Polypeptides having protease activity for use in detergent 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, novel proteases are provided. More specifically, genetically engineered proteases, compositions comprising these enzymes, and methods for preparing and using these enzymes or compositions comprising these enzymes are provided. Background Technology

[0002] Enzymes are increasingly being used in a variety of applications as a sustainable alternative to petrochemicals. Enzymes are biodegradable and can already exert their catalytic activity at lower temperatures, thus reducing energy consumption. Specifically, in the detergent industry, enzymes are applied 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, WO 2011 / 072099 A2, WO 2012 / 151534 A1, WO 2015 / 144932 A1, WO 2017 / 207762 A1, WO 2017 / 192692A1, WO 2018 / 069158 A1 and WO 2022 / 225696 A2 also disclose protease variants.

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

[0006] This invention relates to a variant polypeptide with protease activity or a fragment of said protease-active polypeptide, wherein:

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

[0008] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[0009] This fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0010] The present invention further relates to a polynucleotide encoding the variant polypeptide and a composition comprising the variant polypeptide. Detailed Implementation

[0011] The invention can be more readily understood by referring to the following detailed description of embodiments of the invention and the examples included herein.

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

[0013] definition

[0014] Unless otherwise stated, the terminology used herein should be understood in accordance with the usual usage of those skilled in the art.

[0015] 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.

[0016] 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.

[0017] In the context of this invention, the terms "about" and "approximately" indicate a range of precision that, as will be understood by those skilled in the art, still ensures the technical effect of the features discussed. These terms typically indicate a deviation from the indicated numerical value of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5%.

[0018] 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 embodiments of the invention described herein can operate in orders other than those described or shown herein. Where the terms “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” “i,” “ii,” etc., relate to steps of a method, 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.

[0019] This application references numerous publications throughout. The disclosures of all such publications, along with the references cited therein, are hereby incorporated, in their entirety, to provide a more comprehensive description of the prior art to which this invention pertains.

[0020] 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.

[0021] "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, the substitution of histidine at position 120 with alanine is represented as "His120Ala" or "H120A". Substitution can also be described without specifying the parent amino acid at that position by simply naming the amino acid obtained in the variant, for example, by using "X120A" or "120A" or "Xaa120Ala" or "120Ala".

[0022] Variants containing multiple substitutions are separated by "+", such as "Arg170Tyr+Gly195Glu", "R170Y+G195E", or "X170Y+X195E", indicating that arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively. Alternatively, multiple substitutions can be separated by spaces or commas, for example, "R170Y G195E" or "R170Y,G195E". When different alternative substitutions can be introduced at a position, the different substitutions are separated by commas, for example, "Arg170Tyr,Glu" and "R170T,E" indicating that arginine at position 170 is 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}".

[0023] 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 paragraph 3 in the left column on page 204), according to the numbering of BPN' subtilis protease from Bacillus amyloliquefaciens, the sequence of which is shown in SEQ ID NO: 2 (i.e., according to the numbering of SEQ ID NO: 2 or according to the "BPN' numbering").

[0024] 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:

[0025] Table 1

[0026]

[0027] 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).

[0028] The term “heterogeneous” (or exogenous, foreign, recombinant, non-native, or non-natural) polypeptide is defined herein as a polypeptide that is not natural to the host cell; a polypeptide that is natural to the host cell in which structural modifications (e.g., deletion, substitution, and / or insertion) have been made to alter the natural polypeptide using recombinant DNA technology; or a polypeptide that is natural to the host cell in which the expression of the polypeptide is quantitatively altered due to manipulation of the host cell DNA (e.g., a stronger promoter) using recombinant DNA technology, or the expression of the polypeptide is directed from 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 in which structural modifications (e.g., deletions, substitutions, and / or insertions) have been made to alter the native polynucleotide using recombinant DNA technology; or a polynucleotide native to the host cell in which the expression of the polynucleotide is quantitatively altered due to manipulation of the regulatory elements of the polynucleotide (e.g., a stronger promoter) using recombinant DNA technology; or a polynucleotide native to the host cell but not integrated into its native genetic environment due to genetic manipulation using 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 that the two or more polynucleotide sequences or two or more amino acid sequences do not naturally exist in a specific combination of each other.

[0029] For the purposes of this invention, "recombination" (or transgenic) in relation to cells or organisms means cells or organisms containing heterologous polynucleotides introduced by humans using genetic technology. For polynucleotides, "recombination" includes all constructs produced using genetic technology / recombinant DNA technology, wherein...

[0030] (a) A sequence of a polynucleotide or a portion thereof, or

[0031] (b) One or more genetic control sequences operatively linked to a polynucleotide, including but not limited to promoters, or

[0032] (c) both a) and b)

[0033] It is not located in its wild-type genetic environment or has been artificially modified.

[0034] "Synthetic" compounds are obtained through in vitro chemical and / or enzymatic synthesis.

[0035] Variant polynucleotide and variant polypeptide sequences can be defined by their sequence identity when compared to parental sequences. Sequence identity is typically provided in the form of "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.

[0036] 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 showing the corresponding sequence of the present invention over its full length, multiplied by 100: Identity % = (identical residues / length of the aligned region showing the corresponding sequence of the present invention over its full length) 100.

[0037] A special aspect of amino acid substitution is conserved mutations. These conserved mutations often appear to have minimal impact on protein folding compared to the enzymatic properties of the parent enzyme, resulting in essentially unchanged enzymatic properties for the corresponding enzyme variant. 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's properties. This paper considers the following conserved exchanges:

[0038]

[0039] 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.

[0040] As used herein, a “fragment” or “subsequence” refers to a portion of an amino acid sequence. A polypeptide containing the deletion of one or more amino acids at the N-terminus and / or C-terminus and substantially retaining protease activity is designated herein as a “functional fragment.” Preferably, the functional fragment has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, 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 variant polypeptide. Preferably, the functional fragment comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide. 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 that are consecutive to the original full-length amino acid sequence. In the context of this invention, the "original full-length amino acid sequence" is an amino acid sequence that has at least 65% identity with 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.

[0041] In one embodiment, the polypeptide of the present invention does not contain any internal deletions compared to the amino acid sequence according to SEQ ID NO: 1 or 3. However, as discussed above, the polypeptide of the present invention may contain deletions of one or more amino acids at the N-terminus and / or C-terminus of the polypeptide.

[0042] 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.

[0043] 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.

[0044] The terms “introduction of polynucleotides” or “conversion of polynucleotides” as used herein encompass the transfer of exogenous polynucleotides into host cells, regardless of the method used for the 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 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.

[0045] 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 into a protein or no translation. This process includes DNA transcription and processing of the resulting mRNA product.

[0046] 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 "purified enzyme solution".

[0047] "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). 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 larger 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 (e.g., 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.

[0048] "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 actual function of the enzyme; for example, proteases exert proteolytic activity by catalyzing the hydrolytic cleavage of peptide bonds, lipases exert lipolytic activity by catalyzing the hydrolytic cleavage of ester bonds, amylase activity involves the hydrolysis of glycosidic bonds in polysaccharides, etc. According to the present invention, enzyme activity is proteolytic activity, which can be determined by using succinyl-Ala-Ala-Pro-Phe-p-nitroaniline (Suc-AAPF-pNA; see, for example, 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 yellow light from the free pNA or a change in its fluorescence properties, which can be quantified by measuring the 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.

[0049] 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.

[0050] 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.

[0051] As used in this article, an "enzyme inhibitor" is a compound that slows down or stops enzyme activity. Enzyme inhibitors also typically stabilize the three-dimensional structure of enzymes. Therefore, enzyme inhibitors often also act as "enzyme stabilizers."

[0052] "pH stability" refers to the ability of an enzyme to maintain its activity after being exposed to a certain pH value.

[0053] The term "detergent stability" (also referred to herein as "residual activity in detergent" or "storage stability in detergent composition") refers to the ability of an enzyme to exhibit catalytic activity or cleaning performance after storage in a detergent composition (preferably at 37°C, 45°C, or 50°C for up to 14 days in a detergent composition (preferably, in type B detergent as described herein)). Most preferably, "detergent stability" is determined by measuring the residual activity of the protease after storage at 45°C in a detergent composition (preferably, in type B detergent as described herein) for 18 hours and / or 3 days.

[0054] As used herein, the “washing performance” of an enzyme (also referred to herein as “cleaning performance”) refers to the contribution of the enzyme to the cleaning performance of a detergent composition, i.e., the increased cleaning performance of the detergent 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 indicate the conditions of actual household use in the detergent market segment, particularly washing temperature, time, washing machinery, foam concentration, detergent type, and water hardness. The term “improved washing performance” is used to indicate better end results in stain removal under relevant washing conditions, or, relative to the corresponding control conditions, less enzyme, by weight, is required to obtain the same end results.

[0055] As used herein, the term "specific performance" refers to the ability of each unit of active enzyme to clean and remove specific stains or dirt. In some embodiments, specific performance is determined using stains or dirt such as egg, egg yolk, milk, grass, minced meat, blood, chocolate sauce, baby food, sebum, etc.

[0056] "Detergent composition" or "detergent" means a composition specified for cleaning soiled materials. A detergent composition is a compound formulation as further defined herein. Detergent compositions according to the invention include detergent compositions for various applications, such as laundry and hard surface cleaning. Detergent compositions according to the invention also include biodegradable detergent compositions. The term "detergent component" is defined herein as meaning a class of chemicals that can be used in a detergent composition. A typical detergent component is a surfactant. "Surfactant" (used herein synonymously with "surfactant 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.

[0057] 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 the detergent composition of the present invention. The laundry process can be performed using technical equipment such as household or industrial cleaning machines. Alternatively, the laundry process can be performed manually.

[0058] 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.

[0059] 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 that contain 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 a part of a textile (such as knitted, woven, or non-woven fabrics).

[0060] The term "hard surface cleaning" encompasses both household hard surface cleaning and industrial hard surface cleaning and refers to the process of treating hard surfaces with a solution containing the detergent composition of the present invention. Hard surfaces can include any hard surface in the home or industry, such as floors, furniture, walls, sanitary ceramics, glass, metal surfaces (including cutlery or cutlery), and medical devices such as diagnostic instruments, trays, pans, clamps, shelves, tweezers, scissors, shearing machines, saws (e.g., bone saws and their blades), hemostats, knives, chisels, bone forceps, files, pliers, drill bits, chisels, coarse files, deburrs, applicators, lithotripters, elevators, clamps, needle holders, support frames, clips, hooks, round chisels, scrapers, retractors, straighteners, punches, extractors, spoons, keratomes, tongue depressors, pressers, cannulas, dilators, cages, glassware, pipes, catheters, cannulas, plugs, stents, endoscopes, arthroscopes, and related equipment. A specific form of hard surface cleaning is dishwashing, especially automatic dishwashing (ADW).

[0061] The term "dishwashing" refers to all forms of dishwashing, including 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 serving utensils), as well as ceramic, plastic (such as melamine), metal, porcelain, glass, and acrylic.

[0062] 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.

[0063] 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 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 rinsing.

[0064] In the field of stain removal, the term "stain" is generally used for laundry, such as cleaning textiles, fabrics, or fibers, while the term "dirt" is generally used for cleaning hard surfaces, such as tableware and cutlery. However, the terms "stain" and "dirt" should be used interchangeably in this document.

[0065] 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 combine with all calcium ions in an aqueous solution initially having a hardness of 7 °dH (German hardness) at neutral pH, it does not form a large amount of precipitation. “Strong builder” is classified as a highly effective chelating agent, which can strongly bind divalent cations (such as Ca2+), and the logarithmic stability constant (Log K) of the cation / chelating agent complex is [missing information]. 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.

[0066] "Antimicrobial agents" are chemical 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.

[0067] A composition that is “substantially free” of a compound shall herein mean that the corresponding compound is not intentionally added to the composition, that there is at most an ineffective amount, and most preferably that the composition contains 0% of the compound.

[0068] Detailed description

[0069] In this invention, novel proteases are provided. More specifically, variants of the parental protease, methods for preparing the variant protease, compositions comprising the protease variant, and methods for using the variant protease or compositions comprising the variant protease are provided.

[0070] Protease variants

[0071] This invention relates to a variant polypeptide with protease activity or a fragment of said protease-active polypeptide, wherein:

[0072] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0073] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[0074] This fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0075] This invention relates to a variant polypeptide with protease activity, wherein:

[0076] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0077] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[0078] This invention relates to a variant polypeptide with protease activity or a fragment of said protease-active polypeptide, wherein:

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

[0080] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 18, 38, 59, 212, 261 and 271.

[0081] This fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0082] This invention relates to a variant polypeptide with protease activity, wherein:

[0083] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0084] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 18, 38, 59, 212, 261 and 271.

[0085] This invention relates to a variant polypeptide with protease activity or a fragment of said protease-active polypeptide, wherein:

[0086] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0087] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 38, 59, 212, 261 and 271.

[0088] This fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[0089] This invention relates to a variant polypeptide with protease activity, wherein:

[0090] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0091] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 38, 59, 212, 261 and 271.

[0092] This invention relates to a variant polypeptide with protease activity or a fragment of said protease-active polypeptide, wherein:

[0093] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0094] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[0095] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, with reference to SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0096] This invention relates to a variant polypeptide with protease activity, wherein:

[0097] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0098] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0099] This invention relates to a variant polypeptide with protease activity or a fragment of said protease-active polypeptide, wherein:

[0100] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0101] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 38, 59, 212, 261 and 271.

[0102] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, with reference to SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0103] This invention relates to a variant polypeptide with protease activity, wherein:

[0104] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[0105] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 38, 59, 212, 261 and 271, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0106] The protease-active polypeptides of the present invention are non-naturally occurring proteases. Preferably, the protease-active polypeptides of the present invention are purified, isolated, synthetic, and / or recombinant proteases. Preferably, the protease-active polypeptides of the present invention are purified and recombinant proteases.

[0107] 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 cleavage results in the release of yellow light from free pNA or a change in its fluorescence properties, which can be quantified by measuring the emission at 460 nm after excitation at 380 nm.

[0108] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, including amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[0109] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to SEQ ID NO: 2, including amino acid substitutions at three or more amino acid residues selected from the group consisting of 18, 38, 59, 212, 261 and 271.

[0110] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, and X271D.

[0111] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, and X271D.

[0112] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q, X18Q / E / D, X38R / K / H / W, 38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, and X271D.

[0113] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, and E271D.

[0114] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L and E271D.

[0115] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, includes amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0116] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, and X271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0117] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, and X271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0118] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, and E271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0119] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L and E271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0120] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to SEQ ID NO: 2, includes amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[0121] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to SEQ ID NO: 2, includes amino acid substitutions at three or more amino acid residues selected from the group consisting of 18, 38, 59, 212, 261 and 271.

[0122] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, and X271D.

[0123] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, and X271D.

[0124] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, and X271D.

[0125] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, and E271D.

[0126] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, and E271D.

[0127] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to the number of SEQ ID NO: 2, includes amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0128] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, and X271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0129] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, and X271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0130] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3 is compared with and referenced to the number of SEQ ID NO: 2, wherein, with reference to the number of SEQ ID NO: 2, it contains three or more amino acid substitutions selected from the group consisting of: S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, and E271D, wherein the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0131] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L and E271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[0132] The variant polypeptides of the present invention preferably have an amino acid sequence according to SEQ ID NO: 1 of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95.1%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and 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 at one of the following positions:

[0133] .

[0134] The variant polypeptides of the present invention preferably have an amino acid sequence according to SEQ ID NO: 1 of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95.1%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and 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 at one of the following positions:

[0135] .

[0136] The variant polypeptides of the present invention preferably have an amino acid sequence according to SEQ ID NO: 1 of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95.1%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and 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 at one of the following positions:

[0137] .

[0138] The variant polypeptides of the present invention preferably have an amino acid sequence according to SEQ ID NO: 1 of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95.1%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and 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 at one of the following positions:

[0139]

[0140] Referring to SEQ ID NO: 2, the variant polypeptide contains amino acid residues D or E at position 101, preferably E at position 101.

[0141] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, containing one of the following combinations of amino acid substitutions:

[0142] (a)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W;

[0143] (b)X9A / Q / E / C / D, X18Q / E / D, X59K / E;

[0144] (c)X9A / Q / E / C / D, X18Q / E / D, X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0145] (d)X9A / Q / E / C / D, X18Q / E / D, X261L / M / F / E / W / Y / C;

[0146] (e)X9A / Q / E / C / D, X18Q / E / D, X271D;

[0147] (f)X9A / Q / E / C / D, X38R / K / H / W, X59K / E;

[0148] (g)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0149] (h)X9A / Q / E / C / D、X38R / K / H / W、X261L / M / F / E / W / Y / C;

[0150] (i)X9A / Q / E / C / D、X38R / K / H / W、X271D;

[0151] (j)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0152] (k)X9A / Q / E / C / D、X59K / E、X261L / M / F / E / W / Y / C;

[0153] (l)X9A / Q / E / C / D、X59K / E、X271D;

[0154] (m)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0155] (n)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0156] (o)X9A / Q / E / C / D、X261L / M / F / E / W / Y / C、X271D;

[0157] (p)X18Q / E / D、X38R / K / H / W、X59K / E;

[0158] (q)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0159] (r)X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C;

[0160] (s)X18Q / E / D、X38R / K / H / W、X271D;

[0161] (t)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0162] (u)X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C;

[0163] (v)X18Q / E / D、X59K / E、X271D;

[0164] (w)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0165] (x)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0166] (y)X18Q / E / D、X261L / M / F / E / W / Y / C、X271D;

[0167] (z)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0168] (aa)X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0169] (bb)X38R / K / H / W、X59K / E、X271D;

[0170] (cc)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0171] (dd)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0172] (ee)X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[0173] (ff)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0174] (gg)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0175] (hh)X59K / E、X261L / M / F / E / W / Y / C、X271D;

[0176] (ii)X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0177] (jj)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E;

[0178] (kk)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0179] (ll)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C;

[0180] (mm)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X271D;

[0181] (nn)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0182] (oo)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C;

[0183] (pp)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X271D;

[0184] (qq)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0185] (rr)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0186] (ss)X9A / Q / E / C / D、X18Q / E / D、X261L / M / F / E / W / Y / C、X271D;

[0187] (tt)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0188] (uu)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0189] (vv)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X271D;

[0190] (ww)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0191] (xx)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0192] (yy)X9A / Q / E / C / D、X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[0193] (zz)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0194] (aaa)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0195] (bbb)X9A / Q / E / C / D、X59K / E、X261L / M / F / E / W / Y / C、X271D;

[0196] (ccc)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0197] (ddd)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0198] (eee)X18Q / E / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0199] (fff)X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[0200] (ggg)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0201] (hhh)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0202] (iii)X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[0203] (jjj)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0204] (kkk)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0205] (lll)X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C、X271D;

[0206] (mmm)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0207] (nnn)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0208] (ooo)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0209] (ppp)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0210] (qqq)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0211] (rrr)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0212] (sss)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0213] (ttt)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[0214] (uuu)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0215] (vvv)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0216] (www)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0217] (xxx)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0218] (yyy)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0219] (zzz)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0220] (aaaa)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0221] (bbbb)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0222] (cccc)X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, X271D;

[0223] (dddd)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C;

[0224] (eeee)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X271D;

[0225] (ffff)X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, X271D; or

[0226] (gggg)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, X271D.

[0227] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, containing one of the following combinations of amino acid substitutions:

[0228] (a)X9A / Q / E / C / D, X18Q / E / D, X261L / M / F / E / W / Y / C;

[0229] (b)X9A / Q / E / C / D, X38R / K / H / W, X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0230] (c)X9A / Q / E / C / D, X38R / K / H / W, X271D;

[0231] (d)X9A / Q / E / C / D, X261L / M / F / E / W / Y / C, X271D;

[0232] (e)X18Q / E / D, X38R / K / H / W, X261L / M / F / E / W / Y / C;

[0233] (f)X18Q / E / D, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C;

[0234] (g)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0235] (h)X18Q / E / D、X261L / M / F / E / W / Y / C、X271D;

[0236] (i)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0237] (j)X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0238] (k)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0239] (l)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0240] (m)X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[0241] (n)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0242] (o)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0243] (p)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0244] (q)X9A / Q / E / C / D、X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[0245] (r)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0246] (s)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0247] (t)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0248] (u)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0249] (v)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0250] (w)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0251] (x)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0252] (y)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D。

[0253] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, containing one of the following combinations of amino acid substitutions:

[0254] (a)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W;

[0255] (b)X9A / Q / E / C / D, X18Q / E / D, X59K / E;

[0256] (c)X9A / Q / E / C / D, X18Q / E / D, X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0257] (d)X9A / Q / E / C / D, X18Q / E / D, X261L / M / F / E / W / Y / C;

[0258] (e)X9A / Q / E / C / D, X18Q / E / D, X271D;

[0259] (f)X9A / Q / E / C / D, X38R / K / H / W, X59K / E;

[0260] (g)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0261] (h)X9A / Q / E / C / D、X38R / K / H / W、X261L / M / F / E / W / Y / C;

[0262] (i)X9A / Q / E / C / D、X38R / K / H / W、X271D;

[0263] (j)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0264] (k)X9A / Q / E / C / D、X59K / E、X261L / M / F / E / W / Y / C;

[0265] (l)X9A / Q / E / C / D、X59K / E、X271D;

[0266] (m)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0267] (n)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0268] (o)X9A / Q / E / C / D、X261L / M / F / E / W / Y / C、X271D;

[0269] (p)X18Q / E / D、X38R / K / H / W、X59K / E;

[0270] (q)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0271] (r)X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C;

[0272] (s)X18Q / E / D、X38R / K / H / W、X271D;

[0273] (t)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0274] (u)X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C;

[0275] (v)X18Q / E / D、X59K / E、X271D;

[0276] (w)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0277] (x)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0278] (y)X18Q / E / D、X261L / M / F / E / W / Y / C、X271D;

[0279] (z)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0280] (aa)X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0281] (bb)X38R / K / H / W、X59K / E、X271D;

[0282] (cc)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0283] (dd)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0284] (ee)X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[0285] (ff)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0286] (gg)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0287] (hh)X59K / E、X261L / M / F / E / W / Y / C、X271D;

[0288] (ii)X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0289] (jj)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E;

[0290] (kk)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0291] (ll)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C;

[0292] (mm)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X271D;

[0293] (nn)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0294] (oo)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C;

[0295] (pp)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X271D;

[0296] (qq)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0297] (rr)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0298] (ss)X9A / Q / E / C / D、X18Q / E / D、X261L / M / F / E / W / Y / C、X271D;

[0299] (tt)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0300] (uu)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0301] (vv)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X271D;

[0302] (ww)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0303] (xx)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0304] (yy)X9A / Q / E / C / D、X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[0305] (zz)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0306] (aaa)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0307] (bbb)X9A / Q / E / C / D、X59K / E、X261L / M / F / E / W / Y / C、X271D;

[0308] (ccc)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0309] (ddd)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0310] (eee)X18Q / E / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0311] (fff)X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[0312] (ggg)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0313] (hhh)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0314] (iii)X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[0315] (jjj)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0316] (kkk)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0317] (lll)X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C、X271D;

[0318] (mmm)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0319] (nnn)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0320] (ooo)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0321] (ppp)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0322] (qqq)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0323] (rrr)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0324] (sss)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0325] (ttt)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[0326] (uuu)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0327] (vvv)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0328] (www)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0329] (xxx)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0330] (yyy)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0331] (zzz)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0332] (aaaa)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0333] (bbbb)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0334] (cccc)X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D,

[0335] (dddd)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E,

[0336] (eeee)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E,

[0337] (ffff)X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D,

[0338] (gggg)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E,

[0339] Referring to SEQ ID NO: 2, the variant polypeptide contains amino acid residues D or E at position 101, preferably E at position 101.

[0340] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, containing one of the following combinations of amino acid substitutions:

[0341] (a)X9A / Q, X18Q / E / D, X38K / W;

[0342] (b)X9A / Q, X18Q / E / D, X59K / E;

[0343] (c)X9A / Q, X18Q / E / D, X212P / A / G / R / K / F / Y / M;

[0344] (d)X9A / Q, X18Q / E / D, X261F / W / Y / L;

[0345] (e)X9A / Q, X18Q / E / D, X271D;

[0346] (f)X9A / Q, X38R / K / H / W, X59K / E;

[0347] (g)X9A / Q, X38R / K / H / W, X212P / A / G / R / K / F / Y / M;

[0348] (h)X9A / Q、X38R / K / H / W、X261F / W / Y / L;

[0349] (i)X9A / Q、X38R / K / H / W、X271D;

[0350] (j)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M;

[0351] (k)X9A / Q、X59K / E、X261F / W / Y / L;

[0352] (l)X9A / Q、X59K / E、X271D;

[0353] (m)X9A / Q、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0354] (n)X9A / Q、X212P / A / G / R / K / F / Y / M、X271D;

[0355] (o)X9A / Q、X261F / W / Y / L、X271D;

[0356] (p)X18Q / E / D、X38R / K / H / W、X59K / E;

[0357] (q)X18Q / E / D、X38R / K / H / W、X212P / A / G / R / K / F / Y / M;

[0358] (r)X18Q / E / D、X38R / K / H / W、X261F / W / Y / L;

[0359] (s)X18Q / E / D、X38R / K / H / W、X271D;

[0360] (t)X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M;

[0361] (u)X18Q / E / D、X59K / E、X261F / W / Y / L;

[0362] (v)X18Q / E / D、X59K / E、X271D;

[0363] (w)X18Q / E / D、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0364] (x)X18Q / E / D、X212P / A / G / R / K / F / Y / M、X271D;

[0365] (y)X18Q / E / D、X261F / W / Y / L、X271D;

[0366] (z)X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M!

[0367] (aa)X38R / K / H / W、X59K / E、X261F / W / Y / L;

[0368] (bb)X38R / K / H / W、X59K / E、X271D;

[0369] (cc)X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0370] (dd)X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X271D!

[0371] (ee)X38R / K / H / W、X261F / W / Y / L、X271D!

[0372] (ff)X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0373] (gg)X59K / E、X212P / A / G / R / K / F / Y / M、X271D;

[0374] (hh)X59K / E、X261F / W / Y / L、X271D!

[0375] (ii)X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[0376] (jj)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E!

[0377] (kk)X9A / Q、X18Q / E / D、X38R / K / H / W、X212P / A / G / R / K / F / Y / M!

[0378] (ll)X9A / Q、X18Q / E / D、X38R / K / H / W、X261F / W / Y / L;

[0379] (mm)X9A / Q、X18Q / E / D、X38R / K / H / W、X271D!

[0380] (nn)X9A / Q、X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M!

[0381] (oo)X9A / Q、X18Q / E / D、X59K / E、X261F / W / Y / L!

[0382] (pp)X9A / Q、X18Q / E / D、X59K / E、X271D;

[0383] (qq)X9A / Q、X18Q / E / D、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0384] (rr)X9A / Q、X18Q / E / D、X212P / A / G / R / K / F / Y / M、X271D!

[0385] (ss)X9A / Q、X18Q / E / D、X261F / W / Y / L、X271D!

[0386] (tt)X9A / Q、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M!

[0387] (uu)X9A / Q、X38R / K / H / W、X59K / E、X261F / W / Y / L;

[0388] (vv)X9A / Q、X38R / K / H / W、X59K / E、X271D;

[0389] (ww)X9A / Q、X38R / K / H / WX212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0390] (xx)X9A / Q、X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X271D!

[0391] (yy)X9A / Q、X38R / K / H / W、X261F / W / Y / L、X271D!

[0392] (zz)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0393] (aaa)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M、X271D;

[0394] (bbb)X9A / Q、X59K / E、X261F / W / Y / L、X271D;

[0395] (ccc)X9A / Q、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D;

[0396] (ddd)X18Q / E / D、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M;

[0397] (eee)X18Q / E / D、X38R / K / H / W、X59K / E、X261F / W / Y / L;

[0398] (fff)X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[0399] (ggg)

[0400] (hhh)X18Q / E / D、X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X271D;

[0401] (iii)X18Q / E / D、X38R / K / H / W、X261F / W / Y / L、X271D;

[0402] (jjj)X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0403] (kkk)X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M、X271D;

[0404] (lll)X18Q / E / D、X59K / E、X261F / W / Y / L、X271D;

[0405] (mmm)X18Q / E / D,X212P / A / G / R / K / F / Y / M,X261F / W / Y / L,X271D;

[0406] (nnn)

[0407] (ooo)X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X271D!

[0408] (ppp)X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[0409] (qqq)X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[0410] (rrr)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M;

[0411] (sss)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E、X261F / W / Y / L;

[0412] (ttt)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[0413] (uuu)X9A / Q、X18Q / E / D、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[0414] (vvv)X9A / Q、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L:

[0415] (www)X9A / Q、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X271D!

[0416] (xxx)X9A / Q、X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[0417] (yyy)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[0418] (zzz)X18Q / E / D、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L!

[0419] (aaaa) X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X271D;

[0420] (bbbb) X18Q / E / D, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, X271D;

[0421] (cccc) X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, X271D;

[0422] (dddd) X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L;

[0423] (eeee) X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X271D;

[0424] (ffff) X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, X271D; or

[0425] (gggg) X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, X271D.

[0426] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, containing one of the following combinations of amino acid substitutions:

[0427] (a)X9A / Q, X18Q / E / D, X38K / W;

[0428] (b)X9A / Q, X18Q / E / D, X59K / E;

[0429] (c)X9A / Q, X18Q / E / D, X212P / A / G / R / K / F / Y / M;

[0430] (d)X9A / Q, X18Q / E / D, X261F / W / Y / L;

[0431] (e)X9A / Q, X18Q / E / D, X271D;

[0432] (f)X9A / Q, X38R / K / H / W, X59K / E;

[0433] (g)X9A / Q, X38R / K / H / W, X212P / A / G / R / K / F / Y / M;

[0434] (h)X9A / Q、X38R / K / H / W、X261F / W / Y / L;

[0435] (i)X9A / Q、X38R / K / H / W、X271D;

[0436] (j)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M;

[0437] (k)X9A / Q、X59K / E、X261F / W / Y / L;

[0438] (l)X9A / Q、X59K / E、X271D;

[0439] (m)X9A / Q、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0440] (n)X9A / Q、X212P / A / G / R / K / F / Y / M、X271D;

[0441] (o)X9A / Q、X261F / W / Y / L、X271D;

[0442] (p)X18Q / E / D、X38R / K / H / W、X59K / E;

[0443] (q)X18Q / E / D、X38R / K / H / W、X212P / A / G / R / K / F / Y / M;

[0444] (r)X18Q / E / D、X38R / K / H / W、X261F / W / Y / L;

[0445] (s)X18Q / E / D、X38R / K / H / W、X271D;

[0446] (t)X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M;

[0447] (u)X18Q / E / D、X59K / E、X261F / W / Y / L;

[0448] (v)X18Q / E / D、X59K / E、X271D;

[0449] (w)X18Q / E / D、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0450] (x)X18Q / E / D、X212P / A / G / R / K / F / Y / M、X271D;

[0451] (y)X18Q / E / D、X261F / W / Y / L、X271D;

[0452] (z)X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M!

[0453] (aa)X38R / K / H / W、X59K / E、X261F / W / Y / L;

[0454] (bb)X38R / K / H / W、X59K / E、X271D;

[0455] (cc)X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0456] (dd)X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X271D!

[0457] (ee)X38R / K / H / W、X261F / W / Y / L、X271D!

[0458] (ff)X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0459] (gg)X59K / E、X212P / A / G / R / K / F / Y / M、X271D;

[0460] (hh)X59K / E、X261F / W / Y / L、X271D!

[0461] (ii)X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[0462] (jj)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E!

[0463] (kk)X9A / Q、X18Q / E / D、X38R / K / H / W、X212P / A / G / R / K / F / Y / M!

[0464] (ll)X9A / Q、X18Q / E / D、X38R / K / H / W、X261F / W / Y / L;

[0465] (mm)X9A / Q、X18Q / E / D、X38R / K / H / W、X271D!

[0466] (nn)X9A / Q、X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M!

[0467] (oo)X9A / Q、X18Q / E / D、X59K / E、X261F / W / Y / L!

[0468] (pp)X9A / Q、X18Q / E / D、X59K / E、X271D;

[0469] (qq)X9A / Q、X18Q / E / D、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0470] (rr)X9A / Q、X18Q / E / D、X212P / A / G / R / K / F / Y / M、X271D!

[0471] (ss)X9A / Q、X18Q / E / D、X261F / W / Y / L、X271D!

[0472] (tt)X9A / Q、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M!

[0473] (uu)X9A / Q、X38R / K / H / W、X59K / E、X261F / W / Y / L;

[0474] (vv)X9A / Q、X38R / K / H / W、X59K / E、X271D;

[0475] (ww)X9A / Q、X38R / K / H / WX212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0476] (xx)X9A / Q、X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X271D!

[0477] (yy)X9A / Q、X38R / K / H / W、X261F / W / Y / L、X271D!

[0478] (zz)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0479] (aaa)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M、X271D;

[0480] (bbb)X9A / Q、X59K / E、X261F / W / Y / L、X271D;

[0481] (ccc)X9A / Q、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D;

[0482] (ddd)X18Q / E / D、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M;

[0483] (eee)X18Q / E / D、X38R / K / H / W、X59K / E、X261F / W / Y / L;

[0484] (fff)X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[0485] (ggg)

[0486] (hhh)X18Q / E / D、X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X271D;

[0487] (iii)X18Q / E / D、X38R / K / H / W、X261F / W / Y / L、X271D;

[0488] (jjj)X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0489] (kkk)X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M、X271D;

[0490] (lll)X18Q / E / D、X59K / E、X261F / W / Y / L、X271D;

[0491] (mmm)X18Q / E / D,X212P / A / G / R / K / F / Y / M,X261F / W / Y / L,X271D;

[0492] (nnn)

[0493] (ooo)X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X271D!

[0494] (ppp)X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[0495] (qqq)X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[0496] (rrr)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M;

[0497] (sss)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E、X261F / W / Y / L;

[0498] (ttt)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[0499] (uuu)X9A / Q、X18Q / E / D、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[0500] (vvv)X9A / Q、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L:

[0501] (www)X9A / Q、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X271D!

[0502] (xxx)X9A / Q、X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[0503] (yyy)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[0504] (zzz)X18Q / E / D、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L!

[0505] (aaaa)X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X271D;

[0506] (bbbb)X18Q / E / D, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L,

[0507] (cccc)X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L,

[0508] (dddd)X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M,

[0509] (eeee)X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M,

[0510] (ffff)X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L,

[0511] (gggg)X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M,

[0512] Referring to SEQ ID NO: 2, the variant polypeptide contains amino acid residues D or E at position 101, preferably E at position 101.

[0513] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and 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 at one of the following positions:

[0514] (a)S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W;

[0515] (b)S9A / Q / E / C / D, N18Q / E / D, Q59K / E;

[0516] (c)S9A / Q / E / C / D, N18Q / E / D, S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0517] (d)S9A / Q / E / C / D, N18Q / E / D, N261L / M / F / E / W / Y / C;

[0518] (e)S9A / Q / E / C / D, N18Q / E / D, E271D;

[0519] (f)S9A / Q / E / C / D, T38R / K / H / W, Q59K / E;

[0520] (g)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0521] (h)S9A / Q / E / C / D、T38R / K / H / W、N261L / M / F / E / W / Y / C;

[0522] (i)S9A / Q / E / C / D、T38R / K / H / W、E271D;

[0523] (j)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0524] (k)S9A / Q / E / C / D、Q59K / E、N261L / M / F / E / W / Y / C;

[0525] (l)S9A / Q / E / C / D、Q59K / E、E271D;

[0526] (m)S9A / Q / E / C / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0527] (n)S9A / Q / E / C / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0528] (o)S9A / Q / E / C / D、N261L / M / F / E / W / Y / C、E271D;

[0529] (p)N18Q / E / D、T38R / K / H / W、Q59K / E;

[0530] (q)N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0531] (r)N18Q / E / D、T38R / K / H / W、N261L / M / F / E / W / Y / C;

[0532] (s)N18Q / E / D、T38R / K / H / W、E271D;

[0533] (t)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0534] (u)N18Q / E / D、Q59K / E、N261L / M / F / E / W / Y / C;

[0535] (v)N18Q / E / D、Q59K / E、E271D;

[0536] (w)N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0537] (x)N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0538] (y)N18Q / E / D、N261L / M / F / E / W / Y / C、E271D;

[0539] (z)T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0540] (aa)T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[0541] (bb)T38R / K / H / W、Q59K / E、E271D;

[0542] (cc)T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0543] (dd)T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0544] (ee)T38R / K / H / W、N261L / M / F / E / W / Y / C、E271D;

[0545] (ff)Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0546] (gg)Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0547] (hh)Q59K / E、N261L / M / F / E / W / Y / C、E271D;

[0548] (ii)S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0549] (jj)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E;

[0550] (kk)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0551] (ll)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、N261L / M / F / E / W / Y / C;

[0552] (mm)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、E271D;

[0553] (nn)S9A / Q / E / C / D、N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0554] (oo)S9A / Q / E / C / D、N18Q / E / D、Q59K / E、N261L / M / F / E / W / Y / C;

[0555] (pp)S9A / Q / E / C / D、N18Q / E / D、Q59K / E、E271D;

[0556] (qq)S9A / Q / E / C / D、N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0557] (rr)S9A / Q / E / C / D、N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0558] (ss)S9A / Q / E / C / D、N18Q / E / D、N261L / M / F / E / W / Y / C、E271D;

[0559] (tt)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0560] (uu)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[0561] (vv)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、E271D;

[0562] (ww)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0563] (xx)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0564] (yy)S9A / Q / E / C / D、T38R / K / H / W、N261L / M / F / E / W / Y / C、E271D;

[0565] (zz)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0566] (aaa)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0567] (bbb)S9A / Q / E / C / D、Q59K / E、N261L / M / F / E / W / Y / C、E271D;

[0568] (ccc)S9A / Q / E / C / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0569] (ddd)N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0570] (eee)N18Q / E / D、T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[0571] (fff)N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[0572] (ggg)N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0573] (hhh)N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0574] (iii)N18Q / E / D、T38R / K / H / W、N261L / M / F / E / W / Y / C、E271D;

[0575] (jjj)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0576] (kkk)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0577] (lll)N18Q / E / D、Q59K / E、N261L / M / F / E / W / Y / C、E271D;

[0578] (mmm)N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0579] (nnn)T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0580] (ooo)T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0581] (ppp)T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0582] (qqq)Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0583] (rrr)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0584] (sss)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[0585] (ttt)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[0586] (uuu)S9A / Q / E / C / D、N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0587] (vvv)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0588] (www)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0589] (xxx)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0590] (yyy)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0591] (zzz)N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0592] (aaaa)N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0593] (bbbb)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0594] (cccc)T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D;

[0595] (dddd)S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C;

[0596] (eeee)S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, E271D;

[0597] (ffff)N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D; or

[0598] (gggg)S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D.

[0599] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and 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 at one of the following positions:

[0600] (a)S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W;

[0601] (b)S9A / Q / E / C / D, N18Q / E / D, Q59K / E;

[0602] (c)S9A / Q / E / C / D, N18Q / E / D, S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0603] (d)S9A / Q / E / C / D, N18Q / E / D, N261L / M / F / E / W / Y / C;

[0604] (e)S9A / Q / E / C / D, N18Q / E / D, E271D;

[0605] (f)S9A / Q / E / C / D, T38R / K / H / W, Q59K / E;

[0606] (g)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0607] (h)S9A / Q / E / C / D、T38R / K / H / W、N261L / M / F / E / W / Y / C;

[0608] (i)S9A / Q / E / C / D、T38R / K / H / W、E271D;

[0609] (j)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0610] (k)S9A / Q / E / C / D、Q59K / E、N261L / M / F / E / W / Y / C;

[0611] (l)S9A / Q / E / C / D、Q59K / E、E271D;

[0612] (m)S9A / Q / E / C / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0613] (n)S9A / Q / E / C / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0614] (o)S9A / Q / E / C / D、N261L / M / F / E / W / Y / C、E271D;

[0615] (p)N18Q / E / D、T38R / K / H / W、Q59K / E;

[0616] (q)N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0617] (r)N18Q / E / D、T38R / K / H / W、N261L / M / F / E / W / Y / C;

[0618] (s)N18Q / E / D、T38R / K / H / W、E271D;

[0619] (t)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0620] (u)N18Q / E / D、Q59K / E、N261L / M / F / E / W / Y / C;

[0621] (v)N18Q / E / D、Q59K / E、E271D;

[0622] (w)N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0623] (x)N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0624] (y)N18Q / E / D、N261L / M / F / E / W / Y / C、E271D;

[0625] (z)T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0626] (aa)T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[0627] (bb)T38R / K / H / W、Q59K / E、E271D;

[0628] (cc)T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0629] (dd)T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0630] (ee)T38R / K / H / W、N261L / M / F / E / W / Y / C、E271D;

[0631] (ff)Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0632] (gg)Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0633] (hh)Q59K / E、N261L / M / F / E / W / Y / C、E271D;

[0634] (ii)S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0635] (jj)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E;

[0636] (kk)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0637] (ll)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、N261L / M / F / E / W / Y / C;

[0638] (mm)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、E271D;

[0639] (nn)S9A / Q / E / C / D、N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0640] (oo)S9A / Q / E / C / D、N18Q / E / D、Q59K / E、N261L / M / F / E / W / Y / C;

[0641] (pp)S9A / Q / E / C / D、N18Q / E / D、Q59K / E、E271D;

[0642] (qq)S9A / Q / E / C / D、N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0643] (rr)S9A / Q / E / C / D、N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0644] (ss)S9A / Q / E / C / D、N18Q / E / D、N261L / M / F / E / W / Y / C、E271D;

[0645] (tt)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0646] (uu)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[0647] (vv)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、E271D;

[0648] (ww)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0649] (xx)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0650] (yy)S9A / Q / E / C / D、T38R / K / H / W、N261L / M / F / E / W / Y / C、E271D;

[0651] (zz)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0652] (aaa)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0653] (bbb)S9A / Q / E / C / D、Q59K / E、N261L / M / F / E / W / Y / C、E271D;

[0654] (ccc)S9A / Q / E / C / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0655] (ddd)N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0656] (eee)N18Q / E / D、T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[0657] (fff)N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[0658] (ggg)N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0659] (hhh)N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0660] (iii)N18Q / E / D、T38R / K / H / W、N261L / M / F / E / W / Y / C、E271D;

[0661] (jjj)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0662] (kkk)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0663] (lll)N18Q / E / D、Q59K / E、N261L / M / F / E / W / Y / C、E271D;

[0664] (mmm)N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0665] (nnn)T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0666] (ooo)T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0667] (ppp)T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0668] (qqq)Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0669] (rrr)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[0670] (sss)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[0671] (ttt)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[0672] (uuu)S9A / Q / E / C / D、N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0673] (vvv)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0674] (www)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0675] (xxx)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0676] (yyy)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0677] (zzz)N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[0678] (aaaa)N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[0679] (bbbb)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[0680] (cccc)T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D;

[0681] (dddd)S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C;

[0682] (eeee)S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, E271D;

[0683] (ffff)N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D; or

[0684] (gggg)S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D;

[0685] Referring to SEQ ID NO: 2, the variant polypeptide contains amino acid residues D or E at position 101, preferably E at position 101.

[0686] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and 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 at one of the following positions:

[0687] (a)S9A / Q, N18Q / E / D, T38K / W;

[0688] (b)S9A / Q, N18Q / E / D, Q59K / E;

[0689] (c)S9A / Q, N18Q / E / D, S212P / A / G / R / K / F / Y / M;

[0690] (d)S9A / Q, N18Q / E / D, N261F / W / Y / L;

[0691] (e)S9A / Q, N18Q / E / D, E271D;

[0692] (f)S9A / Q, T38R / K / H / W, Q59K / E;

[0693] (g)S9A / Q, T38R / K / H / W, S212P / A / G / R / K / F / Y / M;

[0694] (h)S9A / Q、T38R / K / H / W、N261F / W / Y / L;

[0695] (i)S9A / Q、T38R / K / H / W、E271D;

[0696] (j)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0697] (k)S9A / Q、Q59K / E、N261F / W / Y / L;

[0698] (l)S9A / Q、Q59K / E、E271D;

[0699] (m)S9A / Q、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0700] (n)S9A / Q、S212P / A / G / R / K / F / Y / M、E271D;

[0701] (o)S9A / Q、N261F / W / Y / L、E271D;

[0702] (p)N18Q / E / D、T38R / K / H / W、Q59K / E;

[0703] (q)N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M;

[0704] (r)N18Q / E / D、T38R / K / H / W、N261F / W / Y / L;

[0705] (s)N18Q / E / D、T38R / K / H / W、E271D;

[0706] (t)N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0707] (u)N18Q / E / D、Q59K / E、N261F / W / Y / L;

[0708] (v)N18Q / E / D、Q59K / E、E271D;

[0709] (w)N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0710] (x)N18Q / E / D、S212P / A / G / R / K / F / Y / M、E271D;

[0711] (y)N18Q / E / D、N261F / W / Y / L、E271D;

[0712] (z)T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0713] (aa)T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[0714] (bb)T38R / K / H / W、Q59K / E、E271D;

[0715] (cc)T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0716] (dd)T38R / K / H / W、S212P / A / G / R / K / F / Y / M、E271D;

[0717] (ee)T38R / K / H / W、N261F / W / Y / L、E271D;

[0718] (ff)Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0719] (gg)Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[0720] (hh)Q59K / E、N261F / W / Y / L、E271D;

[0721] (ii)S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0722] (jj)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E;

[0723] (kk)S9A / Q、N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M;

[0724] (ll)S9A / Q、N18Q / E / D、T38R / K / H / W、N261F / W / Y / L;

[0725] (mm)S9A / Q、N18Q / E / D、T38R / K / H / W、E271D;

[0726] (nn)S9A / Q、N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0727] (oo)S9A / Q、N18Q / E / D、Q59K / E、N261F / W / Y / L;

[0728] (pp)S9A / Q、N18Q / E / D、Q59K / E、E271D;

[0729] (qq)S9A / Q、N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0730] (rr)S9A / Q、N18Q / E / D、S212P / A / G / R / K / F / Y / M、E271D;

[0731] (ss)S9A / Q、N18Q / E / D、N261F / W / Y / L、E271D;

[0732] (tt)S9A / Q、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0733] (uu)S9A / Q、T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[0734] (vv)S9A / Q、T38R / K / H / W、Q59K / E、E271D;

[0735] (ww)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0736] (xx)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、E271D;

[0737] (yy)S9A / Q、T38R / K / H / W、N261F / W / Y / L、E271D;

[0738] (zz)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0739] (aaa)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[0740] (bbb)S9A / Q、Q59K / E、N261F / W / Y / L、E271D;

[0741] (ccc)S9A / Q、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0742] (ddd)N18Q / E / D、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0743] (eee)N18Q / E / D、T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[0744] (fff)N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[0745] (ggg)N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0746] (hhh)N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、E271D;

[0747] (iii)N18Q / E / D、T38R / K / H / W、N261F / W / Y / L、E271D;

[0748] (jjj)N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0749] (kkk)N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[0750] (lll)N18Q / E / D、Q59K / E、N261F / W / Y / L、E271D;

[0751] (mmm)N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0752] (nnn)T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0753] (ooo)T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[0754] (ppp)T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0755] (qqq)Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0756] (rrr)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0757] (sss)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[0758] (ttt)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[0759] (uuu)S9A / Q、N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0760] (vvv)S9A / Q、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0761] (www)S9A / Q、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[0762] (xxx)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0763] (yyy)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0764] (zzz)N18Q / E / D、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0765] (aaaa)N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, E271D;

[0766] (bbbb)N18Q / E / D, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D;

[0767] (cccc)T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D;

[0768] (dddd)S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L;

[0769] (eeee)S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, E271D;

[0770] (ffff)N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D; or

[0771] (gggg)S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D.

[0772] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and 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 at one of the following positions:

[0773] (a)S9A / Q, N18Q / E / D, T38K / W;

[0774] (b)S9A / Q, N18Q / E / D, Q59K / E;

[0775] (c)S9A / Q, N18Q / E / D, S212P / A / G / R / K / F / Y / M;

[0776] (d)S9A / Q, N18Q / E / D, N261F / W / Y / L;

[0777] (e)S9A / Q, N18Q / E / D, E271D;

[0778] (f)S9A / Q, T38R / K / H / W, Q59K / E;

[0779] (g)S9A / Q, T38R / K / H / W, S212P / A / G / R / K / F / Y / M;

[0780] (h)S9A / Q、T38R / K / H / W、N261F / W / Y / L;

[0781] (i)S9A / Q、T38R / K / H / W、E271D;

[0782] (j)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0783] (k)S9A / Q、Q59K / E、N261F / W / Y / L;

[0784] (l)S9A / Q、Q59K / E、E271D;

[0785] (m)S9A / Q、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0786] (n)S9A / Q、S212P / A / G / R / K / F / Y / M、E271D;

[0787] (o)S9A / Q、N261F / W / Y / L、E271D;

[0788] (p)N18Q / E / D、T38R / K / H / W、Q59K / E;

[0789] (q)N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M;

[0790] (r)N18Q / E / D、T38R / K / H / W、N261F / W / Y / L;

[0791] (s)N18Q / E / D、T38R / K / H / W、E271D;

[0792] (t)N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0793] (u)N18Q / E / D、Q59K / E、N261F / W / Y / L;

[0794] (v)N18Q / E / D、Q59K / E、E271D;

[0795] (w)N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0796] (x)N18Q / E / D、S212P / A / G / R / K / F / Y / M、E271D;

[0797] (y)N18Q / E / D、N261F / W / Y / L、E271D;

[0798] (z)T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0799] (aa)T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[0800] (bb)T38R / K / H / W、Q59K / E、E271D;

[0801] (cc)T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0802] (dd)T38R / K / H / W、S212P / A / G / R / K / F / Y / M、E271D;

[0803] (ee)T38R / K / H / W、N261F / W / Y / L、E271D;

[0804] (ff)Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0805] (gg)Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[0806] (hh)Q59K / E、N261F / W / Y / L、E271D;

[0807] (ii)S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0808] (jj)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E;

[0809] (kk)S9A / Q、N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M;

[0810] (ll)S9A / Q、N18Q / E / D、T38R / K / H / W、N261F / W / Y / L;

[0811] (mm)S9A / Q、N18Q / E / D、T38R / K / H / W、E271D;

[0812] (nn)S9A / Q、N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0813] (oo)S9A / Q、N18Q / E / D、Q59K / E、N261F / W / Y / L;

[0814] (pp)S9A / Q、N18Q / E / D、Q59K / E、E271D;

[0815] (qq)S9A / Q、N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0816] (rr)S9A / Q、N18Q / E / D、S212P / A / G / R / K / F / Y / M、E271D;

[0817] (ss)S9A / Q、N18Q / E / D、N261F / W / Y / L、E271D;

[0818] (tt)S9A / Q、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0819] (uu)S9A / Q、T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[0820] (vv)S9A / Q、T38R / K / H / W、Q59K / E、E271D;

[0821] (ww)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0822] (xx)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、E271D;

[0823] (yy)S9A / Q、T38R / K / H / W、N261F / W / Y / L、E271D;

[0824] (zz)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0825] (aaa)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[0826] (bbb)S9A / Q、Q59K / E、N261F / W / Y / L、E271D;

[0827] (ccc)S9A / Q、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0828] (ddd)N18Q / E / D、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0829] (eee)N18Q / E / D、T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[0830] (fff)N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[0831] (ggg)N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0832] (hhh)N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、E271D;

[0833] (iii)N18Q / E / D、T38R / K / H / W、N261F / W / Y / L、E271D;

[0834] (jjj)N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0835] (kkk)N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[0836] (lll)N18Q / E / D、Q59K / E、N261F / W / Y / L、E271D;

[0837] (mmm)N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0838] (nnn)T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0839] (ooo)T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[0840] (ppp)T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0841] (qqq)Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0842] (rrr)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[0843] (sss)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[0844] (ttt)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[0845] (uuu)S9A / Q、N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0846] (vvv)S9A / Q、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0847] (www)S9A / Q、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[0848] (xxx)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0849] (yyy)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[0850] (zzz)N18Q / E / D、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[0851] (aaaa)N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, E271D;

[0852] (bbbb)N18Q / E / D, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D;

[0853] (cccc)T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D;

[0854] (dddd)S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L;

[0855] (eeee)S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, E271D;

[0856] (ffff)N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D; or

[0857] (gggg)S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D;

[0858] Referring to SEQ ID NO: 2, the variant polypeptide contains amino acid residues D or E at position 101, preferably E at position 101.

[0859] The polypeptide of the present invention having protease activity is preferably combined with that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. Compared to the amino acid sequence shown in ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid substitutions contained at one of the following positions:

[0860] .

[0861] The polypeptide of the present invention having protease activity is preferably combined with that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. Compared to the amino acid sequence shown in ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid substitutions contained at one of the following positions:

[0862] .

[0863] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. Compared to the amino acid sequence shown in IDNO: 3 and referring to the number of SEQ ID NO: 2, the amino acid substitutions contained at one of the following positions:

[0864] .

[0865] The polypeptide of the present invention having protease activity is preferably combined with that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in ID NO: 3, compared with and referring to the number in SEQ ID NO: 2, contains one of the following combinations of amino acid substitutions:

[0866] (a)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W;

[0867] (b)X9A / Q / E / C / D, X18Q / E / D, X59K / E;

[0868] (c)X9A / Q / E / C / D, X18Q / E / D, X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0869] (d)X9A / Q / E / C / D, X18Q / E / D, X261L / M / F / E / W / Y / C;

[0870] (e)X9A / Q / E / C / D, X18Q / E / D, X271D;

[0871] (f)X9A / Q / E / C / D、X38R / K / H / W、X59K / E;

[0872] (g)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0873] (h)X9A / Q / E / C / D、X38R / K / H / W、X261L / M / F / E / W / Y / C;

[0874] (i)X9A / Q / E / C / D、X38R / K / H / W、X271D;

[0875] (j)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0876] (k)X9A / Q / E / C / D、X59K / E、X261L / M / F / E / W / Y / C;

[0877] (l)X9A / Q / E / C / D、X59K / E、X271D;

[0878] (m)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0879] (n)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0880] (o)X9A / Q / E / C / D、X261L / M / F / E / W / Y / C、X271D;

[0881] (p)X18Q / E / D、X38R / K / H / W、X59K / E;

[0882] (q)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0883] (r)X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C;

[0884] (s)X18Q / E / D、X38R / K / H / W、X271D;

[0885] (t)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0886] (u)X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C;

[0887] (v)X18Q / E / D、X59K / E、X271D;

[0888] (w)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0889] (x)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0890] (y)X18Q / E / D、X261L / M / F / E / W / Y / C、X271D;

[0891] (z)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0892] (aa)X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0893] (bb)X38R / K / H / W、X59K / E、X271D;

[0894] (cc)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0895] (dd)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0896] (ee)X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[0897] (ff)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0898] (gg)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0899] (hh)X59K / E、X261L / M / F / E / W / Y / C、X271D;

[0900] (ii)X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0901] (jj)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E;

[0902] (kk)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0903] (ll)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C;

[0904] (mm)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X271D;

[0905] (nn)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0906] (oo)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C;

[0907] (pp)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X271D;

[0908] (qq)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0909] (rr)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0910] (ss)X9A / Q / E / C / D、X18Q / E / D、X261L / M / F / E / W / Y / C、X271D;

[0911] (tt)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0912] (uu)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0913] (vv)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X271D;

[0914] (ww)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0915] (xx)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0916] (yy)X9A / Q / E / C / D、X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[0917] (zz)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0918] (aaa)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0919] (bbb)X9A / Q / E / C / D、X59K / E、X261L / M / F / E / W / Y / C、X271D;

[0920] (ccc)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0921] (ddd)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0922] (eee)X18Q / E / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0923] (fff)X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[0924] (ggg)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0925] (hhh)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0926] (iii)X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[0927] (jjj)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0928] (kkk)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0929] (lll)X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C、X271D;

[0930] (mmm)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0931] (nnn)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0932] (ooo)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0933] (ppp)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0934] (qqq)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0935] (rrr)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0936] (sss)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0937] (ttt)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[0938] (uuu)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0939] (vvv)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0940] (www)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0941] (xxx)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0942] (yyy)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0943] (zzz)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0944] (aaaa)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0945] (bbbb) X18Q / E / D, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, X271D;

[0946] (cccc) X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, X271D;

[0947] (dddd) X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C;

[0948] (eeee) X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X271D;

[0949] (ffff) X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, X271D; or

[0950] (gggg) X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, X271D.

[0951] The polypeptide of the present invention having protease activity is preferably combined with that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in ID NO: 3, compared with and referring to the number in SEQ ID NO: 2, contains one of the following combinations of amino acid substitutions:

[0952] (a)X9A / Q / E / C / D, X18Q / E / D, X261L / M / F / E / W / Y / C;

[0953] (b)X9A / Q / E / C / D, X38R / K / H / W, X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0954] (c)X9A / Q / E / C / D, X38R / K / H / W, X271D;

[0955] (d)X9A / Q / E / C / D, X261L / M / F / E / W / Y / C, X271D;

[0956] (e)X18Q / E / D, X38R / K / H / W, X261L / M / F / E / W / Y / C;

[0957] (f)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0958] (g)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0959] (h)X18Q / E / D、X261L / M / F / E / W / Y / C、X271D;

[0960] (i)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0961] (j)X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[0962] (k)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0963] (l)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0964] (m)X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[0965] (n)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[0966] (o)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0967] (p)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0968] (q)X9A / Q / E / C / D、X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[0969] (r)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0970] (s)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0971] (t)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0972] (u)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0973] (v)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[0974] (w)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[0975] (x)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[0976] (y)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D。

[0977] The polypeptide of the present invention having protease activity is preferably combined with that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in ID NO: 3, compared with and referring to the number in SEQ ID NO: 2, contains one of the following combinations of amino acid substitutions:

[0978] (a)X9A / Q, X18Q / E / D, X38K / W;

[0979] (b)X9A / Q, X18Q / E / D, X59K / E;

[0980] (c)X9A / Q, X18Q / E / D, X212P / A / G / R / K / F / Y / M;

[0981] (d)X9A / Q, X18Q / E / D, X261F / W / Y / L;

[0982] (e)X9A / Q, X18Q / E / D, X271D;

[0983] (f)X9A / Q, X38R / K / H / W, / E;

[0984] (g)X9A / Q、X38R / K / H / W、X212P / A / G / R / K / F / Y / M;

[0985] (h)X9A / Q、X38R / K / H / W、X261F / W / Y / L;

[0986] (i)X9A / Q、X38R / K / H / W、X271D;

[0987] (j)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M;

[0988] (k)X9A / Q、X59K / E、X261F / W / Y / L;

[0989] (l)X9A / Q、X59K / E、X271D;

[0990] (m)X9A / Q、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[0991] (n)X9A / Q、X212P / A / G / R / K / F / Y / M、X271D;

[0992] (o)X9A / Q、X261F / W / Y / L、X271D;

[0993] (p)X18Q / E / D、X38R / K / H / W、X59K / E;

[0994] (q)X18Q / E / D、X38R / K / H / W、X212P / A / G / R / K / F / Y / M;

[0995] (r)X18Q / E / D、X38R / K / H / W、X261F / W / Y / L;

[0996] (s)X18Q / E / D、X38R / K / H / W、X271D;

[0997] (t)X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M;

[0998] (u)X18Q / E / D、X59K / E、X261F / W / Y / L;

[0999] (v)X18Q / E / D、X59K / E、X271D;

[1000] (w)X18Q / E / D、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1001] (x)X18Q / E / D、X212P / A / G / R / K / F / Y / M、X271D;

[1002] (y)X18Q / E / D、X261F / W / Y / L、X271D;

[1003] (z)X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M!

[1004] (aa)X38R / K / H / W、X59K / E、X261F / W / Y / L;

[1005] (bb)X38R / K / H / W、X59K / E、X271D;

[1006] (cc)X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1007] (dd)X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X271D!

[1008] (ee)X38R / K / H / W、X261F / W / Y / L、X271D!

[1009] (ff)X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1010] (gg)X59K / E、X212P / A / G / R / K / F / Y / M、X271D;

[1011] (hh)X59K / E、X261F / W / Y / L、X271D!

[1012] (ii)X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[1013] (jj)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E!

[1014] (kk)X9A / Q、X18Q / E / D、X38R / K / H / W、X212P / A / G / R / K / F / Y / M!

[1015] (ll)X9A / Q、X18Q / E / D、X38R / K / H / W、X261F / W / Y / L;

[1016] (mm)X9A / Q、X18Q / E / D、X38R / K / H / W、X271D!

[1017] (nn)X9A / Q、X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M!

[1018] (oo)X9A / Q、X18Q / E / D、X59K / E、X261F / W / Y / L!

[1019] (pp)X9A / Q、X18Q / E / D、X59K / E、X271D;

[1020] (qq)X9A / Q、X18Q / E / D、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1021] (rr)X9A / Q、X18Q / E / D、X212P / A / G / R / K / F / Y / M、X271D!

[1022] (ss)X9A / Q、X18Q / E / D、X261F / W / Y / L、X271D!

[1023] (tt)X9A / Q、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M!

[1024] (uu)X9A / Q、X38R / K / H / W、X59K / E、X261F / W / Y / L;

[1025] (vv)X9A / Q、X38R / K / H / W、X59K / E、X271D;

[1026] (ww)X9A / Q、X38R / K / H / WX212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1027] (xx)X9A / Q、X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X271D!

[1028] (yy)X9A / Q、X38R / K / H / W、X261F / W / Y / L、X271D!

[1029] (zz)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1030] (aaa)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M、X271D;

[1031] (bbb)X9A / Q、X59K / E、X261F / W / Y / L、X271D;

[1032] (ccc)X9A / Q、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D;

[1033] (ddd)X18Q / E / D、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M;

[1034] (eee)X18Q / E / D、X38R / K / H / W、X59K / E、X261F / W / Y / L;

[1035] (fff)X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[1036] (ggg)

[1037] (hhh)X18Q / E / D、X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X271D;

[1038] (iii)X18Q / E / D、X38R / K / H / W、X261F / W / Y / L、X271D;

[1039] (jjj)X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1040] (kkk)X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M、X271D;

[1041] (lll)X18Q / E / D、X59K / E、X261F / W / Y / L、X271D;

[1042] (mmm)X18Q / E / D,X212P / A / G / R / K / F / Y / M,X261F / W / Y / L,X271D;

[1043] (nnn)

[1044] (ooo)X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X271D!

[1045] (ppp)X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[1046] (qqq)X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[1047] (rrr)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M;

[1048] (sss)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E、X261F / W / Y / L;

[1049] (ttt)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[1050] (uuu)X9A / Q、X18Q / E / D、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[1051] (vvv)X9A / Q、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L:

[1052] (www)X9A / Q、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X271D!

[1053] (xxx)X9A / Q、X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[1054] (yyy)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[1055] (zzz)X18Q / E / D、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L!

[1056] (aaaa)X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X271D;

[1057] (bbbb)X18Q / E / D, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, X271D;

[1058] (cccc)X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, X271D;

[1059] (dddd)X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L;

[1060] (eeee)X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X271D;

[1061] (ffff)X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, X271D; or

[1062] (gggg)X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, X271D.

[1063] The polypeptide of the present invention having protease activity is preferably combined with that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. Compared to the amino acid sequence shown in ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid substitutions contained at one of the following positions:

[1064] (a)S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W;

[1065] (b)S9A / Q / E / C / D, N18Q / E / D, Q59K / E;

[1066] (c)S9A / Q / E / C / D, N18Q / E / D, S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1067] (d)S9A / Q / E / C / D, N18Q / E / D, N261L / M / F / E / W / Y / C;

[1068] (e)S9A / Q / E / C / D, N18Q / E / D, E271D;

[1069] (f)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E;

[1070] (g)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1071] (h)S9A / Q / E / C / D、T38R / K / H / W、N261L / M / F / E / W / Y / C;

[1072] (i)S9A / Q / E / C / D、T38R / K / H / W、E271D;

[1073] (j)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1074] (k)S9A / Q / E / C / D、Q59K / E、N261L / M / F / E / W / Y / C;

[1075] (l)S9A / Q / E / C / D、Q59K / E、E271D;

[1076] (m)S9A / Q / E / C / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1077] (n)S9A / Q / E / C / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1078] (o)S9A / Q / E / C / D、N261L / M / F / E / W / Y / C、E271D;

[1079] (p)N18Q / E / D、T38R / K / H / W、Q59K / E;

[1080] (q)N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1081] (r)N18Q / E / D、T38R / K / H / W、N261L / M / F / E / W / Y / C;

[1082] (s)N18Q / E / D、T38R / K / H / W、E271D;

[1083] (t)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1084] (u)N18Q / E / D、Q59K / E、N261L / M / F / E / W / Y / C;

[1085] (v)N18Q / E / D、Q59K / E、E271D;

[1086] (w)N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1087] (x)N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1088] (y)N18Q / E / D、N261L / M / F / E / W / Y / C、E271D;

[1089] (z)T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1090] (aa)T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[1091] (bb)T38R / K / H / W、Q59K / E、E271D;

[1092] (cc)T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1093] (dd)T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1094] (ee)T38R / K / H / W、N261L / M / F / E / W / Y / C、E271D;

[1095] (ff)Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1096] (gg)Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1097] (hh)Q59K / E、N261L / M / F / E / W / Y / C、E271D;

[1098] (ii)S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1099] (jj)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E;

[1100] (kk)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1101] (ll)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、N261L / M / F / E / W / Y / C;

[1102] (mm)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、E271D;

[1103] (nn)S9A / Q / E / C / D、N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1104] (oo)S9A / Q / E / C / D、N18Q / E / D、Q59K / E、N261L / M / F / E / W / Y / C;

[1105] (pp)S9A / Q / E / C / D、N18Q / E / D、Q59K / E、E271D;

[1106] (qq)S9A / Q / E / C / D、N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1107] (rr)S9A / Q / E / C / D、N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1108] (ss)S9A / Q / E / C / D、N18Q / E / D、N261L / M / F / E / W / Y / C、E271D;

[1109] (tt)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1110] (uu)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[1111] (vv)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、E271D;

[1112] (ww)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1113] (xx)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1114] (yy)S9A / Q / E / C / D、T38R / K / H / W、N261L / M / F / E / W / Y / C、E271D;

[1115] (zz)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1116] (aaa)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1117] (bbb)S9A / Q / E / C / D、Q59K / E、N261L / M / F / E / W / Y / C、E271D;

[1118] (ccc)S9A / Q / E / C / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1119] (ddd)N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1120] (eee)N18Q / E / D、T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[1121] (fff)N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[1122] (ggg)N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1123] (hhh)N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1124] (iii)N18Q / E / D、T38R / K / H / W、N261L / M / F / E / W / Y / C、E271D;

[1125] (jjj)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1126] (kkk)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1127] (lll)N18Q / E / D、Q59K / E、N261L / M / F / E / W / Y / C、E271D;

[1128] (mmm)N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1129] (nnn)T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1130] (ooo)T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1131] (ppp)T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1132] (qqq)Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1133] (rrr)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1134] (sss)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[1135] (ttt)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[1136] (uuu)S9A / Q / E / C / D、N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1137] (vvv)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1138] (www)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1139] (xxx)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1140] (yyy)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1141] (zzz)N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1142] (aaaa)N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1143] (bbbb) N18Q / E / D, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D;

[1144] (cccc) T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D;

[1145] (dddd) S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C;

[1146] (eeee) S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, E271D;

[1147] (ffff) N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D; or

[1148] (gggg) S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D.

[1149] The polypeptide of the present invention having protease activity is preferably combined with that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. Compared to the amino acid sequence shown in ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid substitutions contained at one of the following positions:

[1150] (a)S9A / Q, N18Q / E / D, T38K / W;

[1151] (b)S9A / Q, N18Q / E / D, Q59K / E;

[1152] (c)S9A / Q, N18Q / E / D, S212P / A / G / R / K / F / Y / M;

[1153] (d)S9A / Q, N18Q / E / D, N261F / W / Y / L;

[1154] (e)S9A / Q, N18Q / E / D, E271D;

[1155] (f)S9A / Q, T38R / K / H / W, Q59K / E;

[1156] (g)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M;

[1157] (h)S9A / Q、T38R / K / H / W、N261F / W / Y / L;

[1158] (i)S9A / Q、T38R / K / H / W、E271D;

[1159] (j)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1160] (k)S9A / Q、Q59K / E、N261F / W / Y / L;

[1161] (l)S9A / Q、Q59K / E、E271D;

[1162] (m)S9A / Q、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1163] (n)S9A / Q、S212P / A / G / R / K / F / Y / M、E271D;

[1164] (o)S9A / Q、N261F / W / Y / L、E271D;

[1165] (p)N18Q / E / D、T38R / K / H / W、Q59K / E;

[1166] (q)N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M;

[1167] (r)N18Q / E / D、T38R / K / H / W、N261F / W / Y / L;

[1168] (s)N18Q / E / D、T38R / K / H / W、E271D;

[1169] (t)N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1170] (u)N18Q / E / D、Q59K / E、N261F / W / Y / L;

[1171] (v)N18Q / E / D、Q59K / E、E271D;

[1172] (w)N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1173] (x)N18Q / E / D、S212P / A / G / R / K / F / Y / M、E271D;

[1174] (y)N18Q / E / D、N261F / W / Y / L、E271D;

[1175] (z)T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1176] (aa)T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[1177] (bb)T38R / K / H / W、Q59K / E、E271D;

[1178] (cc)T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1179] (dd)T38R / K / H / W、S212P / A / G / R / K / F / Y / M、E271D;

[1180] (ee)T38R / K / H / W、N261F / W / Y / L、E271D;

[1181] (ff)Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1182] (gg)Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[1183] (hh)Q59K / E、N261F / W / Y / L、E271D;

[1184] (ii)S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1185] (jj)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E;

[1186] (kk)S9A / Q、N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M;

[1187] (ll)S9A / Q、N18Q / E / D、T38R / K / H / W、N261F / W / Y / L;

[1188] (mm)S9A / Q、N18Q / E / D、T38R / K / H / W、E271D;

[1189] (nn)S9A / Q、N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1190] (oo)S9A / Q、N18Q / E / D、Q59K / E、N261F / W / Y / L;

[1191] (pp)S9A / Q、N18Q / E / D、Q59K / E、E271D;

[1192] (qq)S9A / Q、N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1193] (rr)S9A / Q、N18Q / E / D、S212P / A / G / R / K / F / Y / M、E271D;

[1194] (ss)S9A / Q、N18Q / E / D、N261F / W / Y / L、E271D;

[1195] (tt)S9A / Q、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1196] (uu)S9A / Q、T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[1197] (vv)S9A / Q、T38R / K / H / W、Q59K / E、E271D;

[1198] (ww)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1199] (xx)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、E271D;

[1200] (yy)S9A / Q、T38R / K / H / W、N261F / W / Y / L、E271D;

[1201] (zz)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1202] (aaa)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[1203] (bbb)S9A / Q、Q59K / E、N261F / W / Y / L、E271D;

[1204] (ccc)S9A / Q、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1205] (ddd)N18Q / E / D、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1206] (eee)N18Q / E / D、T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[1207] (fff)N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[1208] (ggg)N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1209] (hhh)N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、E271D;

[1210] (iii)N18Q / E / D、T38R / K / H / W、N261F / W / Y / L、E271D;

[1211] (jjj)N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1212] (kkk)N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[1213] (lll)N18Q / E / D、Q59K / E、N261F / W / Y / L、E271D;

[1214] (mmm)N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1215] (nnn)T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1216] (ooo)T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[1217] (ppp)T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1218] (qqq)Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1219] (rrr)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1220] (sss)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[1221] (ttt)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[1222] (uuu)S9A / Q、N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1223] (vvv)S9A / Q、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1224] (www)S9A / Q、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[1225] (xxx)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1226] (yyy)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1227] (zzz)N18Q / E / D、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1228] (aaaa)N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, E271D;

[1229] (bbbb)N18Q / E / D, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D;

[1230] (cccc)T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D;

[1231] (dddd)S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L;

[1232] (eeee)S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, E271D;

[1233] (ffff)N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D; or

[1234] (gggg)S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D.

[1235] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to SEQ ID NO: 2, including amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 38, 59, 212, 261 and 271.

[1236] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q / E / C / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, and X271D.

[1237] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, and X271D.

[1238] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q / E / C / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, and E271D.

[1239] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L and E271D.

[1240] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to SEQ ID NO: 2, includes amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 38, 59, 212, 261 and 271.

[1241] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q / E / C / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, and X271D.

[1242] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, and X271D.

[1243] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q / E / C / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, and E271D.

[1244] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, and E271D.

[1245] The polypeptide of the present invention having protease activity is preferably combined with that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. Compared to the amino acid sequence shown in ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid substitutions contained at one of the following positions:

[1246]

[1247] Referring to SEQ ID NO: 2, the variant polypeptide contains amino acid residues D or E at position 101, preferably E at position 101.

[1248] The polypeptide of the present invention having protease activity is preferably combined with that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in ID NO: 3, compared with and referring to the number in SEQ ID NO: 2, contains one of the following combinations of amino acid substitutions:

[1249] (a)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W;

[1250] (b)X9A / Q / E / C / D, X18Q / E / D, X59K / E;

[1251] (c)X9A / Q / E / C / D, X18Q / E / D, X212G / M / R / K / P / A / F / H / W / N / Y / D;

[1252] (d)X9A / Q / E / C / D, X18Q / E / D, X261L / M / F / E / W / Y / C;

[1253] (e)X9A / Q / E / C / D, X18Q / E / D, X271D;

[1254] (f)X9A / Q / E / C / D、X38R / K / H / W、X59K / E;

[1255] (g)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[1256] (h)X9A / Q / E / C / D、X38R / K / H / W、X261L / M / F / E / W / Y / C;

[1257] (i)X9A / Q / E / C / D、X38R / K / H / W、X271D;

[1258] (j)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[1259] (k)X9A / Q / E / C / D、X59K / E、X261L / M / F / E / W / Y / C;

[1260] (l)X9A / Q / E / C / D、X59K / E、X271D;

[1261] (m)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[1262] (n)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[1263] (o)X9A / Q / E / C / D、X261L / M / F / E / W / Y / C、X271D;

[1264] (p)X18Q / E / D、X38R / K / H / W、X59K / E;

[1265] (q)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[1266] (r)X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C;

[1267] (s)X18Q / E / D、X38R / K / H / W、X271D;

[1268] (t)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[1269] (u)X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C;

[1270] (v)X18Q / E / D、X59K / E、X271D;

[1271] (w)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[1272] (x)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[1273] (y)X18Q / E / D、X261L / M / F / E / W / Y / C、X271D;

[1274] (z)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[1275] (aa)X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[1276] (bb)X38R / K / H / W、X59K / E、X271D;

[1277] (cc)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[1278] (dd)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[1279] (ee)X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[1280] (ff)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[1281] (gg)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[1282] (hh)X59K / E、X261L / M / F / E / W / Y / C、X271D;

[1283] (ii)X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[1284] (jj)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E;

[1285] (kk)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[1286] (ll)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C;

[1287] (mm)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X271D;

[1288] (nn)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[1289] (oo)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C;

[1290] (pp)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X271D;

[1291] (qq)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[1292] (rr)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[1293] (ss)X9A / Q / E / C / D、X18Q / E / D、X261L / M / F / E / W / Y / C、X271D;

[1294] (tt)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[1295] (uu)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[1296] (vv)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X271D;

[1297] (ww)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[1298] (xx)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[1299] (yy)X9A / Q / E / C / D、X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[1300] (zz)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[1301] (aaa)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[1302] (bbb)X9A / Q / E / C / D、X59K / E、X261L / M / F / E / W / Y / C、X271D;

[1303] (ccc)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[1304] (ddd)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[1305] (eee)X18Q / E / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[1306] (fff)X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[1307] (ggg)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[1308] (hhh)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[1309] (iii)X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D;

[1310] (jjj)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[1311] (kkk)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[1312] (lll)X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C、X271D;

[1313] (mmm)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[1314] (nnn)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[1315] (ooo)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[1316] (ppp)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[1317] (qqq)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[1318] (rrr)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D;

[1319] (sss)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C;

[1320] (ttt)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[1321] (uuu)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[1322] (vvv)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[1323] (www)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[1324] (xxx)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[1325] (yyy)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D;

[1326] (zzz)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C;

[1327] (aaaa)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D;

[1328] (bbbb)X18Q / E / D, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D,

[1329] (cccc)X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D,

[1330] (dddd)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E,

[1331] (eeee)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E,

[1332] (ffff)X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D,

[1333] (gggg)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E,

[1334] Referring to SEQ ID NO: 2, the variant polypeptide contains amino acid residues D or E at position 101, preferably E at position 101.

[1335] The polypeptide of the present invention having protease activity is preferably combined with that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in ID NO: 3, compared with and referring to the number in SEQ ID NO: 2, contains one of the following combinations of amino acid substitutions:

[1336] (a)X9A / Q, X18Q / E / D, X38K / W;

[1337] (b)X9A / Q, X18Q / E / D, X59K / E;

[1338] (c)X9A / Q, X18Q / E / D, X212P / A / G / R / K / F / Y / M;

[1339] (d)X9A / Q, X18Q / E / D, X261F / W / Y / L;

[1340] (e)X9A / Q, X18Q / E / D, X271D;

[1341] (f)X9A / Q, X38R / K / H / W, / E;

[1342] (g)X9A / Q、X38R / K / H / W、X212P / A / G / R / K / F / Y / M;

[1343] (h)X9A / Q、X38R / K / H / W、X261F / W / Y / L;

[1344] (i)X9A / Q、X38R / K / H / W、X271D;

[1345] (j)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M;

[1346] (k)X9A / Q、X59K / E、X261F / W / Y / L;

[1347] (l)X9A / Q、X59K / E、X271D;

[1348] (m)X9A / Q、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1349] (n)X9A / Q、X212P / A / G / R / K / F / Y / M、X271D;

[1350] (o)X9A / Q、X261F / W / Y / L、X271D;

[1351] (p)X18Q / E / D、X38R / K / H / W、X59K / E;

[1352] (q)X18Q / E / D、X38R / K / H / W、X212P / A / G / R / K / F / Y / M;

[1353] (r)X18Q / E / D、X38R / K / H / W、X261F / W / Y / L;

[1354] (s)X18Q / E / D、X38R / K / H / W、X271D;

[1355] (t)X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M;

[1356] (u)X18Q / E / D、X59K / E、X261F / W / Y / L;

[1357] (v)X18Q / E / D、X59K / E、X271D;

[1358] (w)X18Q / E / D、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1359] (x)X18Q / E / D、X212P / A / G / R / K / F / Y / M、X271D;

[1360] (y)X18Q / E / D、X261F / W / Y / L、X271D;

[1361] (z)X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M!

[1362] (aa)X38R / K / H / W、X59K / E、X261F / W / Y / L;

[1363] (bb)X38R / K / H / W、X59K / E、X271D;

[1364] (cc)X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1365] (dd)X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X271D!

[1366] (ee)X38R / K / H / W、X261F / W / Y / L、X271D!

[1367] (ff)X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1368] (gg)X59K / E、X212P / A / G / R / K / F / Y / M、X271D;

[1369] (hh)X59K / E、X261F / W / Y / L、X271D!

[1370] (ii)X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[1371] (jj)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E!

[1372] (kk)X9A / Q、X18Q / E / D、X38R / K / H / W、X212P / A / G / R / K / F / Y / M!

[1373] (ll)X9A / Q、X18Q / E / D、X38R / K / H / W、X261F / W / Y / L;

[1374] (mm)X9A / Q、X18Q / E / D、X38R / K / H / W、X271D!

[1375] (nn)X9A / Q、X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M!

[1376] (oo)X9A / Q、X18Q / E / D、X59K / E、X261F / W / Y / L!

[1377] (pp)X9A / Q、X18Q / E / D、X59K / E、X271D;

[1378] (qq)X9A / Q、X18Q / E / D、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1379] (rr)X9A / Q、X18Q / E / D、X212P / A / G / R / K / F / Y / M、X271D!

[1380] (ss)X9A / Q、X18Q / E / D、X261F / W / Y / L、X271D!

[1381] (tt)X9A / Q、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M!

[1382] (uu)X9A / Q、X38R / K / H / W、X59K / E、X261F / W / Y / L;

[1383] (vv)X9A / Q、X38R / K / H / W、X59K / E、X271D;

[1384] (ww)X9A / Q、X38R / K / H / WX212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1385] (xx)X9A / Q、X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X271D!

[1386] (yy)X9A / Q、X38R / K / H / W、X261F / W / Y / L、X271D!

[1387] (zz)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1388] (aaa)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M、X271D;

[1389] (bbb)X9A / Q、X59K / E、X261F / W / Y / L、X271D;

[1390] (ccc)X9A / Q、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D;

[1391] (ddd)X18Q / E / D、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M;

[1392] (eee)X18Q / E / D、X38R / K / H / W、X59K / E、X261F / W / Y / L;

[1393] (fff)X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[1394] (ggg)

[1395] (hhh)X18Q / E / D、X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X271D;

[1396] (iii)X18Q / E / D、X38R / K / H / W、X261F / W / Y / L、X271D;

[1397] (jjj)X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L;

[1398] (kkk)X18Q / E / D、X59K / E、X212P / A / G / R / K / F / Y / M、X271D;

[1399] (lll)X18Q / E / D、X59K / E、X261F / W / Y / L、X271D;

[1400] (mmm)X18Q / E / D,X212P / A / G / R / K / F / Y / M,X261F / W / Y / L,X271D;

[1401] (nnn)

[1402] (ooo)X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X271D!

[1403] (ppp)X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[1404] (qqq)X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[1405] (rrr)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M;

[1406] (sss)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E、X261F / W / Y / L;

[1407] (ttt)X9A / Q、X18Q / E / D、X38R / K / H / W、X59K / E、X271D;

[1408] (uuu)X9A / Q、X18Q / E / D、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[1409] (vvv)X9A / Q、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L:

[1410] (www)X9A / Q、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X271D!

[1411] (xxx)X9A / Q、X38R / K / H / W、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[1412] (yyy)X9A / Q、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L、X271D!

[1413] (zzz)X18Q / E / D、X38R / K / H / W、X59K / E、X212P / A / G / R / K / F / Y / M、X261F / W / Y / L!

[1414] (aaaa)X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X271D;

[1415] (bbbb)X18Q / E / D, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L,

[1416] (cccc)X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L,

[1417] (dddd)X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M,

[1418] (eeee)X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M,

[1419] (ffff)X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L,

[1420] (gggg)X9A / Q, X18Q / E / D, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M,

[1421] Referring to SEQ ID NO: 2, the variant polypeptide contains amino acid residues D or E at position 101, preferably E at position 101.

[1422] The polypeptide of the present invention having protease activity is preferably combined with that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. Compared to the amino acid sequence shown in ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid substitutions contained at one of the following positions:

[1423] (a)S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W;

[1424] (b)S9A / Q / E / C / D, N18Q / E / D, Q59K / E;

[1425] (c)S9A / Q / E / C / D, N18Q / E / D, S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1426] (d)S9A / Q / E / C / D, N18Q / E / D, N261L / M / F / E / W / Y / C;

[1427] (e)S9A / Q / E / C / D, N18Q / E / D, E271D;

[1428] (f)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E;

[1429] (g)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1430] (h)S9A / Q / E / C / D、T38R / K / H / W、N261L / M / F / E / W / Y / C;

[1431] (i)S9A / Q / E / C / D、T38R / K / H / W、E271D;

[1432] (j)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1433] (k)S9A / Q / E / C / D、Q59K / E、N261L / M / F / E / W / Y / C;

[1434] (l)S9A / Q / E / C / D、Q59K / E、E271D;

[1435] (m)S9A / Q / E / C / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1436] (n)S9A / Q / E / C / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1437] (o)S9A / Q / E / C / D、N261L / M / F / E / W / Y / C、E271D;

[1438] (p)N18Q / E / D、T38R / K / H / W、Q59K / E;

[1439] (q)N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1440] (r)N18Q / E / D、T38R / K / H / W、N261L / M / F / E / W / Y / C;

[1441] (s)N18Q / E / D、T38R / K / H / W、E271D;

[1442] (t)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1443] (u)N18Q / E / D、Q59K / E、N261L / M / F / E / W / Y / C;

[1444] (v)N18Q / E / D、Q59K / E、E271D;

[1445] (w)N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1446] (x)N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1447] (y)N18Q / E / D、N261L / M / F / E / W / Y / C、E271D;

[1448] (z)T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1449] (aa)T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[1450] (bb)T38R / K / H / W、Q59K / E、E271D;

[1451] (cc)T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1452] (dd)T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1453] (ee)T38R / K / H / W、N261L / M / F / E / W / Y / C、E271D;

[1454] (ff)Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1455] (gg)Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1456] (hh)Q59K / E、N261L / M / F / E / W / Y / C、E271D;

[1457] (ii)S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1458] (jj)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E;

[1459] (kk)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1460] (ll)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、N261L / M / F / E / W / Y / C;

[1461] (mm)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、E271D;

[1462] (nn)S9A / Q / E / C / D、N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1463] (oo)S9A / Q / E / C / D、N18Q / E / D、Q59K / E、N261L / M / F / E / W / Y / C;

[1464] (pp)S9A / Q / E / C / D、N18Q / E / D、Q59K / E、E271D;

[1465] (qq)S9A / Q / E / C / D、N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1466] (rr)S9A / Q / E / C / D、N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1467] (ss)S9A / Q / E / C / D、N18Q / E / D、N261L / M / F / E / W / Y / C、E271D;

[1468] (tt)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1469] (uu)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[1470] (vv)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、E271D;

[1471] (ww)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1472] (xx)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1473] (yy)S9A / Q / E / C / D、T38R / K / H / W、N261L / M / F / E / W / Y / C、E271D;

[1474] (zz)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1475] (aaa)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1476] (bbb)S9A / Q / E / C / D、Q59K / E、N261L / M / F / E / W / Y / C、E271D;

[1477] (ccc)S9A / Q / E / C / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1478] (ddd)N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1479] (eee)N18Q / E / D、T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[1480] (fff)N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[1481] (ggg)N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1482] (hhh)N18Q / E / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1483] (iii)N18Q / E / D、T38R / K / H / W、N261L / M / F / E / W / Y / C、E271D;

[1484] (jjj)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1485] (kkk)N18Q / E / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1486] (lll)N18Q / E / D、Q59K / E、N261L / M / F / E / W / Y / C、E271D;

[1487] (mmm)N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1488] (nnn)T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1489] (ooo)T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1490] (ppp)T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1491] (qqq)Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1492] (rrr)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D;

[1493] (sss)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E、N261L / M / F / E / W / Y / C;

[1494] (ttt)S9A / Q / E / C / D、N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[1495] (uuu)S9A / Q / E / C / D、N18Q / E / D、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1496] (vvv)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1497] (www)S9A / Q / E / C / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1498] (xxx)S9A / Q / E / C / D、T38R / K / H / W、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1499] (yyy)S9A / Q / E / C / D、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C、E271D;

[1500] (zzz)N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、N261L / M / F / E / W / Y / C;

[1501] (aaaa)N18Q / E / D、T38R / K / H / W、Q59K / E、S212G / M / R / K / P / A / F / H / W / N / Y / D、E271D;

[1502] (bbbb)N18Q / E / D, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D;

[1503] (cccc)T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D;

[1504] (dddd)S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C;

[1505] (eeee)S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, E271D;

[1506] (ffff)N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D; or

[1507] (gggg)S9A / Q / E / C / D, N18Q / E / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C, E271D;

[1508] Referring to SEQ ID NO: 2, the variant polypeptide contains amino acid residues D or E at position 101, preferably E at position 101.

[1509] The variant polypeptides of the present invention having protease activity are preferably combined with those according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. Compared to the amino acid sequence shown in ID NO: 3 and referring to the number of SEQ ID NO: 2, the amino acid substitutions contained at one of the following positions:

[1510] (a)S9A / Q, N18Q / E / D, T38K / W;

[1511] (b)S9A / Q, N18Q / E / D, Q59K / E;

[1512] (c)S9A / Q, N18Q / E / D, S212P / A / G / R / K / F / Y / M;

[1513] (d)S9A / Q, N18Q / E / D, N261F / W / Y / L;

[1514] (e)S9A / Q, N18Q / E / D, E271D;

[1515] (f)S9A / Q, T38R / K / H / W, Q59K / E;

[1516] (g)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M;

[1517] (h)S9A / Q、T38R / K / H / W、N261F / W / Y / L;

[1518] (i)S9A / Q、T38R / K / H / W、E271D;

[1519] (j)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1520] (k)S9A / Q、Q59K / E、N261F / W / Y / L;

[1521] (l)S9A / Q、Q59K / E、E271D;

[1522] (m)S9A / Q、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1523] (n)S9A / Q、S212P / A / G / R / K / F / Y / M、E271D;

[1524] (o)S9A / Q、N261F / W / Y / L、E271D;

[1525] (p)N18Q / E / D、T38R / K / H / W、Q59K / E;

[1526] (q)N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M;

[1527] (r)N18Q / E / D、T38R / K / H / W、N261F / W / Y / L;

[1528] (s)N18Q / E / D、T38R / K / H / W、E271D;

[1529] (t)N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1530] (u)N18Q / E / D、Q59K / E、N261F / W / Y / L;

[1531] (v)N18Q / E / D、Q59K / E、E271D;

[1532] (w)N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1533] (x)N18Q / E / D、S212P / A / G / R / K / F / Y / M、E271D;

[1534] (y)N18Q / E / D、N261F / W / Y / L、E271D;

[1535] (z)T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1536] (aa)T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[1537] (bb)T38R / K / H / W、Q59K / E、E271D;

[1538] (cc)T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1539] (dd)T38R / K / H / W、S212P / A / G / R / K / F / Y / M、E271D;

[1540] (ee)T38R / K / H / W、N261F / W / Y / L、E271D;

[1541] (ff)Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1542] (gg)Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[1543] (hh)Q59K / E、N261F / W / Y / L、E271D;

[1544] (ii)S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1545] (jj)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E;

[1546] (kk)S9A / Q、N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M;

[1547] (ll)S9A / Q、N18Q / E / D、T38R / K / H / W、N261F / W / Y / L;

[1548] (mm)S9A / Q、N18Q / E / D、T38R / K / H / W、E271D;

[1549] (nn)S9A / Q、N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1550] (oo)S9A / Q、N18Q / E / D、Q59K / E、N261F / W / Y / L;

[1551] (pp)S9A / Q、N18Q / E / D、Q59K / E、E271D;

[1552] (qq)S9A / Q、N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1553] (rr)S9A / Q、N18Q / E / D、S212P / A / G / R / K / F / Y / M、E271D;

[1554] (ss)S9A / Q、N18Q / E / D、N261F / W / Y / L、E271D;

[1555] (tt)S9A / Q、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1556] (uu)S9A / Q、T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[1557] (vv)S9A / Q、T38R / K / H / W、Q59K / E、E271D;

[1558] (ww)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1559] (xx)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、E271D;

[1560] (yy)S9A / Q、T38R / K / H / W、N261F / W / Y / L、E271D;

[1561] (zz)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1562] (aaa)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[1563] (bbb)S9A / Q、Q59K / E、N261F / W / Y / L、E271D;

[1564] (ccc)S9A / Q、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1565] (ddd)N18Q / E / D、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1566] (eee)N18Q / E / D、T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[1567] (fff)N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[1568] (ggg)N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1569] (hhh)N18Q / E / D、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、E271D;

[1570] (iii)N18Q / E / D、T38R / K / H / W、N261F / W / Y / L、E271D;

[1571] (jjj)N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1572] (kkk)N18Q / E / D、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[1573] (lll)N18Q / E / D、Q59K / E、N261F / W / Y / L、E271D;

[1574] (mmm)N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1575] (nnn)T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1576] (ooo)T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[1577] (ppp)T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1578] (qqq)Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1579] (rrr)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M;

[1580] (sss)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E、N261F / W / Y / L;

[1581] (ttt)S9A / Q、N18Q / E / D、T38R / K / H / W、Q59K / E、E271D;

[1582] (uuu)S9A / Q、N18Q / E / D、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1583] (vvv)S9A / Q、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1584] (www)S9A / Q、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、E271D;

[1585] (xxx)S9A / Q、T38R / K / H / W、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1586] (yyy)S9A / Q、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L、E271D;

[1587] (zzz)N18Q / E / D、T38R / K / H / W、Q59K / E、S212P / A / G / R / K / F / Y / M、N261F / W / Y / L;

[1588] (aaaa)N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, E271D;

[1589] (bbbb)N18Q / E / D, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D;

[1590] (cccc)T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D;

[1591] (dddd)S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L;

[1592] (eeee)S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, E271D;

[1593] (ffff)N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D; or

[1594] (gggg)S9A / Q, N18Q / E / D, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, E271D;

[1595] Referring to SEQ ID NO: 2, the variant polypeptide contains amino acid residues D or E at position 101, preferably E at position 101.

[1596] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, includes amino acid substitutions at three or more amino acid residues selected from the group consisting of: 9, 38, 59, 212, 261 and 271, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1597] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q / E / C / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, and X271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1598] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, and X271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1599] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q / E / C / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C and E271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1600] The polypeptide of the present invention having protease activity preferably has an amino acid sequence according to SEQ ID NO: 1 having a content of at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, and at least 95%. %, at least 95.2%, at least 95.3%, 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%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, but less than 100% sequence identity, and with SEQ The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, and E271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1601] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to the number of SEQ ID NO: 2, includes amino acid substitutions at three or more amino acid residues selected from the group consisting of: 9, 38, 59, 212, 261 and 271, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1602] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q / E / C / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, X261L / M / F / E / W / Y / C, and X271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1603] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in IDNO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: X9A / Q, X38R / K / H / W, X59K / E, X212P / A / G / R / K / F / Y / M, X261F / W / Y / L, and X271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1604] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q / E / C / D, T38R / K / H / W, Q59K / E, S212G / M / R / K / P / A / F / H / W / N / Y / D, N261L / M / F / E / W / Y / C and E271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1605] The variant polypeptide of the present invention is preferably related to that according to SEQ ID NO: The amino acid sequence of 1 has 65% to 98%, 66% to 98%, 67% to 98%, 68% to 98%, 69% to 98%, 70% to 98%, 71% to 98%, 72% to 98%, 73% to 98%, 74% to 98%, 75% to 98%, 76% to 98%, 77% to 98%, 78% to 98%, 79% to 98%, 80% to 98%, 82% 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%, 88% to 98%, 88.5% to 98%, and 89%... The sequence identity is 98%, 89.5%, 98%, 90%, 98%, 90.3%, 98%, 90.5%, 98%, 90.7%, 98%, 91%, 98%, 91.3%, 98%, 91.5%, 98%, 91.7%, 92%, 98%, 92.3%, 98%, 92.5%, 98%, 92.7%, 93%, 98%, 93.3%, 98%, 93.5%, 98%, 93.7%, 94%, 98%, 94.3%, 98%, 94.5%, 98%, 94.7%, 95%, 98%, 95.1%, 95.2%, 98%, 95.3%, 98%, 95.4%, and 95.5% to 98%, and is consistent with SEQ. The amino acid sequence shown in ID NO: 3, compared with and referring to the number of SEQ ID NO: 2, contains three or more amino acid substitutions selected from the group consisting of: S9A / Q, T38R / K / H / W, Q59K / E, S212P / A / G / R / K / F / Y / M, N261F / W / Y / L, and E271D, wherein, referring to the number of SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1606] 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 of the present invention or the fragment of the polypeptide having protease activity further includes at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 43, 78, 160, 183, 194, 204, 210, 218, 228, 259 and 260.

[1607] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1608] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1609] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[1610] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 43, 78, 160, 183, 194, 204, 210, 218, 228, 259 and 260.

[1611] This fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[1612] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1613] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1614] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[1615] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 43, 78, 160, 183, 194, 204, 210, 218, 228, 259 and 260.

[1616] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, with reference to SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1617] 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 of the present invention or the fragment of the polypeptide having protease activity further comprises at least one amino acid substitution selected from the group consisting of: X21I / V / F / W / Y, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X160H / S / D / E / P, X183D / E / C / Q / A / M, X194E / C / D, X204D / E / C / G, X210I / V, X218T / S / D, X228S, X259E / D, and X260D / E / K.

[1618] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1619] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1620] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[1621] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains at least one amino acid substitution selected from the group consisting of: X21I / V / F / W / Y, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X160H / S / D / E / P, X183D / E / C / Q / A / M, X194E / C / D, X204D / E / C / G, X210I / V, X218T / S / D, X228S, X259E / D, and X260D / E / K,

[1622] This fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[1623] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1624] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1625] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[1626] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains at least one amino acid substitution selected from the group consisting of: X21I / V / F / W / Y, X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, X78N / D / R / W / F / H / K / E / L / Y / M / C / Q, X160H / S / D / E / P, X183D / E / C / Q / A / M, X194E / C / D, X204D / E / C / G, X210I / V, X218T / S / D, X228S, X259E / D, and X260D / E / K,

[1627] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, with reference to SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1628] 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 of the present invention or the fragment of the polypeptide having protease activity further comprises at least one amino acid substitution selected from the group consisting of: X21V / I, X43K / R, X78N / D, X160H, X183D / E, X194E / D, X204D / E, X210I, X218S / T, X228S, X259D, and X260D / E.

[1629] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1630] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1631] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[1632] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains at least one amino acid substitution selected from the group consisting of: X21V / I, X43K / R, X78N / D, X160H, X183D / E, X194E / D, X204D / E, X210I, X218S / T, X228S, X259D and X260D / E,

[1633] This fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[1634] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1635] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1636] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[1637] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains at least one amino acid substitution selected from the group consisting of: X21V / I, X43K / R, X78N / D, X160H, X183D / E, X194E / D, X204D / E, X210I / V, X218S / T, X228S, X259D, and X260D / E,

[1638] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, with reference to SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1639] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number in SEQ ID NO: 2, the variant polypeptide of the present invention or the fragment of the polypeptide having protease activity further comprises at least one amino acid substitution selected from the group consisting of: L21I / V / F / W / Y, N43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, S78N / D / R / W / F / H / K / E / L / Y / M / C / Q, S160H / S / D / E / P, N183D / E / C / Q / A / M, A194E / C / D, N204D / E / C / G, P210I / V, N218T / S / D, A228S, S259E / D, and T260D / E / K.

[1640] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to SEQ ID NO: 2, the variant polypeptide of the present invention or the fragment of the polypeptide having protease activity further comprises at least one amino acid substitution selected from the group consisting of: L21V / I, N43K / R, S78N / D, S160H, N183D / E, A194E / D, N204D / E, P210I, N218S / T, A228S, S259D, and T260D / E.

[1641] 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 of the present invention or the fragment of the polypeptide having protease activity further includes at least one amino acid substitution at an amino acid residue selected from the group consisting of 24, 25, 103, 109, 144, 182 and 248.

[1642] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1643] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1644] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[1645] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 24, 25, 103, 109, 144, 182 and 248.

[1646] This fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[1647] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1648] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1649] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[1650] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 24, 25, 103, 109, 144, 182 and 248.

[1651] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, with reference to SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1652] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number SEQ ID NO: 2, the variant polypeptide of the present invention or the fragment of the polypeptide having protease activity further comprises at least one amino acid substitution selected from the group consisting of: X24K, X25D, X103S, X109K / A, X144N / R, X182K / R / E and X248Q / R.

[1653] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to SEQ ID NO: 2, the variant polypeptide of the present invention or the fragment of the polypeptide having protease activity further comprises at least one amino acid substitution selected from the group consisting of: S24K, G25D, A103S, Q109K / A, S144N / R, Q182K / R / E and 182K / R / E and N248Q / R.

[1654] 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 of the present invention or the fragment of the polypeptide having protease activity further includes at least one amino acid substitution at an amino acid residue selected from the group consisting of 3, 76 and 256.

[1655] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1656] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1657] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[1658] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 3, 76, and 256.

[1659] This fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[1660] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1661] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1662] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[1663] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 3, 76, and 256.

[1664] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, with reference to SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1665] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1666] (i) The polypeptide has an amino acid sequence that is 95.5% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1667] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[1668] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 3, 76, and 256.

[1669] This fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[1670] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1671] (i) The polypeptide has an amino acid sequence that is 95.5% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1672] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[1673] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 3, 76, and 256.

[1674] The fragment of the variant polypeptide comprises 100 to 259 consecutive amino acids of the full-length variant polypeptide, and wherein, with reference to SEQ ID NO: 2, the variant polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

[1675] 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 of the present invention or the fragment of the polypeptide having protease activity further comprises at least one amino acid substitution selected from the group consisting of X3T / Q / V, X76D and X256E / T / D / R / P.

[1676] 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 of the present invention or the fragment of the polypeptide having protease activity further comprises at least one amino acid substitution selected from the group consisting of S3T / Q / V, N76D and S256E / T / D / R / P.

[1677] In one embodiment, the variant polypeptide of the present invention or the fragment of the polypeptide having protease activity, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, includes amino acid substitutions at positions 18, 21, 24, 25, 38, 78, 103, 109, 144, 160, 182, 183, 194, 204, 210, 212, 218, 228, 248, 260, and 271, and refers to the number of SEQ ID NO: 2, includes amino acid residue D or E at position 101, preferably E at position 101.

[1678] In one embodiment, the variant polypeptide of the present invention or the fragment of the polypeptide having protease activity, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, contains amino acid substitutions X18Q / E / D, X21V, X24K, X25D, X38R / K / H / W, X78N / D, X103S, X109K / A, X144N / R, X160H, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X228S, X248Q / R, X260D / E, and X271D, and refers to the number of SEQ ID NO: 2, contains amino acid residue D or E at position 101, preferably E at position 101.

[1679] In one embodiment, the variant polypeptide of the present invention or the fragment of the polypeptide having protease activity, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, contains amino acid substitutions N18Q / E / D, L21V, S24K, G25D, T38R / K / H / W, S78N / D, A103S, Q109K / A, S144N / R, S160H, Q182K / R / E, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, A228S, N248Q / R, T260D / E, and E271D, and refers to the number of SEQ ID NO: 2, contains amino acid residue D or E at position 101, preferably E at position 101.

[1680] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1681] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1682] (ii) The polypeptide or a fragment thereof, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, contains amino acid substitutions at positions 18, 21, 24, 25, 38, 78, 103, 109, 144, 160, 182, 183, 194, 204, 210, 212, 218, 228, 248, 260 and 271, and, referring to the number of SEQ ID NO: 2, contains amino acid residue D or E at position 101, preferably containing E at position 101.

[1683] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1684] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1685] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or fragment thereof contains amino acid substitutions X18Q / E / D, X21V, X24K, X25D, X38R / K / H / W, X78N / D, X103S, X109K / A, X144N / R, X160H, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X228S, X248Q / R, X260D / E and X271D, and referring to the number of SEQ ID NO: 2, contains amino acid residue D or E at position 101, preferably E at position 101.

[1686] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1687] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1688] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or fragment thereof contains amino acid substitutions N18Q / E / D, L21V, S24K, G25D, T38R / K / H / W, S78N / D, A103S, Q109K / A, S144N / R, S160H, Q182K / R / E, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, A228S, N248Q / R, T260D / E and E271D, and referring to the number of SEQ ID NO: 2, contains amino acid residue D or E at position 101, preferably E at position 101.

[1689] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1690] (i) The polypeptide has an amino acid sequence that is 95.5% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1691] (ii) The polypeptide or a fragment thereof, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, contains amino acid substitutions at positions 18, 21, 24, 25, 38, 78, 103, 109, 144, 160, 182, 183, 194, 204, 210, 212, 218, 228, 248, 260 and 271, and, referring to the number of SEQ ID NO: 2, contains amino acid residue D or E at position 101, preferably containing E at position 101.

[1692] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1693] (i) The polypeptide has an amino acid sequence that is 95.5% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1694] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or fragment thereof contains amino acid substitutions X18Q / E / D, X21V, X24K, X25D, X38R / K / H / W, X78N / D, X103S, X109K / A, X144N / R, X160H, X182K / R / E, X183D / E, X194E / D, X204D / E, X210I, X212P / A / G / R / K / F / Y / M, X218S / T, X228S, X248Q / R, X260D / E and X271D, and referring to the number of SEQ ID NO: 2, contains amino acid residue D or E at position 101, preferably E at position 101.

[1695] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1696] (i) The polypeptide has an amino acid sequence that is 95.5% to 98% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1697] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or fragment thereof contains amino acid substitutions N18Q / E / D, L21V, S24K, G25D, T38R / K / H / W, S78N / D, A103S, Q109K / A, S144N / R, S160H, Q182K / R / E, N183D / E, A194E / D, N204D / E, P210I, S212P / A / G / R / K / F / Y / M, N218S / T, A228S, N248Q / R, T260D / E and E271D, and referring to the number of SEQ ID NO: 2, contains amino acid residue D or E at position 101, preferably E at position 101.

[1698] In one embodiment, the amino acid substitution at amino acid residue 9 is X9A, and / or the amino acid substitution at amino acid residue 18 is X18Q; and / or the amino acid substitution at amino acid residue 21 is X21V; and / or the amino acid substitution at amino acid residue 38 is X38K / W; and / or the amino acid substitution at amino acid residue 43 is X43K / R; and / or the amino acid substitution at amino acid residue 59 is X59K; and / or the amino acid substitution at amino acid residue 78 is X78N; and / or the amino acid substitution at amino acid residue 109 is X109K; and / or the amino acid substitution at amino acid residue 144 is X144N; and / or the amino acid substitution at amino acid residue 182 is X182K; and / or The amino acid substitution at amino acid residue 183 is X183D; and / or the amino acid substitution at amino acid residue 194 is X194E; and / or the amino acid substitution at amino acid residue 204 is X204D; and / or the amino acid substitution at amino acid residue 210 is X210I; and / or the amino acid substitution at amino acid residue 212 is X212P; and / or the amino acid substitution at amino acid residue 218 is X218S; and / or the amino acid substitution at amino acid residue 248 is X248Q; and / or the amino acid substitution at amino acid residue 259 is X259D; and / or the amino acid substitution at amino acid residue 260 is X260D; and / or the amino acid substitution at amino acid residue 261 is X261F.

[1699] 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 of the present invention or the fragment of the polypeptide having protease activity further includes at least one amino acid substitution at an amino acid residue selected from the group consisting of 8, 108, 133, 137 and 215.

[1700] Therefore, in one embodiment, the present invention relates to a variant polypeptide having protease activity or a fragment of said protease-active polypeptide, wherein:

[1701] (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and

[1702] (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271.

[1703] (iii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains at least one amino acid substitution at an amino acid residue selected from the group consisting of 8, 108, 133, 137 and 215.

[1704] This fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

[1705] In one embodiment, the variant polypeptide of the present invention has 95.5% to 98% sequence identity with the amino acid sequence according to SEQ ID NO: 1, and, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number in SEQ ID NO: 2, includes amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261, and 271.

[1706] The variant polypeptide, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, further includes at least one amino acid substitution at an amino acid residue selected from the group consisting of 8, 108, 133, 137 and 215.

[1707] In one embodiment, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the SEQ ID NO: 2 number, the variant polypeptide of the present invention or the fragment of the polypeptide having protease activity further comprises at least one amino acid substitution selected from the group consisting of: X8V, X108V, X133L, X137 / LH and X215K / R / Q / N / S / T / W.

[1708] 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 contains amino acids selected from 4, 15, 35, 40, 45, 55, 58, 61, 68, 69, 72, 87, 97, 98, 104, 106, 114, 115, 117, 118, 120, 124, 128, 129, 130, 136, 138, 143, 145, 147, 149, 156, 158, 161, 1 At least one amino acid substitution at an amino acid residue in the group consisting of 63, 166, 167, 169, 170, 171, 172, 177, 185, 187, 188, 189, 191, 195, 199, 205, 206, 209, 211, 216, 217, 222, 225, 235, 238, 244, 245, 255 and / or 262.

[1709] 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 at least one amino acid substitution selected from the group consisting of: X4I, X15T, X35XD, X40E, X45E / D / Q, X55N, X58W / L / N, X61E / D / R, X68A, X69S, X72V / A, X87E / D / N / R, X97AD / XE / D / F, X98D / E / R, X104F / V, X106A / W, X114V, X115T / W, X117E, X118R / V, 128A / L / R, X129D / Q / E, X130A / G, X136Q, X138Q, X143W, , X156D / G / Q, X158E, X161E / T / C, X163A, X166Q, X167A / T, 72S, X177L, X185E / Q / C, X187D, X188A / C / E / D, X189R, X191R / N, 5I, X206L / A / S / T / C / E / I / K / V, X209W / V / K, X211P, S / Q / C / R, X225A, X235L / N, X238H, X244T / R, X245R / K, X255C / E and or X262E / D / Q / N / C.

[1710] 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:

[1711] (a) X9A; X18Q; X21V; X24K; X25D; X38W; X182K;

[1712] (b)X9A;X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X260D;X271D;

[1713] (c)X9A;X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X260D;X261F;X271D;

[1714] (d)X9A;X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X260D;X261F;X271D;

[1715] (e)X18Q;X21V;X24K;X25D;X38K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X260D;X261F;X271D;

[1716] (f)X18Q;X21V;X24K;X38W;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X260D;X261F;X271D;

[1717] (g)X9A;X18Q;X21V;X24K;X25D;X38K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X260D;X261F;X271D;

[1718] (h)X9A;X18Q;X21V;X24K;X38K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X260D;X261F;X271D;

[1719] (i)X9A;X18Q;X21V;X24K;X38K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X260D;X261F;X271D;

[1720] (j)X18Q;X21V;X24K;X38K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X260D;X261F;X271D;

[1721] (k)X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X218S;X228S;X248Q;X260D;X261F;X271D;

[1722] (l)X9A;X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X218S;X228S;X248Q;X260D;X261F;X271D;

[1723] (m)X9A;X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X204D;X210I;X218S;X228S;X248Q;X260D;X261F;X271D;

[1724] (n)X9A;X18Q;X21V;X24K;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X218S;X228S;X248Q;X260D;X261F;X271D;

[1725] (o)X9A;X18Q;X21V;X24K;X38K;X56D;X78N;X109K;X144N;X160H;X182K;X183D;X204D;X210I;X218S;X228S;X248Q;X260D;X271D;

[1726] (p)X18Q;X21V;X24K;X25D;X38K;X43R;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X261F;

[1727] (q)X18Q;X21V;X24K;X25D;X38K;X43R;X56D;X78N;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;

[1728] (r)X9A;X18Q;X21V;X24K;X25D;X38K;X43R;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X261F;X271D;

[1729] (s)X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X261F;

[1730] (t)X9A;X18Q;X21V;X24K;X25D;X38W;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X261F;X271D;

[1731] (u)X9A;X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X271D;

[1732] (v)X9A;X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X261F;

[1733] (w)X9A;X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X271D;

[1734] (x)X9A;X18Q;X21V;X24K;X25D;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X261F;X271D;

[1735] (y)X9A;X18Q;X21V;X24K;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X261F;

[1736] (z)X9A\X18Q\X21V\X24K,X38K;X43K;X56D\X78N;X103S\X109K;X144N; X160H;X18 2K;X183D\X194E\X204D,X210I\X212P;X218S\X228S;X248Q\X259D\X261F;X271D;

[1737] (aa)X9A;X18Q;X21V;X24K;X38K;X43K;X56D;X78N;X103S;X109K;X144N;X160H \X182K\X183D\X204D,X210I\X212P;X218S\X228S;X248Q\X259D\X261F;X271D;

[1738] (bb)X18Q;X21V;X24K;X38W;X56D;X59K;X78N;X103S;X109K;X144N;X160H;X182 K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X260D;X261F;X271D?

[1739] (cc)X9A\X18Q\X21V\X24K,X25D\X38W;X56D\X59K;X78N;X103S\X109K;X144N;X160H? X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X260D;X261F;X271D?

[1740] (dd)X9A;X18Q\X21V\X24K:X25D\X38W;X56D\X59K;X78N;X103S\X109K;X144N;X160H? X182K;X183D;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X260D;X261F;X271D?

[1741] (e)X9A\X18Q\X21V\X24K;X25D\X38W,X56D\X59K;X78N;X109K\X144N;X16 0H;X182K;X183D;X204D;X210I;X212P;X218S;X228S;X248Q;X260D;X271D?

[1742] (ff)X9A;X18Q;X21V;X24K;X25D;X38K;X56D;X59K;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X260D;X271D;

[1743] (gg)X9A;X18Q;X21V;X24K;X25D;X38K;X56D;X59K;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X260D;X261F;X271D;

[1744] (hh)X9A;X18Q;X21V;X24K;X25D;X38K;X56D;X59K;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X260D;X271D;

[1745] (ii)X9A;X18Q;X21V;X24K;X25D;X38K;X56D;X59K;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X194E;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X260D;X261F;X271D;

[1746] (jj)X9A;X18Q;X21V;X24K;X25D;X38K;X56D;X59K;X78N;X103S;X109K;X144N;X160H;X182K;X183D;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X260D;X261F;X271D;

[1747] (kk)X9A;X18Q;X21V;X24K;X25D;X38K;X56D;X59K;X78N;X109K;X144N;X160H;X182K;X183D;X204D;X210I;X212P;X218S;X228S;X248Q;X259D;X260D;X271D;

[1748] (ll)X9A; 82K;

[1749] (mm) X18Q; X21V; X24K; X38K; X56D; X59K; X78N; X103S; X109K; X144N; X160H; X182K; X183D; X194E; X204D; X210I; X212P; X218S; X228S; X248Q; X260D; X271D; and

[1750] (nn)X18Q; K; X183D; X194E; X204D; X210I;

[1751] 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:

[1752] (b)S9A; N18Q; L21V; S24K; G25D; T38W; N43K; S56D; S78N; A103S; Q109K; S144N; S160H; Q182K; N183D; A194E; N204D; P210I; S212P; N218S; A228S; N248Q; T260D; N261F; E271D;

[1753] (b)S9A; N18Q; L21V; S24K; G25D; T38K; N43K; S56D; S78N; A103S; Q109K; S144N; S16 0H; Q182K; N183D; A194E; N204D; P210I; S212P; N218S; A228S; N248Q; T260D; E271D;

[1754] (c)S9A;N18Q;L21V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;T260D;N261F;E271D;

[1755] (d)S9A;N18Q;L21V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;T260D;N261F;E271D;

[1756] (e)N18Q;L21V;S24K;G25D;T38K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;T260D;N261F;E271D;

[1757] (f)N18Q;L21V;S24K;T38W;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;T260D;N261F;E271D;

[1758] (g)S9A;N18Q;L21V;S24K;G25D;T38K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;T260D;N261F;E271D;

[1759] (h)S9A;N18Q;L21V;S24K;T38K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;T260D;N261F;E271D;

[1760] (i)S9A;N18Q;L21V;S24K;T38K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N1 83D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;T260D;N261F;E271D;

[1761] (j)N18Q;L21V;S24K;T38K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N 183D;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;T260D;N261F;E271D;

[1762] (k)N18Q;L21V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H ;Q182K;N183D;A194E;N204D;P210I;N218S;A228S;N248Q;T260D;N261F;E271D;

[1763] (l)S9A;N18Q;L21V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;N218S;A228S;N248Q;T260D;N261F;E271D;

[1764] (m)S9A;N18Q;L21V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N; S160H;Q182K;N183D;N204D;P210I;N218S;A228S;N248Q;T260D;N261F;E271D;

[1765] (n)S9A;N18Q;L21V;S24K;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;N218S;A228S;N248Q;T260D;N261F;E271D;

[1766] (o)S9A;N18Q;L21V;S24K;T38K;S56D;S78N;Q109K;S144N;S160H;Q182K;N183D;N204D;P210I;N218S;A228S;N248Q;T260D;E271D;

[1767] (p)N18Q;L21V;S24K;G25D;T38K;N43R;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;N261F;

[1768] (q)N18Q;L21V;S24K;G25D;T38K;N43R;S56D;S78N;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;

[1769] (r)S9A;N18Q;L21V;S24K;G25D;T38K;N43R;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;N261F;E271D;

[1770] (s)N18Q;L21V;S24K;G25D;T38K;N43K;S56D;S78N;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;N261F;

[1771] (t)S9A;N18Q;L21V;S24K;G25D;T38W;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;N261F;E271D;

[1772] (u)S9A;N18Q;L21V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;E271D;

[1773] (v)S9A;N18Q;L21V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S16 0H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;N261F;

[1774] (w)S9A;N18Q;L21V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N; S160H;Q182K;N183D;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;E271D;

[1775] (x)S9A;N18Q;L21V;S24K;G25D;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S16 0H;Q182K;N183D;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;N261F;E271D;

[1776] (y)S9A;N18Q;L21V;S24K;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;N261F;

[1777] (z)S9A;N18Q;L21V;S24K;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;Q18 2K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;N261F;E271D;

[1778] (aa)S9A;N18Q;L21V;S24K;T38K;N43K;S56D;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;N261F;E271D;

[1779] (bb)N18Q;L21V;S24K;T38W;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;Q182 K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;T260D;N261F;E271D;

[1780] (cc)S9A;N18Q;L21V;S24K;G25D;T38W;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H; Q182K;N183D;A194E;N204D;P210I;S212P;;N218S;A228S;N248Q;T260D;N261F;E271D;

[1781] (dd)S9A;N18Q;L21V;S24K;G25D;T38W;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H; Q182K;N183D;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;T260D;N261F;E271D;

[1782] (ee)S9A;N18Q;L21V;S24K;G25D;T38W;S56D;Q59K;S78N;Q109K;S144N;S16 0H;Q182K;N183D;N204D;P210I;S212P;N218S;A228S;N248Q;T260D;E271D;

[1783] (ff)S9A;N18Q;L21V;S24K;G25D;T38K;S56D;Q59K;S78N;A103S;Q109K;S144N;S16 0H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;T260D;E271D;

[1784] (gg)S9A;N18Q;L21V;S24K;G25D;T38K;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;T260D;N261F;E271D;

[1785] (hh)S9A;N18Q;L21V;S24K;G25D;T38K;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;T260D;E271D;

[1786] (ii)S9A;N18Q;L21V;S24K;G25D;T38K;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;A194E;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;T260D;N261F;E271D;

[1787] (jj)S9A;N18Q;L21V;S24K;G25D;T38K;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;T260D;N261F;E271D;

[1788] (kk)S9A;N18Q;L21V;S24K;G25D;T38K;S56D;Q59K;S78N;Q109K;S144N;S160H;Q182K;N183D;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;T260D;E271D;

[1789] (ll)S9A;N18Q;L21V;S24K;T38W;S56D;Q59K;S78N;A103S;Q109K;S144N;S160H;Q182K;N183D;N204D;P210I;S212P;N218S;A228S;N248Q;S259D;T260D;N261F;E271D;

[1790] (mm) N18Q; L21V; S24K; T38K; S56D; Q59K; S78N; A103S; Q109K; S144N; S160H; Q182K; N183D; A194E; N204D; P210I; S212P; N218S; A228S; N248Q; T260D; E271D; and

[1791] (nn)N18Q; L21V; S24K; T38K; S56D; Q59K; S78N; A103S; Q109K; S144N; S160H; Q182 K; N183D; A194E; N204D; P210I; S212P; N218S; A228S; N248Q; T260D; N261F; E271D.

[1792] 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% identical to the amino acid sequence according to any one of the following: 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, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO: 27. SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40. SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44.

[1793] In one embodiment, the variant protease has sequence identity of at least 95%, at least 95.5%, 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% with an amino acid sequence according to any one of the following: SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, and SEQ ID NO: 105.

[1794] In one embodiment, the protease variant of the present invention exhibits one or more improved properties compared to the parent protease, preferably compared to the protease according to SEQ ID NO: 3 or 4, and more preferably compared to the protease according to SEQ ID NO: 4.

[1795] Preferably, the variant polypeptides of the present invention exhibit increased stability, particularly storage stability, compared to the parent protease according to SEQ ID NO: 3 or 4, and more preferably compared to the protease according to SEQ ID NO: 4. The increased stability preferably indicates residual activity after a stability challenge. Preferably, storage stability indicates residual activity after storage under appropriate storage conditions, preferably in a detergent composition (preferably in a laundry detergent or dishwashing detergent, more 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 SEQ ID NO: 3 or 4, preferably according to SEQ ID NO: 4. Preferably, compared with the residual activity of the parental protease, preferably compared with protease SEQ ID NO: 3 or 4, preferably compared with the protease according to SEQ ID NO: 4, the residual activity of the stored protease variant increases 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%.

[1796] Preferably, the improved property is one or more properties selected from the group consisting of: increased stability, thermal stability, performance in detergents, performance in laundry detergents, and performance in ADW detergents. Preferably, the improved activity is improved cleaning performance, the cleaning performance of laundry detergents, and / or the cleaning performance of ADW detergents.

[1797] Preferably, the improved characteristic is improved stability in a detergent composition, preferably in a laundry detergent composition, preferably in a detergent composition containing 1%-20%, more preferably 5%-10% of anionic surfactant (preferably LAS) and / or more than 50% water, and preferably in type B detergents as described herein. In one embodiment, the improved characteristic is improved stability in a detergent composition, preferably in a laundry detergent composition, preferably in a detergent composition containing 0.1%-5%, preferably 0.3%-3% of a strong chelating builder. In this embodiment, the detergent composition preferably contains 1%-20%, more preferably 5%-10% of anionic surfactant (preferably LAS) and / or more than 50% water.

[1798] Nucleic acid constructs

[1799] The present invention also relates to a polynucleotide encoding a polypeptide of the present invention having protease activity. Preferably, the polynucleotide is a codon-optimized polynucleotide for improving expression in specific host cells (preferably Bacillus cells).

[1800] The present invention also relates to a nucleic acid construct (preferably an expression cassette) comprising polynucleotides as described herein.

[1801] Typically, an expression cassette contains three elements: a promoter sequence, an open reading frame (OPF), and a 3' untranslated region (UTR). In eukaryotes, the 3' UTR usually contains a polyadenylation site. Additional regulatory elements may include transcriptional enhancers and translational enhancers. Intron sequences can also be added to the 5' UTR or coding sequence to increase the amount of maturation messengers accumulating in the cytosol. Expression cassettes can be part of a vector or can be integrated into the host cell's genome and replicated along with the host cell's genome. Expression cassettes are generally capable of increasing or decreasing expression.

[1802] This invention also relates to an expression vector comprising a polynucleotide or nucleic acid construct as described herein. The expression vector may be a low copy number vector or a high copy number vector.

[1803] After transformation into a host cell or host cell organelle, a vector, as used herein, can provide segments for the transcription and translation of foreign polynucleotides. Such additional segments may include regulatory nucleotide sequences, one or more origins of replication required for maintenance and / or replication in a particular cell type, one or more optional markers, polyadenylation signals, suitable sites for the insertion of foreign coding sequences, such as multiple cloning sites, etc. One example is when it is necessary to maintain the vector as an additional genetic element (e.g., a plasmid or granular molecule) in bacterial cells. Non-limiting examples of suitable origins of replication include f1-ori and colE1.

[1804] Without integrating into the host cell genome, a vector can replicate, for example, as a plasmid in a bacterial host cell, or it can integrate some or all of its DNA into the host cell genome, thus causing its DNA to be replicated and expressed.

[1805] Polynucleotides encoding the protease-active polypeptides of this invention can be introduced into a vector using standard recombinant DNA techniques. Once introduced into the vector, the polynucleotides containing the coding sequence are suitable for introduction (transformation, transduction, transfection, etc.) into host cells or host organelles. Cloning vectors suitable for polynucleotide sequence expression in host cells or host cell organelles can be selected.

[1806] host cells

[1807] This invention also relates to a host cell comprising a polynucleotide encoding a polypeptide of the present invention having protease activity, a nucleic acid construct as described herein, or an expression vector as described herein. In one embodiment of the invention, the vector is used for transformation of the host cell. Preferably, the host cell is capable of expressing a polynucleotide encoding a polypeptide of the present invention having protease activity.

[1808] Polynucleotides encoding the protease-active polypeptides of the present invention can be transiently or stably introduced into host cells. The polynucleotides can remain non-integrated, for example, as plasmids. Typically, stable transformation occurs due to the integration of the nucleic acid containing the foreign coding sequence into the host cell chromosome or as an appendage (a separate fragment of nuclear DNA). Transient transformation typically occurs due to the failure to integrate the nucleic acid containing the foreign nucleic acid sequence into the host cell chromosome or as an appendage. Methods for introducing nucleic acids into host cells are well known in the art.

[1809] Various host cells can be used to express the nucleic acid constructs described herein. The host cells of this invention do not naturally express the protease-active polypeptides of this invention. Therefore, the host cells are recombinant host cells; the nucleic acid constructs described herein are heterologous to the host cells.

[1810] In one embodiment, the host cell is a prokaryotic or eukaryotic organism. In another embodiment, the host cell is a bacterial, archaea, fungal, yeast, or eukaryotic cell. In yet another embodiment, the host cell is a non-human host cell.

[1811] In one embodiment, the host cell is a bacterial cell. The bacterial host cell can be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Brevibacterium, Corynebacterium, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, and Oceanobacillus. Gram-negative bacteria include, but are not limited to, *Escherichia*, *Pseudomonas*, *Salmonella*, *Campylobacter*, *Helicobacter*, *Acetobacter*, *Flavobacterium*, *Fusobacterium*, and *Gluconobacter*. In a particular embodiment, the bacterial host cell is an *Escherichia coli* cell. In one embodiment, the host cell is a bacterial cell. In a particular embodiment, the host cell is a cell of *Escherichia* or *Bacillus*.

[1812] In the method of the present invention, the bacterial host cell can be any Bacillus genus cell. In practice, useful Bacillus cells include, but are not limited to, *Bacillus alkalophilus*, *Bacillus amyloliquefaciens*, *Bacillus brevis*, *Bacillus circulans*, *Bacillus clausii*, *Bacillus scoagulans*, *Bacillus firmus*, *Bacillus lautus*, *Bacillus lentus*, *Bacillus licheniformis*, *Bacillus megaterium*, *Bacillus pumilus*, *Bacillus stearothermophilus*, *Bacillus methylotrophicus*, and *Bacillus cereus*. The bacterial host cells are *Bacillus cereus*, *Bacillus paralicheniformis*, *Bacillus subtilis*, and *Bacillus thuringiensis* cells. In one embodiment, the bacterial host cell is *Bacillus amyloliquefaciens*, *Bacillus pumilus*, *Bacillus tarda*, *Bacillus licheniformis*, *Bacillus thermophilus*, or *Bacillus subtilis* cells. In a preferred embodiment, the bacterial host cell is *Bacillus licheniformis*, *Bacillus pumilus*, or *Bacillus subtilis* cells. Preferably, the bacterial host cell is *Bacillus licheniformis* cells.

[1813] Preparation method

[1814] Another embodiment of the present invention is a method for obtaining a protease variant of a parent protease, the method comprising the following steps:

[1815] a) Introducing a parental protease, preferably according to the protease of SEQ ID NO: 3, by comparing the amino acid sequence shown in SEQ ID NO: 3 and referring to the number in SEQ ID NO: 2, by substituting amino acids at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261, and 271, thereby providing a variant polypeptide of the parental protease.

[1816] The variant has at least 65% but less than 100% sequence identity with the amino acid sequence of the polypeptide of SEQ ID NO: 1, and the variant polypeptide has protease activity, and preferably the variant polypeptide has improved properties relative to the parent protease.

[1817] The introduction of amino acid substitutions into protein sequences is well known in the art. Variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[1818] The protease-active variant peptides of this invention can be produced on an industrial scale and subsequently purified. Industrial production of enzymes is typically accomplished by culturing host cells expressing the enzyme (also known as fermentation). Suitable host cells are described herein. The nucleic acid sequence encoding the protease-active variant peptide of this invention can be transformed into host cells, which are then cultured under conditions suitable for the host cells to produce the protease-active variant peptide of this invention. In a preferred embodiment, the protease-active variant peptide of this invention is purified from host cells.

[1819] Therefore, in yet another embodiment, the present invention relates to a method for generating a variant polypeptide with protease activity, the method comprising the following steps:

[1820] (a) Providing a host cell containing a heterologous nucleic acid construct comprising a polynucleotide encoding a variant polypeptide having protease activity;

[1821] (b) Culturing the recombinant host cells from step (a) under conditions favorable to the expression of the polynucleotide; and

[1822] (c) Optionally, the variant polypeptide encoded by the polynucleotide with protease activity is recovered.

[1823] Host cell culture is typically carried out in a suitable nutrient medium that allows recombinant cells to grow and express the desired protein. At the end of fermentation, the fermentation broth, containing both liquid and solid fractions, is collected and can be further processed. Variant peptides with protease activity can be further purified from the fermentation broth.

[1824] The protease-active variant peptides described herein may be secreted (into the liquid portion of the fermentation broth) or may not be secreted from the microbial cells (and thus contained within the cells of the fermentation broth). Accordingly, the protease-active variant peptides can be recovered from the liquid portion of the fermentation broth or from cell lysates. Preferably, the protease-active variant peptide is secreted from the cells into the fermentation broth, preferably by adding a secretion signal peptide to the N-terminus of the amino acid sequence, thereby forming a propeptide of the peptide. After cleavage of the propeptide, the peptide folds, and the mature protease is released as the active variant. During cultivation, measures can be taken to actively protect the protein from autocatalysis, as described in WO 2024 / 028340 or WO2024 / 028338. Recovery of the protease-active variant peptides can be achieved by methods known to those skilled in the art. Suitable methods for recovering proteins from the fermentation broth include, but are not limited to, collection, centrifugation, filtration, extraction, and precipitation. If the target protein precipitates or crystallizes in the fermentation broth or is at least partially bound to particulate matter in the fermentation broth, additional processing steps may be required to release the target protein from the biomass or dissolve the target protein crystals and precipitates. WO 00 / 43502 A1, WO 2008 / 110498 A1, and WO 2017 / 097869 A1 describe methods for recovering a target protein from a fermentation broth that precipitates and / or crystallizes during fermentation. If the desired protein is contained within cells in the fermentation broth, it may be necessary to release the target protein from the cells. Release from cells can be achieved, for example, but not limited to, by cell lysis using techniques well known to those skilled in the art (e.g., lysozyme treatment, sonication, French press, or combinations thereof).

[1825] Variant peptides with protease activity can be purified from fermentation broth using methods known in the art. For example, variant peptides with protease activity can be isolated from fermentation broth using conventional procedures, including but not limited to centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. The isolated peptides can then be further purified using a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatographic focusing, and size exclusion), electrophoresis procedures (e.g., preparative isoelectric focusing (IEF)), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, for example, *Protein Purification*, J.-C. Janson and Lars Ryden, eds., VCH Publishers, New York, 1989). The purified peptides can then be concentrated using procedures known in the art, including but not limited to ultrafiltration and evaporation, particularly membrane evaporation.

[1826] Protease composition

[1827] The purified variant polypeptides of the present invention with protease activity can be readily further processed to form compositions containing proteases. Therefore, a composition comprising the variant polypeptides of the present invention with protease activity and at least one additional component is also claimed herein.

[1828] Therefore, the present invention also relates to a method for preparing a composition, the method comprising the steps of mixing the following:

[1829] i. variant peptides with protease activity as described herein; and

[1830] ii. One or more components described herein.

[1831] Furthermore, the present invention also relates to a method for improving the stability of a protease in a composition, the method comprising the steps of mixing the following:

[1832] (a) variant peptides with protease activity as described herein; and

[1833] (b) One or more components described herein.

[1834] As described herein, the composition may be a non-complex formulation (e.g., a protease formulation) or a complex formulation (e.g., a detergent composition).

[1835] 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 variant formulation may be solid or liquid. The protein formulation can be obtained using techniques known in the art. For example, but not limited to, solid enzyme formulations can be obtained by extrusion or granulation. Suitable extrusion and granulation techniques are known in the art and are described, for example, in WO 94 / 19444 A1 and WO 97 / 43482 A1.

[1836] Liquid protease formulations may contain 2% to 40%, 2% to 30%, 2% to 25%, 2% to 12%, or preferably 2% to 7.5% of protease by weight, all relative to the total weight of the enzyme formulation.

[1837] In one embodiment, the protease formulation, particularly the 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.

[1838] In one embodiment, the present invention relates to a composition comprising a variant polypeptide of the present invention having protease activity 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 the variant polypeptide of the present invention having protease activity 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).

[1839] Therefore, the present invention also provides compositions comprising the protease-active variant polypeptide of the present invention, neopentyl glycol, and glycerol. The present invention also provides compositions comprising the protease-active variant polypeptide of the present invention, neopentyl glycol, and MPG. The present invention also provides compositions comprising the protease-active variant polypeptide of the present invention, neopentyl glycol, glycerol, and MPG. The present invention also provides compositions comprising the protease-active variant polypeptide of the present invention, 1,6-hexanediol, and glycerol. The present invention also provides compositions comprising the protease-active variant polypeptide of the present invention, 1,6-hexanediol, and MPG. The present invention also provides compositions comprising the protease-active variant polypeptide of the present invention, 1,6-hexanediol, glycerol, and MPG. The present invention also provides compositions comprising the protease-active variant polypeptide of the present invention, glycerol, and MPG.

[1840] In one embodiment, the protease formulation contains at least one preservative. Preferably, a preservative means a substance added to the liquid composition for preservative 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 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 relative to the total weight of the liquid composition, such as ranging from 2 ppm to 5% by weight. Preferably, the protease formulation is preservative-free, meaning the preservative content is less than 1 ppm, preferably 0 ppm.

[1841] Preferably, 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, ethylene glycol, glycerol, 1,2-propanediol, 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 acid (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, boric acid compounds (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 borates, boric acid, boronic acid (preferably 4-FPBA), peptide aldehydes (preferably peptide aldehydes like Z-VAL-H or Z-GAY-H), peptide acetals, and peptide aldehyde bisulfite adducts, preferably 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.

[1842] In one embodiment, the protease formulation comprises a protease inhibitor, preferably a peptidaldehyde, in an amount relative to the total weight of the liquid protease variant formulation ranging from about 0.04% to 0.8%, 0.04% to 0.7%, 0.04% to 0.6%, 0.04% to 0.5%, 0.04% to 0.4%, or 0.04% to 0.3% by weight. Preferably, the peptidaldehyde content ranges from about 0.1% to 0.6%, about 0.1% to 0.5%, about 0.1% to 0.4%, or about 0.1% to 0.35% by weight, all relative to the total weight of the liquid protease formulation.

[1843] Preferably, the liquid protease formulation comprises or consists of the following: a variant polypeptide of the present invention having protease activity, a solvent, an enzyme stabilizing system, and optionally a preservative and an additional enzyme optionally different from the protease variant. Preferably, the formulation of the variant polypeptide of the present invention is substantially free of surfactant, i.e., the protease formulation contains less than 1% surfactant, preferably less than 0.5% surfactant.

[1844] Therefore, the present invention also relates to a method for preparing a protease formulation, preferably a concentrated protease formulation, the method comprising the steps of mixing the following:

[1845] a) Variant polypeptides of the present invention possessing protease activity; and

[1846] b) One or more components selected from the group consisting of: solvents, enzyme stabilizing systems, preservatives, and other enzymes different from proteases.

[1847] Furthermore, the present invention relates to a method for improving the stability of a protease in a composition, the method comprising the steps of mixing the following:

[1848] a) Variant polypeptides of the present invention possessing protease activity; and

[1849] b) One or more components selected from the group consisting of: solvents, enzyme stabilizing systems, preservatives, and other enzymes different from proteases.

[1850] In one embodiment, the variant polypeptide of the present invention having protease activity is part of a microorganism (live, attenuated, or inactivated), probiotic, or prebiotic.

[1851] Other enzymes

[1852] 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 the protease. Preferably, the additional enzymes are selected from the group consisting of: amylase, lipase, proteases other than the polypeptide of the present invention having protease activity, cellulase, mannanase, hemicellulase, phospholipase, esterase, pectinase, lactase, peroxidase, xylanase, keratinase, pectate lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, amylopectinase, tanninase, pentosanase, malarananase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, and nucleic acids. Enzymes, including ribonucleases (RNases), deoxyribonucleases (DNases), phosphodiesterases, phytases, carbohydrate enzymes, galactanases, xanthan gumases, xyloglucanases, oxidoreductases, hydrolases, aminopeptidases, asparaginases, carbohydrate enzymes, carboxypeptidases, catalases, chitinases, cyclodextrin glycosyltransferases, α-galactosidases, β-galactosidases, glucosylamylases, α-glucosidases, β-glucosidases, invertases, transglutaminases, and dispersins, as well as combinations of at least two of the foregoing types. More preferably, the additional enzymes are selected from the group consisting of: amylases, lipases, proteases other than the polypeptides with protease activity of the present invention, cellulases, mannanases, esterases, xylanases, DNases, dispersins, pectinases, oxidoreductases, and keratinases, as well as 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.

[1853] 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 of the present invention having protease activity).

[1854] protease

[1855] Proteases other than the variant polypeptides with protease activity of the present invention may comprise the following amino acid sequence having preferred increasing sequence identity with SEQ ID NO: 45 of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100%, and containing amino acid substitutions at one or more of the following positions: 3, 4, 9, 15, 27, 33, 36, 57, 68, 77, 87, 95, 96, 9 7, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 252, and 274 (according to BPN' numbers), these proteases possess proteolytic activity. In one embodiment, such proteases have no mutations at positions Asp32, His64, and Ser221 (according to BPN' numbers). Preferably, the protease used in combination with the polypeptide having protease activity of the present invention comprises an amino acid sequence having a preferred increasing order of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with SEQ ID NO: 45, and is further characterized by having the amino acid glutamic acid (E), or aspartic acid (D), or asparagine (N), or glutamine (Q), or alanine (A), or glycine (G), or serine (S), preferably glutamic acid (E), at position 101 (according to BPN' number), and having proteolytic activity. Most preferably, the protease has a sequence identity of at least 80% but less than 100% with SEQ ID NO: 45, and is characterized by having the amino acid glutamic acid (E) at position 101 (according to BPN' number), and having proteolytic activity. This protease may be contained alone or in combination with one or more substitutions at positions 3, 4, 9, 15, 27, 33, 36, 57, 68, 77, 87, 95, 96, 97, 98, 99, 100, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 252 and / or 274 (according to BPN' number) as an amino acid substitution at position 101 (e.g., R101E), and has proteolytic activity.In one embodiment, the protease comprises one or more additional substitutions: (a) threonine at position 3 (3T), (b) isoleucine at position 4 (4I), (c) alanine, threonine, or arginine at position 63 (63A, 63T, or 63R), (d) aspartic acid or glutamic acid at position 156 (156D or 156E), (e) proline at position 194 (194P), (f) methionine at position 199 (199M), (g) isoleucine at position 205 (205I), (h) aspartic acid, glutamic acid, or glycine at position 217 (217D, 217E, or 217G), and (i) combinations of two or more amino acids according to (a) through (h), as per BPN' numbering. A suitable protease may have at least 80% identity with SEQ ID NO: 45 and is characterized by comprising one amino acid (according to (a)-(h)) or a combination thereof according to (i) and amino acids 101E, 101D, 101N, 101Q, 101A, 101G or 101S (according to BPN' number), and having proteolytic activity. In one embodiment, the protease has at least 80% identity with SEQ ID NO: 45 and is characterized by comprising the mutant (according to BPN' number) R101E, or S3T + V4I + V205I, or S3T + V4I + R101E + V205I or S3T + V4I + V199M + V205I + L217D, and having proteolytic activity. In another embodiment, the protease comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 45, and is further characterized by comprising S3T + V4I + S9R + A15T + V68A + D99S + R101S + A103S + I104V + N218D (according to BPN' number) and having proteolytic activity.In another embodiment, the protease may have an amino acid sequence that is at least 80% identical to SEQ ID NO:45, and is further characterized by comprising R101E and one or more substitutions selected from the group consisting of: S156D, L262E, Q137H, S3T, R45E, D, Q, P55N, T58W, Y, L, Q59D, M, N, T, G61D, R, S87E, G97S, A98D, E, R, S106 A,W,N117E,H120V,D,K,N,S125M,P129D,E136Q,S144W,S161T,S163A,G,Y171L,A172S,N185Q,V199M,Y209W,M222Q,N238H,V244T,N261T,D andL262N,Q,D (according to BPN' number), and possess proteolytic activity.

[1856] In this embodiment, preferably, the protease used in combination with the polypeptide having protease activity comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% identity with SEQ ID NO: 45, and compared with SEQ ID NO: 45, the protease comprises amino acid substitutions R101E and S156D and / or L262E, and optionally at least one additional mutation (according to BPN' number) selected from I104T, H120D, Q137H, S141H, R145H, and S163G.

[1857] In another embodiment, the protease used in combination with a polypeptide having protease activity has at least 60%, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with SEQ ID NO: 45, and compared with SEQ ID NO: 45, comprises amino acid substitutions R101D or R101E, preferably R101E, and one or more amino acid substitutions selected from the group consisting of S3T, V4I, and V205I, preferably all of S3T, V4I, and V205I (according to BPN' number), and amino acid substitutions selected from 76, 138, 145, 156, 166, 167, 169, 177, 187, 189, 191, 206, 209, 215, 218, and 2 One or more substitutions at the position of group 62 (according to BPN' number), preferably selected from the group consisting of N76D / E / Q, A138Q, R145L / W / Y, S156D / Q, S166G, Y167T, A169G, V177L, A187D, F189R, Q191R, Q206A / L / S / T, Y209K / V / W, A215K / W, N218S / T, and L262D / E / Q.

[1858] In this embodiment, preferably, the protease used in combination with the polypeptide having protease activity has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with SEQ ID NO: 45, and is also similar to SEQ ID NO: Compared to 45, it includes amino acid substitutions R101D or R101E, preferably R101E, and one or more amino acid substitutions selected from the group consisting of S3T, V4...

Claims

1. A variant polypeptide with protease activity or a fragment of said protease-active polypeptide, wherein: (i) The polypeptide has an amino acid sequence that is at least 65% but less than 100% identical to the amino acid sequence shown in SEQ ID NO: 1, and (ii) Compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof contains amino acid substitutions at three or more amino acid residues selected from the group consisting of 9, 18, 38, 59, 212, 261 and 271. This fragment of the variant polypeptide contains 100 to 259 consecutive amino acids of the full-length variant polypeptide.

2. The variant polypeptide of claim 1, wherein: (a) The amino acid substitution at amino acid residue 9 is X9A / Q / E / C / D, preferably X9A / Q; and / or (b) The amino acid substitution at amino acid residue 18 is X18Q / E / D; and / or (c) The amino acid substitution at amino acid residue 38 is X38R / K / H / W; and / or (d) The amino acid substitution at amino acid residue 59 is X59K / E; and / or (e) 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 / R / K / F / Y / M; and / or (f) The amino acid substitution at amino acid residue 261 is X261L / M / F / E / W / Y / C, preferably X261F / W / Y / L; and / or (g) The amino acid substitution at amino acid residue 271 is X271D.

3. The variant polypeptide of claim 1 or 2, wherein, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, the polypeptide or a fragment thereof comprises one of a combination of the following amino acid substitutions: (a)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W; (b)X9A / Q / E / C / D, X18Q / E / D, X59K / E; (c)X9A / Q / E / C / D, X18Q / E / D, X212G / M / R / K / P / A / F / H / W / N / Y / D; (d)X9A / Q / E / C / D, X18Q / E / D, X261L / M / F / E / W / Y / C; (e)X9A / Q / E / C / D, X18Q / E / D, X271D; (f)X9A / Q / E / C / D, X38R / K / H / W, X59K / E; (g)X9A / Q / E / C / D, X38R / K / H / W, X212G / M / R / K / P / A / F / H / W / N / Y / D; (h)X9A / Q / E / C / D, X38R / K / H / W, X261L / M / F / E / W / Y / C; (i)X9A / Q / E / C / D、X38R / K / H / W、X271D; (j)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D; (k)X9A / Q / E / C / D、X59K / E、X261L / M / F / E / W / Y / C; (l)X9A / Q / E / C / D、X59K / E、X271D; (m)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C; (n)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D; (o)X9A / Q / E / C / D、X261L / M / F / E / W / Y / C、X271D; (p)X18Q / E / D、X38R / K / H / W、X59K / E; (q)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D; (r)X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C; (s)X18Q / E / D、X38R / K / H / W、X271D; (t)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D; (u)X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C; (v)X18Q / E / D、X59K / E、X271D; (w)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C; (x)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D; (y)X18Q / E / D、X261L / M / F / E / W / Y / C、X271D; (z)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D; (aa)X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C; (bb)X38R / K / H / W、X59K / E、X271D; (cc)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C; (dd)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D; (ee)X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D; (ff)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C; (gg)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D; (hh)X59K / E、X261L / M / F / E / W / Y / C、X271D; (ii)X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D; (jj)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E; (kk)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D; (ll)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C; (mm)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X271D; (nn)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D; (oo)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C; (pp)X9A / Q / E / C / D、X18Q / E / D、X59K / E、X271D; (qq)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C; (rr)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D; (ss)X9A / Q / E / C / D、X18Q / E / D、X261L / M / F / E / W / Y / C、X271D; (tt)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D; (uu)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C; (vv)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X271D; (ww)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C; (xx)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D; (yy)X9A / Q / E / C / D、X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D; (zz)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C; (aaa)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D; (bbb)X9A / Q / E / C / D、X59K / E、X261L / M / F / E / W / Y / C、X271D; (ccc)X9A / Q / E / C / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D; (ddd)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D; (eee)X18Q / E / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C; (fff)X18Q / E / D、X38R / K / H / W、X59K / E、X271D; (ggg)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C; (hhh)X18Q / E / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D; (iii)X18Q / E / D、X38R / K / H / W、X261L / M / F / E / W / Y / C、X271D; (jjj)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C; (kkk)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D; (lll)X18Q / E / D、X59K / E、X261L / M / F / E / W / Y / C、X271D; (mmm)X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D; (nnn)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C; (ooo)X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D; (ppp)X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D; (qqq)X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D; (rrr)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D; (sss)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X261L / M / F / E / W / Y / C; (ttt)X9A / Q / E / C / D、X18Q / E / D、X38R / K / H / W、X59K / E、X271D; (uuu)X9A / Q / E / C / D、X18Q / E / D、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D; (vvv)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C; (www)X9A / Q / E / C / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D; (xxx)X9A / Q / E / C / D、X38R / K / H / W、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D; (yyy)X9A / Q / E / C / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D; (zzz)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C; (aaaa)X18Q / E / D、X38R / K / H / W、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X271D; (bbbb)X18Q / E / D、X59K / E、X212G / M / R / K / P / A / F / H / W / N / Y / D、X261L / M / F / E / W / Y / C、X271D; (cccc)X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, (dddd)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E, (eeee)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E, (ffff)X18Q / E / D, X38R / K / H / W, X59K / E, X212G / M / R / K / P / A / F / H / W / N / Y / D, (gggg)X9A / Q / E / C / D, X18Q / E / D, X38R / K / H / W, X59K / E, 4. A variant polypeptide as claimed in any of the preceding claims, wherein the polypeptide further comprises at least one amino acid substitution at an amino acid residue selected from the group consisting of 21, 43, 78, 160, 183, 194, 204, 210, 218, 228, 259 and 260.

5. The variant polypeptide of claim 4, wherein: (a) The amino acid substitution at amino acid residue 21 is X21I / V / F / W / Y, preferably X21V / I; and / or (b) The amino acid substitution at amino acid residue 43 is X43K / R / C / H / D / L / S / W / A / M / Y / Q / F / I, preferably X43K / R; and / or (c) 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 (d) The amino acid substitution at amino acid residue 160 is X160H / S / D / E / P, preferably X160H; and / or (e) The amino acid substitution at amino acid residue 183 is X183D / E / C / Q / A / M, preferably X183D / E; and / or (f) The amino acid substitution at amino acid residue 194 is X194E / C / D, preferably X194E / D; and / or (g) The amino acid substitution at amino acid residue 204 is X204D / E / C / G, preferably X204D / E; and / or (h) The amino acid substitution at amino acid residue 210 is X210I / V, preferably X210I; and / or (i) The amino acid substitution at amino acid residue 218 is X218T / S / D, preferably X218S / T; and / or (j) The amino acid substitution at amino acid residue 228 is X228S; and / or (k) The amino acid substitution at amino acid residue 259 is X259E / D, preferably X259D; and / or (l) The amino acid substitution at amino acid residue 260 is X260D / E / K, preferably X260D / E.

6. A variant polypeptide as claimed in any of the preceding claims, wherein the polypeptide further comprises at least one amino acid substitution at an amino acid residue selected from the group consisting of 24, 25, 103, 109, 144, 182 and 248.

7. The variant polypeptide of claim 6, wherein: (a) The amino acid substitution at amino acid residue 24 is X24K; and / or (b) The amino acid substitution at amino acid residue 25 is X25D; and / or (c) The amino acid substitution at amino acid residue 103 is X103S; and / or (d) The amino acid substitution at amino acid residue 109 is X109K / A; and / or (e) The amino acid substitution at amino acid residue 144 is X144N / R; and / or (f) The amino acid substitution at amino acid residue 182 is X182K / R / E; and / or (g) The amino acid substitution at amino acid residue 248 is X248Q / R, preferably X248Q.

8. A variant polypeptide as claimed in any of the preceding claims, wherein the polypeptide, compared with the amino acid sequence shown in SEQ ID NO: 3 and referring to the number of SEQ ID NO: 2, further comprises at least one amino acid substitution at an amino acid residue selected from the group consisting of 3, 76 and 256.

9. The variant polypeptide of claim 8, wherein: (a) The amino acid substitution at amino acid residue 3 is X3T / Q / V, preferably X3T; and / or (b) The amino acid substitution at amino acid residue 76 is X76D; and / or (c) The amino acid substitution at amino acid residue 256 is X256E / T / D / R / P, preferably X256D.

10. A variant polypeptide as claimed in any of the preceding claims, wherein, with reference to SEQ ID NO: 2, the polypeptide contains amino acid residue D or E at position 101, preferably E at position 101.

11. A variant polypeptide as claimed in any of the preceding claims, wherein the polypeptide exhibits one or more improved properties compared to the proteases according to SEQ ID NO: 3 and / or SEQ ID NO: 4, preferably wherein such improved properties are selected from: (i) Increased stability (ii) Increased storage stability, and (iii) Increased storage stability in detergent compositions.

12. A polynucleotide encoding a variant polypeptide as described in any of the preceding claims.

13. A composition comprising a variant polypeptide as described in any one of claims 1 to 11 and at least one additional component, preferably comprising an enzyme stabilizing system, wherein the enzyme stabilizing system preferably comprises at least one compound selected from the group consisting of: polyols (preferably 1,3-propanediol, ethylene glycol, glycerol, 1,2-propanediol 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, boric acid (preferably 4-formylphenylboronic acid (4-FPBA)), peptide aldehydes (preferably 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).

14. The composition of claim 13, wherein the composition comprises one or more second enzymes different from the variant polypeptides of any of the preceding claims, 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.

15. The composition of any one of claims 13 or 14, wherein the composition is a detergent composition, preferably a laundry detergent composition or a hard surface cleaning detergent composition, preferably a biodegradable detergent composition, preferably wherein the composition comprises one or more surfactants and / or one or more builder agents, preferably a strongly chelating builder agent.

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