protease variants not according to the present invention (proteases according to SEQ ID NO: 14 and SEQ ID NO: 15), and shows comparable cleaning results compared with the reference protease according to SEQ ID NO: 3.

[0963] Table 7: Washing Conditions

[0964]

[0965] 1) Obtained from the Testing Materials Center BV, NL-3130 AC Fraldingen

[0966] Table 8: Full-size washing performance in ES1-C preparations under the washing conditions given in Table 5. The data shows the summary performance for all six egg stain types, expressed as ΔE:

[0967]

[0968] As can be seen from Table 8 above, the protease variant of the present invention (SEQ ID NO: 4) also provides the best cleaning results for egg stains compared with other protease variants not according to the present invention (proteases according to SEQ ID NO: 14 and SEQ ID NO: 15) in different detergent formulations (ES1-C detergent formulations).

[0969] Determination of washing performance in a Launder-O-Meter (LOM) color fastness tester:

[0970] The purchased stained fabrics were washed in LOM at 25°C for 40 minutes in liquid laundry detergents containing different proteases (ASEAN2, see Table 3a). After washing, the stained fabric samples were rinsed three times with water and then laid flat to air dry. The washing performance against egg stains was determined by measuring the CIE-Lab values ​​of the stained fabrics after washing using a flatbed scanner and converting the RGB values ​​using Mathematica software. The washing performance was determined as ΔE calculated between detergents with and without proteases. The sum of the egg stain performance was divided by the sum of the washing performance of the reference proteases against egg stains (set as 100%), as listed in Table 10.

[0971] Evaluation of washing performance in detergent formulations of ASEAN2 (Table 3a).

[0972] Table 9: Washing Conditions

[0973]

[0974] 1) Obtained from the Test Materials Center BV, NL-3130 AC Fraldingen

[0975] Table 10. LOM-scale washing performance in ASEAN2 formulations under the washing conditions given in Table 9. The data show the summative and normalized performance for the three egg stains, expressed as a percentage of the reference control after deducting the detergent control.

[0976]

[0977] As can be seen from Table 10 above, in LOM-scale washing experiments, the protease variants of the present invention (SEQ ID NO: 4 and SEQ ID NO: 16) provided the best cleaning results for egg stains compared with other protease variants not according to the present invention (proteases according to SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17).

Claims

1. A method for cleaning an object, the method comprising contacting the object with a cleaning composition at a temperature of 15°C to 65°C, the cleaning composition comprising a variant polypeptide having protease activity or a fragment of said variant polypeptide having protease activity. The variant polypeptide having protease activity described above: (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1; (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9; (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261 and 271; and (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260. The fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

2. The method of claim 1, wherein, referring to SEQ ID NO: 2, the variant polypeptide or a fragment thereof contains glutamine at position 59.

3. The method according to claim 1 or 2, wherein the variant polypeptide or a fragment thereof (i) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 38, 212, 261 and 271; and (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 18 and 218.

4. The method according to any one of the preceding claims, wherein the variant polypeptide or a fragment thereof (i) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, at least one amino acid substitution is contained at the amino acid residue selected from the group consisting of 21, 38, 160, 210 and 228; and (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 109, 144, 182, 183, 204, 218 and 248.

5. The method according to any one of the preceding claims, wherein: (a) The amino acid substitution at amino acid residue 18 is X18Q / E / D; and / or (b) The amino acid substitution at amino acid residue 21 is X21I / V / F / W / Y, preferably X21V / I; and / or (c) The amino acid substitution at amino acid residue 24 is X24K; and / or (d) The amino acid substitution at amino acid residue 25 is X25D; and / or (e) The amino acid substitution at amino acid residue 38 is X38R / K / H / W; and / or (f) The amino acid substitution at amino acid residue 56 is X56D; and / or (g) 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; and / or (h) The amino acid substitution at amino acid residue 103 is X103S; and / or The substitution of this amino acid at amino acid residue 109 is X109K / A; and / or (i) The amino acid substitution at amino acid residue 144 is X144N / R; and / or (j) The amino acid substitution at amino acid residue 160 is X160H / S / D / E / P, preferably X160H; (k) The amino acid substitution at amino acid residue 182 is X182K / R / E; and / or (l) The amino acid substitution at amino acid residue 183 is X183D / E / C / Q / A / M, preferably X183D / E; and / or (m) The amino acid substitution at amino acid residue 194 is X194E / C / D, preferably X194E / D; and / or (n) The amino acid substitution at amino acid residue 204 is X204D / E / C / G, preferably X204D / E; and / or (o) The amino acid substitution at amino acid residue 210 is X210I / V, preferably X210I; and / or (p) The amino acid substitution at amino acid residue 212 is X212G / M / R / K / P / A / F / H / W / N / Y / D, preferably X212P / A / G / RK / F / Y / M; and / or (q) The amino acid substitution at amino acid residue 218 is X218S / T / D, preferably X218S / T; and / or (r) The amino acid substitution at amino acid residue 228 is X228S; and / or (s) The amino acid substitution at amino acid residue 248 is X248Q / R; and / or (t) The amino acid substitution at amino acid residue 260 is X260D / E / K, preferably X260D / E; and / or (u) The amino acid substitution at amino acid residue 261 is X261L / M / F / E / W / Y / C, preferably X261F / W / Y / L; and / or (v) The amino acid substitution at amino acid residue 271 is X271D.

6. The method according to any one of the preceding claims, wherein the variant polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO:

13.

7. The method according to any one of the preceding claims, wherein the object is a textile or a hard surface, preferably tableware.

8. The method according to any one of the preceding claims, wherein the object has protease-sensitive stains.

9. The method according to any one of the preceding claims, wherein the polypeptide has washing properties comparable to those of the protease according to SEQ ID NO: 3 and increased storage stability compared to the protease according to SEQ ID NO: 3, preferably wherein the polypeptide has washing properties comparable to those of the protease according to SEQ ID NO: 3 against stains selected from the group consisting of eggs, blood / milk / ink, milk / chocolate and grass / spinach.

10. The method according to any one of the preceding claims, wherein the cleaning composition is a liquid detergent composition.

11. The method according to any one of the preceding claims, wherein the cleaning composition comprises one or more additional enzymes different from the variant polypeptides of any one of the preceding claims, preferably selected from the group consisting of: amylase, additional protease, lipase, cellulase, hemicellulase, mannanase, xylanase, DNase, dispersin, pectinase, oxidoreductase and keratinase, preferably selected from amylase, mannanase, cellulase and lipase, most preferably amylase.

12. The method according to any one of the preceding claims, wherein the cleaning composition comprises one or more surfactants and / or one or more builder agents, preferably strongly chelating builder agents.

13. The method according to any one of the preceding claims, wherein the cleaning 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% phenoxyethanol by weight of the composition, and / or preferably comprising 0.001% to 3%, preferably 0.002% to 1%, more preferably 0.01% to 0.6% of 4,4'-dichloro-2-hydroxydiphenyl ether, each by weight of the composition.

14. A composition comprising: (a) A polypeptide having protease activity, wherein the polypeptide having protease activity is: (i) Having an amino acid sequence that is at least 80% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1; (ii) Referring to SEQ ID NO: 2, a serine residue is contained at position 9; (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 25, 38, 160, 210, 212, 228, 261 and 271; and (iv) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, it contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 18, 24, 56, 78, 103, 109, 144, 182, 183, 194, 204, 218, 248 and 260. The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide; and (b) at least one solubilizer selected from the group consisting of neopentyl glycol, γ-valerol, 1,6-hexanediol and diethylene glycol.

15. The composition of claim 14, wherein the composition comprises a protease inhibitor, and the molar ratio of the protease inhibitor to the protease in the composition is not more than 3:1, preferably not more than 1:1, or wherein the composition does not contain a protease inhibitor, preferably wherein the composition does not contain a peptidaldehyde protease inhibitor, or a salt or bisulfite adduct thereof.

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