Cutinase variants and polynucleotides encoding same

Abstract

Variants having cutinase activity of a parent cutinase, the variants comprising alterations at one or more (e.g., several) positions corresponding to positions 181, 182, 115, 161, 1, 2, 43, 55, 79, or 5 of SEQ ID NO: 2, where the alterations are substitutions for positions 181, 115, 161, 43, 55, 79, and 5, and deletions for positions 1, 2, and 182, and where the variants have at least 75% but less than 100% sequence identity to a mature polypeptide of SEQ ID NO: 2. Polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells including the polynucleotides; as well as methods for obtaining the variants and methods for producing the variants. Comprising the variants, and methods for using the variants.

Description

[0001] This application is a divisional application of the invention patent application filed on December 10, 2014, with application number 201480065727.6 and entitled "Keratinase variant and polynucleotide encoding it".

[0002] References to sequence lists

[0003] This application contains a sequence list in a computer-readable form, which is incorporated herein by reference. Background of the Invention Invention Field

[0004] This invention relates to keratinase variants, polynucleotides encoding these variants, methods for generating these variants, and methods for using these variants.

[0005] Related technical specifications

[0006] Poly(ethylene terephthalate) (PET) fibers constitute the majority of polyester used in the textile industry. These fibers are produced by the polycondensation of terephthalic acid and ethylene glycol and by drawing fibers from the melt.

[0007] Polyester possesses certain core advantages, including high strength, a soft hand feel, tensile resistance, stain resistance, machine washability, wrinkle resistance, and abrasion resistance. However, polyester is not optimal in terms of its hydrophobicity, pilling, static electricity, dyeability, the passive surface of its adhesive medium (i.e., softening or wetting-enhancing compounds), lack of breathability, and less-than-ideal luminous or glossy appearance.

[0008] Because of their strength, polyester fabrics and / or clothing are susceptible to pilling, and the processes applied to finishing polyester staple fiber materials are perhaps most importantly those designed for pilling control. All staple fiber materials tend to form small balls or "beads" with entangled fibers on the fabric surface when subjected to minor abrasion during washing and wear. If the fabric contains a significant proportion of fibers with high resistance to flexural abrasion, these beads will remain on the fabric surface in a number insufficient to produce an unpleasant feel and appearance.

[0009] Another problem with polyester is that during the synthesis of PET, cyclic or linear oligomers of poly(ethylene terephthalate) are formed, such as bis-2-benzoyloxyethyl terephthalate (BETEB) and / or cyclic tri(ethylene terephthalate). These oligomers are partially deposited mechanically and partially remain on / within these fibers. The oligomers tend to give the fabric a light gray appearance. This is attributed to the deposition of oligomers on the fabric surface, specifically, frankly, after high-temperature wet processing such as high-temperature dyeing. These oligomers can be removed by harsh alkaline treatments, which result in significant loss of fibrous material. Organic extraction of these oligomers is a technical possibility, but not industrially feasible.

[0010] Among other things, the industry has made great efforts to improve the characteristics of polyesters by applying keratinase.

[0011] Keratinases are known to originate from various fungi, such as filamentous fungal keratins, for example strains from the genera *Pyrophyllus* or *Fusarium*, particularly specific *Pyrophyllus* strains such as *Pyrophyllus* DSM1800 (US 5827719), or *Fusarium solani*. Methods for reducing the pilling tendency of polyester fabrics and / or garments using a diethyl terephthalate hydrolase (ETE hydrolase) and / or an ethylene glycol dibenzyl ester hydrolase (BEB hydrolase) have been disclosed (WO 99 / 001604), methods for modifying polyester by treating it with an esterase (WO 2001 / 34899), and enzymatic hydrolysis of cyclic oligomers of poly(ethylene terephthalate) comprising subjecting the cyclic oligomer to the action of one or more carboxylic acid ester hydrolases (WO 97 / 27237).

[0012] Keratinase variants have been described, for example, in WO 0192502, which discloses the use of specific humic mold variants for treating polyester textiles.

[0013] However, there is a need for continued improvements in the treatment of enzymatic polyester fabrics and / or clothing, including enhanced efficiency of these enzymes on their substrates. Therefore, it is ideal to identify enzymes with such improved properties for use in methods for treating fabrics. Invention Overview

[0014] The present invention relates to variants having keratinase activity of a parent keratinase, which include alterations at one or more (e.g., several) positions corresponding to positions 181, 182, 115, 161, 1, 2, 43, 55, 79, or 5 of SEQ ID NO:2, wherein the alteration is a substitution for positions 181, 115, 161, 43, 55, 79, and 5, and wherein the variants have at least 75%, 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 the mature polypeptide of SEQ ID NO:2.

[0015] The present invention also relates to polynucleotides encoding these variants; nucleic acid constructs, vectors, and host cells including these polynucleotides; and methods for obtaining these variants and methods for generating these variants.

[0016] The present invention further relates to compositions including this variant, and to methods including modifying polyesters using the variant; methods including hydrolyzing cyclic oligomers of polyethylene terephthalate using the variant; and methods using the variant to reduce the pilling tendency of fabrics comprising or composed of polyesters.

[0017] definition

[0018] Keratinase: The term "keratinase" refers to a lipase with keratinase activity (EC3.1.1.74) that catalyzes the following reaction: Keratin + H2O Keratin monomer. For the purposes of this invention, as described in Example 3, keratinase activity was determined using the oligomer bis-2-benzoyloxyethyl terephthalate (BETEB) as a substrate. BETEB is a byproduct of PET synthesis and is typically retained in the fabric or garment during textile manufacturing. BETEB is produced by, for example, the condensation of terephthalic acid, benzoic acid, and ethylene glycol, which has the same benzoyloxyethyl ester unit as PET.

[0019] In one aspect, variants of the invention have at least 20%, for example 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%, or at least 100% of the keratinase activity of the mature polypeptide of SEQ ID NO: 2.

[0020] In some embodiments, the keratinase may be a variant comprising one or more amino acid substitutions and / or deletions of SEQ ID NO: 2. Preferably, the total number of amino acid substitutions, deletions, and / or insertions of SEQ ID NO: 2 is 1-20, for example 1-10 or 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0021] Allelic variants: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variations arise naturally from mutations and can lead to polymorphism within a population. Gene mutations can be silent (without alteration in the encoded polypeptide) or can encode a polypeptide with a modified amino acid sequence. Allelic variants of a polypeptide are polypeptides encoded by allelic variants of a gene.

[0022] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules derived from eukaryotic or prokaryotic cells. cDNA lacks the intron sequences that can be present in the corresponding genomic DNA. Early initial RNA transcripts are precursors to mRNA, undergoing a series of processing steps, including splicing, before becoming mature, spliced ​​mRNA.

[0023] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly identifies the amino acid sequence of a variant. The boundaries of a coding sequence are generally determined by an open reading frame, which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0024] Control Sequences: The term "control sequence" refers to the nucleic acid sequence necessary for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) for the polynucleotide encoding that variant, or native or exogenous relative to each other. Such control sequences include, but are not limited to, pre-leaders, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction enzyme sites that facilitate the linking of these control sequences to the coding regions of the polynucleotides encoding the variant.

[0025] Expression: The term “expression” includes any step involved in variant generation, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0026] Expression vector: The term “expression vector” refers to a linear or circular DNA molecule that includes a polynucleotide encoding a variant and that the polynucleotide is operatively linked to a control sequence provided for its expression.

[0027] Fragment: The term "fragment" refers to a polypeptide in which one or more (e.g., several) amino acids are deleted from the amino and / or carboxyl termini of a mature polypeptide. The fragment possesses keratinase activity. In one aspect, a fragment consists of or comprises at least 172 amino acid residues (e.g., amino acids 17 to 188 of SEQ ID NO: 2). In another aspect, the fragment comprises 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% of the number of amino acids in SEQ ID NO: 2, but less than 100%. In another aspect, the fragment comprises at least 172 amino acid residues of the number of amino acids in SEQ ID NO: 2 (e.g., amino acids 17 to 188 of SEQ ID NO: 2) and 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%.

[0028] Highly stringent conditions: The term "highly stringent conditions" refers to following a standard DNA blotting procedure for probes of at least 100 nucleotides in length, pre-hybridizing and hybridizing at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 65°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0029] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "host cell" also encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication.

[0030] Improved properties: The term "improved properties" refers to characteristics associated with variants that are improved compared to the parent. Such improved properties include, but are not limited to, specific activity, substrate cleavage, substrate specificity, thermal stability, and reduced pilling tendency.

[0031] Isolated: The term “isolated” means a substance in a form or environment that does not exist in nature. Non-limiting examples of isolated substances include (1) any substance that is not naturally occurring; (2) any substance removed at least partially from one or more naturally occurring components associated with it in nature, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor; (3) any substance that has been artificially modified relative to such a substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., multiple copies of the gene encoding the substance; use of a promoter stronger than that naturally associated with the gene encoding the substance). Isolated substances may be present in fermentation broth samples.

[0032] Low stringency conditions: The term "low stringency conditions" refers to following a standard DNA blotting procedure for probes of at least 100 nucleotides in length, pre-hybridizing and hybridizing at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 50°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0033] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 1 to 194 of SEQ ID NO: 2, amino acids -35 to -13 of SEQ ID NO: 2 is a signal peptide, and amino acids -12 to -1 of SEQ ID NO: 2 is a propeptide. It is known in the art that host cells can produce mixtures of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide.

[0034] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide encoding a mature polypeptide with keratinase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 106 to 687 of SEQ ID NO: 1. Nucleotides 1 to 69 of SEQ ID NO: 1 encode a signal peptide. Nucleotides 70 to 105 of SEQ ID NO: 1 encode a propeptide.

[0035] Medium-tough conditions: The term "medium-tough conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 55°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0036] Medium-high stringent conditions: The term "medium-high stringent conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 60°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0037] Mutant: The term “mutant” refers to a polynucleotide that encodes a variant.

[0038] Nucleic acid constructs: The term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a way that does not normally exist in nature to contain nucleic acid segments, or is synthesized, and that the nucleic acid molecule includes one or more control sequences.

[0039] Operable ligation: The term “operable ligation” refers to a construction in which a control sequence is positioned relative to the coding sequence of a polynucleotide so that the control sequence directs the expression of the coding sequence.

[0040] Parent or parental keratinase: The term "parent" or "parental keratinase" refers to a keratinase that is modified to produce the enzyme variant of the present invention. The parent can be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof.

[0041] Polyester textiles: As used herein, "polyester" means a linear polymeric molecule comprising a lactone group and derived from the condensation of a diacid and a diol or from the polymerization of a hydroxy acid. The invention applies to both aliphatic and aromatic polyesters. However, aromatic polyester articles used to produce fibers and resins are particularly preferred, and include synthetically produced long-chain polymers comprising at least 85%, preferably at least 90%, and most preferably at least 95% by weight an ester of a substituted aromatic carboxylic acid, such as substituted terephthalic acid or para-substituted hydroxybenzoic acid esters. Other useful polyester articles include those made from bulk polymers, yarns, fabrics, films, resins, and powders. These basic polyesters used in industrial applications include polyethylene terephthalate (PET), tetramethylene terephthalate (PTMT), polybutylene terephthalate (PBT), polypropylene terephthalate (PTT), polyethylene naphthalate (PEN), polycyclohexanedimethyl terephthalate (CHDMT), poly(ethylene-4-oxybenzoate) A-Tell, polyglycolic acid lactide, PHBA, and 2GN. However, PET is the most commonly produced linear polymer and accounts for the majority of polyesters used in industry today.

[0042] Polyester textiles as used herein means fibers, yarns, fabrics, and clothing containing polyester. The polyester yarn, fabric, or clothing may be any yarn, fabric, or clothing made of pure polyethylene terephthalate (PET), or a blend of PET fibers and any other materials conventionally used in the manufacture of textiles.

[0043] In one aspect, the polyester fabric is a fabric blend comprising at least 5% (w / w) polyester, such as at least 10%, at least 20%, at least 30%, at least 40%, 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%, or at least 95% polyester. In another aspect, the process of the present invention is applied to fabrics or garments composed essentially of poly(ethylene terephthalate) polyester material, i.e., pure poly(ethylene terephthalate) polyester material.

[0044] Sequence consistency: The parameter “sequence consistency” is used to describe the correlation between two amino acid sequences or two nucleotide sequences.

[0045] For the purposes of this invention, the Niedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) implemented in the Niedle program of the EMBOSS package (EMBOSS: European Open Software Suite for Molecular Biology, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 5.0.0 or later) is used to determine sequence consistency between two amino acid sequences. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the Niedle-annotated “longest consistency” (obtained using the -non-simplification option) is used as the percentage consistency and is calculated as follows:

[0046] (Consistent residues x 100) / (Alignment length - Total number of vacancies in the alignment)

[0047] For the purposes of this invention, the Niederman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) implemented in the Nieder program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 5.0.0 or later) is used to determine sequence consistency between two deoxyribonucleotide sequences. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Nieder-annotated "longest consistency" output (obtained using the -non-simplified option) is used as the percentage consistency and calculated as follows:

[0048] (Consistent deoxyribonucleotides x 100) / (Alignment length - Total number of vacancies in the alignment)

[0049] Subsequence: The term "subsequence" refers to a polynucleotide in which one or more (e.g., several) nucleotides are deleted from the 5' and / or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having keratinase activity. In one aspect, a subsequence consists of or includes at least 516 nucleotides (e.g., nucleotides 154 to 669 of SEQ ID NO: 1). In another aspect, a subsequence comprises 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% of the number of nucleotides in SEQ ID NO: 1, but less than 100%. In one respect, the fragment comprises at least 516 nucleotides of the number of nucleotides in SEQ ID NO: 1 (e.g., nucleotides 154 to 669 of SEQ ID NO: 1) and 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%.

[0050] Variant: The term "variant" means a polypeptide having keratinase activity that includes alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions. Substitution means that an amino acid occupying a position is replaced by a different amino acid; deletion means the removal of an amino acid occupying a position; and insertion means the addition of an amino acid adjacent to and immediately following an amino acid occupying a position. Variant of the present invention has at least 20%, at least 30%, at least 40%, 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%, or at least 100% of the keratinase activity of the mature polypeptide of SEQ ID NO: 2.

[0051] Very High Tough Conditions: The term "very high tough conditions" means that for probes at least 100 nucleotides in length, standard DNA blotting procedures are followed, including pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 70°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0052] Very low stringency conditions: The term "very low stringency conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 45°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0053] Wild-type keratinase: The term "wild-type" keratinase refers to keratinase expressed by naturally occurring microorganisms, such as bacteria, yeast, or filamentous fungi found in nature.

[0054] Variant Naming Conventions

[0055] For the purposes of this invention, the mature polypeptide disclosed in SEQ ID NO: 2 is used to determine the corresponding amino acid residues in another keratinase. The amino acid sequence of the other keratinase is aligned with the mature polypeptide disclosed in SEQ ID NO: 2, and based on this alignment, the Niederman-Onsch algorithm (Niederman and Onsch, 1970, Journal of Molecular Biology 48: 443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: European Open Software Suite for Molecular Biology, Rice et al., 2000, Trends in Genetics 16: 276-277) (preferably version 5.0.0 or later) is used to determine the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO: 2. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0056] The identification of the corresponding amino acid residues in another type of keratinase can be determined by comparing multiple polypeptide sequences using several computer programs with their corresponding default parameters. These computer programs include, but are not limited to, MUSCLE (multiple sequence comparisons by logarithmic expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797); MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Kato et al., 2005, Nucleic Acids Research 33: 511-518; Kato and Toh, 2007, Bioinformatics 23: 372-374; Kato et al., 2009, Methods in Molecular Biology 537: 39-64; Kato and Asato, 2010, Bioinformatics 26: 1899–1900); and EMBOSS EMMA using ClustalW (version 1.83 or later; Thompson et al., 1994, Nucleic Acid Research 22: 4673–4680).

[0057] When other enzymes deviate from the mature polypeptide of SEQ ID NO: 2, making traditional sequence-based comparison methods unable to detect their relationship (Lindahl and Elofsson, 2000, J.Mol. Biol. 295: 613-615), other pairwise sequence comparison algorithms can be applied. Greater sensitivity in sequence-based searches can be achieved using search programs that utilize probabilistic representations (profiles) of polypeptide families to search a database. For example, the PSI-BLAST program generates multiple profiles through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402). Even greater sensitivity can be achieved if the polypeptide family or superfamily has one or more representatives in a protein structure database. Procedures such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) utilize information from various sources (PSI-BLAST, secondary structure prediction, structural alignment spectra, and solvation potential) as input to a neural network that predicts the structural folding of a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of such models can be assessed using a variety of tools developed for this purpose.

[0058] For proteins with known structures, several tools and resources are available for retrieving and generating structure alignments. For example, the SCOP superfamily of proteins has already been structurally aligned, and those alignments are accessible and downloadable. Various algorithms, such as distance alignment matrices (Holm and Sander, 1998, Proteins 33: 88-96) or combined extensions (Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747), can be used to align two or more protein structures, and implementations of these algorithms can also be used to query structure databases with structures of interest to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

[0059] In the description of variations of the invention, the following nomenclature is provided for ease of reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are used.

[0060] replace. For amino acid substitutions, the following nomenclature is used: initial amino acid, position, substituted amino acid. Therefore, the substitution of threonine at position 226 with alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), for example, "Gly205Arg + Ser411Phe" or "G205R + S411F" represent the substitution of glycine (G) with arginine (R) at positions 205 and 411, respectively, and the substitution of serine (S) with phenylalanine (F).

[0061] Missing For amino acid deletions, use the following nomenclature: initial amino acid, position, * Therefore, the deletion of glycine at position 195 is represented as "Gly195". * "or G195" * Multiple missing items are separated by a plus sign ("+"), for example, "Gly195". * + Ser411 * "or "G195 * + S411 * ".

[0062] Different changes.When different changes can be introduced at a single position, these changes are separated by commas. For example, "Arg170Tyr,Glu" means that arginine at position 170 is replaced by either tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" has the following variants: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".

[0063] Detailed description of the invention

[0064] variants

[0065] The present invention provides variants having the keratinase activity of a parental keratinase, which include changes at one or more (e.g., several) positions corresponding to positions 181, 182, 115, 161, 1, 2, 43, 55, 79, or 5 of SEQ ID NO:2, wherein the change is a substitution for positions 181, 115, 161, 43, 55, 79, and 5, and wherein the variants have at least 75% but less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 2.

[0066] On the one hand, the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence similarity to the amino acid sequence of the parental keratinase.

[0067] On the one hand, the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 2 or amino acids 1-194 of SEQ ID NO: 2.

[0068] In one respect, the number of changes in the variations of the present invention is 1 to 20, for example 1 to 10 and 1 to 5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 changes.

[0069] In another aspect, the variant includes changes at one or more (e.g., several) positions corresponding to positions 181, 182, 115, 161, 1, 2, 43, 55, 79, and 5. In another aspect, the variant includes changes at two positions corresponding to positions 181, 182, 115, 161, 1, 2, 43, 55, 79, and 5. In another aspect, the variant includes changes at three positions corresponding to any one of positions 181, 182, 115, 161, 1, 2, 43, 55, 79, and 5. In another aspect, the variant includes changes at four positions corresponding to any one of positions 181, 182, 115, 161, 1, 2, 43, 55, 79, and 5. In another aspect, the variant includes changes at five positions corresponding to any one of positions 181, 182, 115, 161, 1, 2, 43, 55, 79, and 5. In another aspect, the variant includes changes at six positions corresponding to any one of positions 181, 182, 115, 161, 1, 2, 43, 55, 79, and 5. In another aspect, the variant includes changes at seven positions corresponding to any one of positions 181, 182, 115, 161, 1, 2, 43, 55, 79, and 5. In another aspect, the variant includes changes at eight positions corresponding to any one of positions 181, 182, 115, 161, 1, 2, 43, 55, 79, and 5. In another aspect, the variant includes changes at nine positions corresponding to any one of positions 181, 182, 115, 161, 1, 2, 43, 55, 79, and 5. In another aspect, the variant includes changes at each position corresponding to positions 181, 182, 115, 161, 1, 2, 43, 55, 79, and 5.

[0070] In another aspect, this variant includes or is composed of a substitution at the position corresponding to position 181. In another aspect, the amino acid at position 181 is substituted with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Pro. In another aspect, this variant includes or is composed of a substituted R181P of the mature polypeptide of SEQ ID NO: 2.

[0071] In another aspect, this variant includes a deletion or is composed of a term corresponding to position 182. In another aspect, an amino acid is deleted at position 182. In another aspect, this variant includes a deletion G182* of the mature polypeptide of SEQ ID NO: 2 or is composed of a term corresponding to that.

[0072] In another aspect, the variant includes or is composed of a substitution at the position corresponding to position 115. In another aspect, the amino acid at the position corresponding to position 115 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably substituted with Ile. In another aspect, the variant includes or is composed of a substituted V115I of the mature polypeptide of SEQ ID NO: 2.

[0073] In another aspect, this variant includes or consists of a substitution at the position corresponding to position A161L. In yet another aspect, the amino acid at the position corresponding to position A161L is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Leu. In yet another aspect, this variant includes or consists of a substitution of A161L in the mature polypeptide of SEQ ID NO: 2.

[0074] In another aspect, this variant includes a deletion or is composed of a term corresponding to position 1. In another aspect, an amino acid is deleted at the position corresponding to position 1. In another aspect, this variant includes a deletion of Q1* of the mature polypeptide of SEQ ID NO: 2 or is composed of a term thereof.

[0075] In another aspect, this variant includes a deletion or is composed of a term corresponding to position 2. In another aspect, an amino acid is deleted at position 2. In another aspect, this variant includes a deletion L2* of the mature polypeptide of SEQ ID NO: 2 or is composed of a term thereof.

[0076] In another aspect, this variant includes or is composed of a substitution at the position corresponding to position 43. In another aspect, the amino acid at the position corresponding to position 43 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Cys. In another aspect, this variant includes or is composed of a substituted A43C of the mature polypeptide of SEQ ID NO: 2.

[0077] In another aspect, this variant includes or is composed of a substitution at position 55. In another aspect, the amino acid at position 55 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Cys. In another aspect, this variant includes or is composed of a substituted I55C of the mature polypeptide of SEQ ID NO: 2.

[0078] In another aspect, this variant includes or consists of a substitution at the position corresponding to position 79. In another aspect, the amino acid at the position corresponding to position 79 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably substituted with Ala. In another aspect, this variant includes or consists of a substituted N79A of the mature polypeptide of SEQ ID NO: 2.

[0079] In another aspect, this variant includes or is composed of a substitution at position 5. In another aspect, the amino acid at position 5 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Val. In another aspect, this variant includes or is composed of a substitution I5V of the mature polypeptide of SEQ ID NO: 2.

[0080] On the other hand, the variant includes the following positions corresponding to 181+182, 181+115, 181+161, 181+1, 181+2, 181+43, 181+55, 181+79, 181+5, 182+115, 182+161, 182+1, 182+2, 182+43, 182+55, 182+79, 182+5, 115+161, 115+1, 115+2, 115 The changes or composition of the positions of +43, 115+55, 115+79, 115+5, 161+1, 161+2, 161+43, 161+55, 161+79, 161+5, 1+2, 1+43, 1+55, 1+79, 1+5, 2+43, 2+55, 2+79, 2+5, 43+55, 43+79, 43+5, 55+79, 55+5, 79+5 are as described above.

[0081] On the other hand, the variant includes the following corresponding positions: 181+182+115, 181+182+161, 181+182+1, 181+182+2, 181+182+43, 181+182+55, 181+182+79, 181+182+5, 181+115+161, 181+115+1, 181+115+2, 181+115+43, 181+115+55, 181+115+79, 181+115+5, 181+161+1, 181+161+2, 181+161+43, 181+161+55, 181+161+79, 181+161+5, 181+1+2 181+1+43, 181+1+55, 181+1+79, 181+1+5, 181+2+43, 181+2+55, 181+2+79, 181+2+5, 181+43+55, 181+43+79, 181+43+5, 181+55+79, 181+55+5, 181+7 9+5, 182+115+161, 182+115+1, 182+115+2, 182+115+43, 182+115+55, 182+115+79, 182+115+5, 182+161+1, 182+161+2, 182+161+43, 182+161+55, 182+ 161+79, 182+161+5, 182+1+2, 182+1+43, 182+1+55, 182+1+79, 182+1+5, 182+2+43, 182+2+55, 182+2+79, 182+2+5, 182+43+55, 182+43+79, 182+43+5 182+55+79, 182+55+5, 182+79+5, 115+161+1, 115+161+2, 115+161+43, 115+161+55, 115+161+79, 115+161+5, 115+1+2, 115+1+43, 115+1+55, 115+1+79 115+1+5, 115+2+43, 115+2+55, 115+2+79, 115+2+5, 115+43+55, 115+43+79, 115+43+5, 115+55+79, 115+55+5, 115+79+5, 161+1+2, 161+1+43, 161+1+5 5. 161+1+79, 161+1+5, 161+2+43, 161+2+55, 161+2+79, 161+2+5, 161+43+55, 161+43+79, 161+43+5, 161+55+79, 161+55+5, 161+79+5, 1+2+43, 1+2+55The changes or composition of the positions of 1+2+79, 1+2+5, 1+43+55, 1+43+79, 1+43+5, 1+55+79, 1+55+5, 1+79+5, 2+43+55, 2+43+79, 2+43+5, 2+55+79, 2+55+5, 2+79+5, 43+55+79, 43+55+5, 43+79+5, 55+79+5, as described above.

[0082] On the other hand, the variant includes the following corresponding positions: 181+182+115+161, 181+182+115+1, 181+182+115+2, 181+182+115+43, 181+182+115+55, 181+182+115+79, 181+182+115+5, 181+182+161+1, 181+182+161+2, 181+182+161+43, 181+182+161+55, 181+182+161+79, 181+182+161+5, 181+182+1+2, 181+182+1+43, 181+182+1+55, 18 1+182+1+79, 181+182+1+5, 181+182+2+43, 181+182+2+55, 181+182+2+79, 181+182+2+5, 181+182+43+55, 181+182+43+79, 181+182+43+5, 181+182+5 5+79, 181+182+55+5, 181+182+79+5, 181+115+161+1, 181+115+161+2, 181+115+161+43, 181+115+161+55, 181+115+161+79, 181+115+161+5, 181+11 5+1+2, 181+115+1+43, 181+115+1+55, 181+115+1+79, 181+115+1+5, 181+115+2+43, 181+115+2+55, 181+115+2+79, 181+115+2+5, 181+115+43+55, 18 1+115+43+79, 181+115+43+5, 181+115+55+79, 181+115+55+5, 181+115+79+5, 181+161+1+2, 181+161+1+43, 181+161+1+55, 181+161+1+79, 181+161+ 1+5, 181+161+2+43, 181+161+2+55, 181+161+2+79, 181+161+2+5, 181+161+43+55, 181+161+43+79, 181+161+43+5, 181+161+55+79, 181+161+55+5, 1 81+161+79+5, 181+1+2+43, 181+1+2+55, 181+1+2+79, 181+1+2+5, 181+1+43+55, 181+1+43+79, 181+1+43+5, 181+1+55+79, 181+1+55+5, 181+1+79+5181+2+43+55、181+2+43+79、181+2+43+5、181+2+55+79、181+2+55+5、181+2+79+5、181+43+55+79、181+43+55+5、181+43+79+5、181+55+79+5、182+115+161+1、182+115+161+2、182+115+161+43、182+115+161+55、182+115+161+79、182+115+161+5、182+115+1+2、182+115+1+43、182+115+1+55、182+115+1+79、182+115+1+5、182+115+2+43、182+115+2+55、182+115+2+79、182+115+2+5、182+115+43+55、182+115+43+79、182+115+43+5、182+115+55+79、182+115+55+5、182+115+79+5、182+161+1+2、182+161+1+43、182+161+1+55、182+161+1+79、182+161+1+5、182+161+2+43、182+161+2+55、182+161+2+79、182+161+2+5、182+161+43+55、182+161+43+79、182+161+43+5、182+161+55+79、182+161+55+5、182+161+79+5、182+1+2+43、182+1+2+55、182+1+2+79、182+1+2+5、182+1+43+55、182+1+43+79、182+1+43+5、182+1+55+79、182+1+55+5、182+1+79+5、182+2+43+55、182+2+43+79、182+2+43+5、182+2+55+79、182+2+55+5、182+2+79+5、182+43+55+79、182+43+55+5、182+43+79+5、182+55+79+5、115+161+1+2、115+161+1+43、115+161+1+55、115+161+1+79、115+161+1+5、115+161+2+43、115+161+2+55、115+161+2+79、115+161+2+5、115+161+43+55、115+161+43+79、115+161+43+5、115+161+55+79、115+161+55+5, 115+161+79+5, 115+1+2+43, 115+1+2+55, 115+1+2+79, 115+1+2+5, 115+1+43+55, 115+1+43+79, 115+1+43+5, 115+1+55+79, 115+1+55+5, 115+1+79+5, 115+2+43+55, 115+2+43+79, 11 5+2+43+5, 115+2+55+79, 115+2+55+5, 115+2+79+5, 115+43+55+79, 115+43+55+5, 115+43+79+5, 115+55+79+5, 161+1+2+43, 161+1+2+55, 161+1+2+79, 161+1+2+5, 161+1+43+55, 161+1+43+79, 161+1 +43+5, 161+1+55+79, 161+1+55+5, 161+1+79+5, 161+2+43+55, 161+2+43+79, 161+2+43+5, 161+2+55+79, 161+2+55+5, 161+2+79+5, 161+43+55+79, 161+43+55+5, 161+43+79+5, 1+2+43 The changes or composition of the positions of +55, 1+2+43+79, 1+2+43+5, 1+2+55+79, 1+2+55+5, 1+2+79+5, 1+43+55+79, 1+43+55+5, 1+43+79+5, 1+55+79+5, 2+43+55+79, 2+43+55+5, 2+43+79+5, 2+55+79+5, 43+55+79+5, as described above, are examples of such changes.

[0083] On the other hand, the variant includes the following corresponding positions: 181+182+115+161+1, 181+182+115+161+2, 181+182+115+161+43, 181+182+115+161+55, 181+182+115+161+79, 181+182+115+161+5, 181+182+115+1+2, 181+182+115+1+43, 181+182+115+1+55, 181+182+115+1+79, 181+182+115+1+5, 181+182+115+2+43, 181+182+115+2+55, 181+ 182+115+2+79, 181+182+115+2+5, 181+182+115+43+55, 181+182+115+43+79, 181+182+115+43+5, 181+182+115+55+79, 181+182+115+55+5, 181+182+ 115+79+5, 181+182+161+1+2, 181+182+161+1+43, 181+182+161+1+55, 181+182+161+1+79, 181+182+161+1+5, 181+182+161+2+43, 181+182+161+2+55 181+182+161+2+79, 181+182+161+2+5, 181+182+161+43+55, 181+182+161+43+79, 181+182+161+43+5, 181+182+161+55+79, 181+182+161+55+5, 18 1+182+161+79+5, 181+182+1+2+43, 181+182+1+2+55, 181+182+1+2+79, 181+182+1+2+5, 181+182+1+43+55, 181+182+1+43+79, 181+182+1+43+5, 181+ 182+1+55+79, 181+182+1+55+5, 181+182+1+79+5, 181+182+2+43+55, 181+182+2+43+79, 181+182+2+43+5, 181+182+2+55+79, 181+182+2+55+5, 181+1 82+2+79+5, 181+182+43+55+79, 181+182+43+55+5, 181+182+43+79+5, 181+182+55+79+5, 181+115+161+1+2, 181+115+161+1+43, 181+115+161+1+55,181+115+161+1+79、181+115+161+1+5、181+115+161+2+43、181+115+161+2+55、181+115+161+2+79、181+115+161+2+5、181+115+161+43+55、181+115+161+43+79、181+115+161+43+5、181+115+161+55+79、181+115+161+55+5、181+115+161+79+5、181+115+1+2+43、181+115+1+2+55、181+115+1+2+79、181+115+1+2+5、181+115+1+43+55、181+115+1+43+79、181+115+1+43+5、181+115+1+55+79、181+115+1+55+5、181+115+1+79+5、181+115+2+43+55、181+115+2+43+79、181+115+2+43+5、181+115+2+55+79、181+115+2+55+5、181+115+2+79+5、181+115+43+55+79、181+115+43+55+5、181+115+43+79+5、181+115+55+79+5、181+161+1+2+43、181+161+1+2+55、181+161+1+2+79、181+161+1+2+5、181+161+1+43+55、181+161+1+43+79、181+161+1+43+5、181+161+1+55+79、181+161+1+55+5、181+161+1+79+5、181+161+2+43+55、181+161+2+43+79、181+161+2+43+5、181+161+2+55+79、181+161+2+55+5、181+161+2+79+5、181+161+43+55+79、181+161+43+55+5、181+161+43+79+5、181+161+55+79+5、181+1+2+43+55、181+1+2+43+79、181+1+2+43+5、181+1+2+55+79、181+1+2+55+5、181+1+2+79+5、181+1+43+55+79、181+1+43+55+5、181+1+43+79+5、181+1+55+79+5、181+2+43+55+79、181+2+43+55+5、181+2+43+79+5、181+2+55+79+5、181+43+55+79+5、182+115+161+1+2、182+115+161+1+43、182+115+161+1+55、182+115+161+1+79、182+115+161+1+5、182+115+161+2+43、182+115+161+2+55、182+115+161+2+79、182+115+161+2+5、182+115+161+43+55、182+115+161+43+79、182+115+161+43+5、182+115+161+55+79、182+115+161+55+5、182+115+161+79+5、182+115+1+2+43、182+115+1+2+55、182+115+1+2+79、182+115+1+2+5、182+115+1+43+55、182+115+1+43+79、182+115+1+43+5、182+115+1+55+79、182+115+1+55+5、182+115+1+79+5、182+115+2+43+55、182+115+2+43+79、182+115+2+43+5、182+115+2+55+79、182+115+2+55+5、182+115+2+79+5、182+115+43+55+79、182+115+43+55+5、182+115+43+79+5、182+115+55+79+5、182+161+1+2+43、182+161+1+2+55、182+161+1+2+79、182+161+1+2+5、182+161+1+43+55、182+161+1+43+79、182+161+1+43+5、182+161+1+55+79、182+161+1+55+50、182+161+1+79+5、182+161+2+43+55、182+161+2+43+79、182+161+2+43+5、182+161+2+55+79、182+161+2+55+5、182+161+2+79+5、182+161+43+55+79、182+161+43+55+5、182+161+43+79+5、182+161+55+79+5、182+1+2+43+55、182+1+2+43+79、182+1+2+43+5、182+1+2+55+79、182+1+2+55+5、182+1+2+79+5、182+1+43+55+79, 182+1+43+55+5, 182+1+43+79+5, 182+1+55+79+5, 182+2+43+55+79, 182+2+43+55+5, 182+2+43+79+5, 182+2+55+79+5, 182+43+55+79+5, 115+161+1+2+43, 115+161+1+2+55, 115+161+1+2+79, 115+161+1+2+5, 115+161+1+43+55, 115+161+1+43+79, 115+161+1+43+5, 115 +161+1+55+79, 115+161+1+55+5, 115+161+1+79+5, 115+161+2+43+55, 115+161+2+43+79, 115+161+2+43+5, 115+161+2+55+79, 115+161+2+55+5, 115+161+2+79+5, 115+161+43+55+79, 115+1+2+43+55, 115+1+2+43+79, 115+1+2+43 +5, 115+1+2+55+79, 115+1+2+55+5, 115+1+2+79+5, 115+1+43+55+79, 115+1+43+55+5, 115+1+43+79+5, 115+1+55+79+5, 115+2+43+55+79, 115+2+43+55+5, 115+2+43+79+5, 115+2+55+79+5, 115+43+55+79+5, 161+1+2+43+55, 161+1+2+43+79, 161+1+2+43+5, 161+1+2+55+79, 161+1+2+55+5 The changes or composition of the positions of 161+1+2+79+5, 161+1+43+55+79, 161+1+43+55+5, 161+1+55+79+5, 161+2+43+55+79, 161+2+43+55+5, 161+2+43+79+5, 161+2+55+79+5, 161+43+55+79+5, 1+2+43+55+79, 1+2+43+55+5, 1+2+43+79+5, 1+2+55+79+5, 1+43+55+79+5, 2+43+55+79+5, as described above.

[0084] On the other hand, the variant includes the following corresponding positions: 181+182+115+161+1+2, 181+182+115+161+1+43, 181+182+115+161+1+55, 181+182+115+161+1+79, 181+182+115+161+1+5, 181+182+115+161+2+43, 181+182+115+161+2+55, 181+182+115+161+2+79, 181+182+115+161+2+5, 181+182+115+161+43+55, 181+182+115+161+43+79, 1 81+182+115+161+43+5, 181+182+115+161+55+79, 181+182+115+161+55+5, 181+182+115+161+79+5, 181+182+115+1+2+43, 181+182+115+1+2+55, 18 1+182+115+1+2+79, 181+182+115+1+2+5, 181+182+115+1+43+55, 181+182+115+1+43+79, 181+182+115+1+43+5, 181+182+115+1+55+79, 181+182+11 5+1+55+5, 181+182+115+1+79+5, 181+182+115+2+43+55, 181+182+115+2+43+79, 181+182+115+2+43+5, 181+182+115+2+55+79, 181+182+115+2+55+ 5. 181+182+115+2+79+5, 181+182+115+43+55+79, 181+182+115+43+55+5, 181+182+115+43+79+5, 181+182+115+55+79+5, 181+182+161+1+2+43, 181 +182+161+1+2+55, 181+182+161+1+2+79, 181+182+161+1+2+5, 181+182+161+1+43+55, 181+182+161+1+43+79, 181+182+161+1+43+5, 181+182+161+ 1+55+79, 181+182+161+1+55+5, 181+182+161+1+79+5, 181+182+161+2+43+55, 181+182+161+2+43+79, 181+182+161+2+43+5, 181+182+161+2+55+79181+182+161+2+55+5、181+182+161+2+79+5、181+182+161+43+55+79、181+182+161+43+55+5、181+182+161+43+79+5、181+182+161+55+79+5、181+182+1+2+43+55、181+182+1+2+43+79、181+182+1+2+43+5、181+182+1+2+55+79、181+182+1+2+55+5、181+182+1+2+79+5、181+182+1+43+55+79、181+182+1+43+55+5、181+182+1+43+79+5、181+182+1+55+79+5、181+182+2+43+55+79、181+182+2+43+55+5、181+182+2+43+79+5、181+182+2+55+79+5、181+182+43+55+79+5、181+115+161+1+2+43、181+115+161+1+2+55、181+115+161+1+2+79、181+115+161+1+2+5、181+115+161+1+43+55、181+115+161+1+43+79、181+115+161+1+43+5、181+115+161+1+55+79、181+115+161+1+55+5、181+115+161+1+79+5、181+115+161+2+43+55、181+115+161+2+43+79、181+115+161+2+43+5、181+115+161+2+55+79、181+115+161+2+55+5、181+115+161+2+79+5、181+115+161+43+55+79、181+115+161+43+55+5、181+115+161+43+79+5、181+115+161+55+79+5、181+115+1+2+43+55、181+115+1+2+43+79、181+115+1+2+43+5、181+115+1+2+55+79、181+115+1+2+55+5、181+115+1+2+79+5、181+115+1+43+55+79、181+115+1+43+55+5、181+115+1+43+79+5、181+115+1+55+79+5、181+115+2+43+55+79、181+115+2+43+55+5、181+115+2+43+79+5、181+115+2+55+79+5、181+115+43+55+79+5、181+161+1+2+43+55、181+161+1+2+43+79、181+161+1+2+43+5、181+161+1+2+55+79、181+161+1+2+55+5、181+161+1+2+79+5、181+161+1+43+55+79、181+161+1+43+55+5、181+161+1+43+79+5、181+161+1+55+79+5、181+161+2+43+55+79、181+161+2+43+55+5、181+161+2+43+79+5、181+161+2+55+79+5、181+161+43+55+79+5、181+1+2+43+55+79、181+1+2+43+55+5、181+1+2+43+79+5、181+1+2+55+79+5、181+1+43+55+79+5、181+2+43+55+79+5、182+115+161+1+2+43、182+115+161+1+2+55、182+115+161+1+2+79、182+115+161+1+2+5、182+115+161+1+43+55、182+115+161+1+43+79、182+115+161+1+43+5、182+115+161+1+55+79、182+115+161+1+55+5、182+115+161+1+79+5、182+115+161+2+43+55、182+115+161+2+43+79、182+115+161+2+43+5、182+115+161+2+55+79、182+115+161+2+55+5、182+115+161+2+79+5、182+115+161+43+55+79、182+115+161+43+55+5、182+115+161+43+79+5、182+115+161+55+79+5、182+115+1+2+43+55、182+115+1+2+43+79、182+115+1+2+43+5、182+115+1+2+55+79、182+115+1+2+55+5、182+115+1+2+79+5、182+115+1+43+55+79、182+115+1+43+55+5、182+115+1+43+79+5、182+115+1+55+79+5、182+115+2+43+55+79, 182+115+2+43+55+5, 182+115+2+43+79+5, 182+115+2+55+79+5, 182+115+43+55+79+5, 182+161+1+2+43+55, 182+161+1+2+43+79, 182+161+1+2+43+5, 182+161+1+2+55+79, 182+161+1+2+79+5, 182+161+1+43+55+79, 182+161+1+43+55+5, 182+161 +1+43+79+5, 182+161+1+55+79+5, 182+161+2+43+55+79, 182+161+2+43+55+5, 182+161+2+43+79+5, 182+161+2+55+79+5, 182+1+2+43+55+79+5, 182+1+2+43+55+5, 182+1+2+43+79+5, 182+1+2+55+79+5, 182+1+43+55+79+5, 182+2+43+55+79+5, 115+161+1+2+43+55, 115+ 161+1+2+43+79, 115+161+1+2+43+5, 115+161+1+2+55+79, 115+161+1+2+55+5, 115+161+1+2+79+5, 115+161+1+43+55+79, 115+161+1+43+55+5, 115+161+1+43+79+5, 115+161+1+55+79+5, 115+161+2+43+55+79, 115+161+2+43+79+5, 115+161+2+55+79+5, 115+161+43+5 The changes or composition of the positions of 5+79+5, 115+1+2+43+55+790, 115+1+2+43+55+5, 115+1+2+43+79+5, 115+1+2+55+79+5, 115+1+43+55+79+5, 115+2+43+55+79+5, 161+1+2+43+55+79, 161+1+2+43+55+79+5, 161+1+2+55+79+5, 161+1+43+55+79+5, 161+2+43+55+79+5, and 1+2+43+55+79+5, as described above, are examples of such changes or compositions.

[0085] On the other hand, the variant includes the following corresponding positions: 181+182+115+161+1+2+43, 181+182+115+161+1+2+55, 181+182+115+161+1+2+79, 181+182+115+161+1+2+5, 181+182+115+161+1+43+55, 181+182+115+161+1+43+79, 181+182+115+161+1+43+5, 181+182+115+161+1+55+79, 181+182+115+161+1+55+5, 181+182+115+161+1+79+ 5. 181+182+115+161+2+43+55, 181+182+115+161+2+43+79, 181+182+115+161+2+43+5, 181+182+115+161+2+55+79, 181+182+115+161+2+55+5, 181+ 182+115+161+2+79+5, 181+182+115+161+43+55+79, 181+182+115+161+43+55+5, 181+182+115+161+43+79+5, 181+182+115+161+55+79+5, 181+182+ 115+1+2+43+55, 181+182+115+1+2+43+79, 181+182+115+1+2+43+5, 181+182+115+1+2+55+79, 181+182+115+1+2+55+5, 181+182+115+1+2+79+5, 181 +182+115+1+43+55+79, 181+182+115+1+43+55+5, 181+182+115+1+43+79+5, 181+182+115+1+55+79+5, 181+182+115+2+43+55+79, 181+182+115+2+4 3+55+5, 181+182+115+2+43+79+5, 181+182+115+2+55+79+5, 181+182+115+43+55+79+5, 181+182+161+1+2+43+55, 181+182+161+1+2+43+79, 181+18 2+161+1+2+43+5, 181+182+161+1+2+55+79, 181+182+161+1+2+55+5, 181+182+161+1+2+79+5, 181+182+161+1+43+55+79, 181+182+161+1+43+55+5181+182+161+1+43+79+5、181+182+161+1+55+79+5、181+182+161+2+43+55+79、181+182+161+2+43+55+5、181+182+161+2+43+79+5、181+182+161+2+55+79+5、181+182+161+43+55+79+5、181+182+1+2+43+55+79、181+182+1+2+43+55+5、181+182+1+2+43+79+5、181+182+1+2+55+79+5、181+182+1+43+55+79+5、181+182+2+43+55+79+5、181+115+161+1+2+43+55、181+115+161+1+2+43+79、181+115+161+1+2+43+5、181+115+161+1+2+55+79、181+115+161+1+2+55+5、181+115+161+1+2+79+5、181+115+161+1+43+55+79、181+115+161+1+43+55+5、181+115+161+1+43+79+5、181+115+161+1+55+79+5、181+115+161+2+43+55+79、181+115+161+2+43+55+5、181+115+161+2+43+79+5、181+115+161+2+55+79+5、181+115+161+43+55+79+5、181+115+1+2+43+55+79、181+115+1+2+43+55+5、181+115+1+2+43+79+5、181+115+1+2+55+79+5、181+115+1+43+55+79+5、181+115+2+43+55+79+5、181+161+1+2+43+55+79、181+161+1+2+43+55+5、181+161+1+2+43+79+5、181+161+1+2+55+79+5、181+161+1+43+55+79+5、181+161+2+43+55+79+5、181+1+2+43+55+79+5、182+115+161+1+2+43+55、182+115+161+1+2+43+79、182+115+161+1+2+43+5、182+115+161+1+2+55+79、182+115+161+1+2+55+5、182+115+161+1+2+79+5, 182+115+161+1+43+55+79, 182+115+161+1+43+55+5, 182+115+161+1+43+79+5, 182+115+161+1+55+79+5, 182+115+161+2+43+55+79, 182+115+161+2+43+55+5, 182+115+161+ 2+43+79+5, 182+115+161+2+55+79+5, 182+115+161+43+55+79+5, 182+115+1+2+43+55+79, 182+115+1+2+43+55+5, 182+115+1+2+43+79+5, 182+115+1+2+55+79+5, 182+115+1+43+55+79+5, 182+115+2+ 43+55+79+5, 182+161+1+2+43+55+79, 182+161+1+2+43+55+5, 182+161+1+2+43+79+5, 182+161+1+2+55+79+5, 182+161+1+43+55+79+5, 182+1+2+43+55+79+5, 115+161+1+2+43 The changes or composition of the positions of +55+79, 115+161+1+2+43+55+5, 115+161+1+2+43+79+5, 115+161+1+2+55+79+5, 115+161+1+43+55+79+5, 115+161+2+43+55+79+5, 115+1+2+43+55+79+5, 161+1+2+43+55+79+5, as described above, are as follows.

[0086] On the other hand, the variant includes the following corresponding positions: 181+182+115+161+1+2+43+55, 181+182+115+161+1+2+43+79, 181+182+115+161+1+2+43+5, 181+182+115+161+1+2+55+79, 181+182+115+161+1+2+55+5, 181+182+115+161+1+2+79+5, 181+182+115+161+1+43+55+79, 181+182+115+161+1+43+55+5, 181+182+115+161+1+43+79+ 5. 181+182+115+161+1+55+79+5, 181+182+115+161+2+43+55+79, 181+182+115+161+2+43+55+5, 181+182+115+161+2+43+79+5, 181+182+115+161+2 +55+79+5, 181+182+115+161+43+55+79+5, 181+182+115+1+2+43+55+79, 181+182+115+1+2+43+55+5, 181+182+115+1+2+43+79+5, 181+182+115+1+2 +55+79+5, 181+182+115+1+43+55+79+5, 181+182+115+2+43+55+79+5, 181+182+161+1+2+43+55+79, 181+182+161+1+2+43+55+5, 181+182+161+1+2 +43+79+5, 181+182+161+1+2+55+79+5, 181+182+161+1+43+55+79+5, 181+182+161+2+43+55+79+5, 181+182+1+2+43+55+79+5, 181+115+161+1+2+43 +55+79, 181+115+161+1+2+43+55+5, 181+115+161+1+2+43+79+5, 181+115+161+1+2+55+79+5, 181+115+161+1+43+55+79+5, 181+115+161+2+43+55+ 79+5, 181+115+1+2+43+55+79+5, 181+161+1+2+43+55+79+5, 182+115+161+1+2+43+55+79, 182+115+161+1+2+43+55+5, 182+115+161+1+2+43+79+5The changes or composition of the positions of 182+115+161+1+2+55+79+5, 182+115+161+1+43+55+79+5, 182+115+161+2+43+55+79+5, 182+115+1+2+43+55+79+5, 182+161+1+2+43+55+79+5, and 115+161+1+2+43+55+79+5, as described above, are examples of such changes.

[0087] On the other hand, the variant includes the following corresponding positions: 181+182+115+161+1+2+43+55+79, 181+182+115+161+1+2+43+55+5, 181+182+115+161+1+2+43+79+5, 181+182+115+161+1+2+55+79+5, 181+182+115+161+1+43+55+79+5 The changes or composition of the positions of 181+182+115+161+2+43+55+79+5, 181+182+115+1+2+43+55+79+5, 181+182+161+1+2+43+55+79+5, 181+115+161+1+2+43+55+79+5, and 182+115+161+1+2+43+55+79+5 are as described above.

[0088] On the other hand, the variant includes changes or composition thereof at positions corresponding to 181+182+115+161+1+2+43+55+79+5, as described above.

[0089] On the other hand, the variant includes one or more (e.g., several) replacements or components selected from the group consisting of R181P, G182*, V115I, A161L, Q1*, L2*, A43C, I55C, N79A, and I5V.

[0090] On the other hand, this variant includes substitutions of the mature peptide of SEQ ID NO: 2 with R181P+G182*, R181P+V115I, R181P+A161L, R181P+Q1*, R181P+L2*, R181P+A43C, R181P+I55C, R181P+N79A, R181P+I5V, G182*+V115I, G182*+A161L, G182*+Q1*, G182*+L2*, G182*+A43C, G182*+I55C, G182*+N79A, G182*+I5V, V115I+A161L, V115I+Q1*, V115I+L2*, and V115I+A43. C. V115I+I55C, V115I+N79A, V115I+I5V, A161L+Q1*, A161L+L2*, A161L +A43C、A161L+I55C、A161L+N79A、A161L+I5V、Q1*+L2*、Q1*+A43C、Q1*+ I55C, Q1*+N79A, Q1*+I5V, L2*+A43C, L2*+I55C, L2*+N79A, L2*+I5V, A4 3C+I55C, A43C+N79A, A43C+I5V, I55C+N79A, I55C+I5V, N79A+I5V or composed of them.

[0091] On the other hand, this variant includes SEQ ID NO: Substitution of mature peptides of 2: R181P+G182*+V115I, R181P+G182*+A161L, R181P+G182*+Q1*, R181P+G182*+L2*, R181P+G182*+A43C, R181P+G182*+I55C, R181P+G182*+N79A, R181P+G182*+I5V, R181P+V115I+A161L, R181P+V115I+Q1*, R181P+V115I+L2*, R181P+V115I+A43C, R181P+V115I+I55C, R181P+V115I+N 79A, R181P+V115I+I5V, R181P+A161L+Q1*, R181P+A161L+L2*, R181P+A1 61L+A43C, R181P+A161L+I55C, R181P+A161L+N79A, R181P+A161L+I5V, R 181P+Q1*+L2*, R181P+Q1*+A43C, R181P+Q1*+I55C, R181P+Q1*+N79A, R1 81P+Q1*+I5V, R181P+L2*+A43C, R181P+L2*+I55C, R181P+L2*+N79A, R181 P+L2*+I5V, R181P+A43C+I55C, R181P+A43C+N79A, R181P+A43C+I5V, R18 1P+I55C+N79A, R181P+I55C+I5V, R181P+N79A+I5V, G182*+V115I+A161L , G182*+V115I+Q1*, G182*+V115I+L2*, G182*+V115I+A43C, G182*+V115 I+I55C, G182*+V115I+N79A, G182*+V115I+I5V, G182*+A161L+Q1*, G182* +A161L+L2*, G182*+A161L+A43C, G182*+A161L+I55C, G182*+A161L+N79 A. G182*+A161L+I5V, G182*+Q1*+L2*, G182*+Q1*+A43C, G182*+Q1*+I55C , G182*+Q1*+N79A, G182*+Q1*+I5V, G182*+L2*+A43C, G182*+L2*+I55C, G182*+L2*+N79A, G182*+L2*+I5V, G182*+A43C+I55C, G182*+A43C+N79A,G182*+A43C+I5V, G182*+I55C+N79A, G182*+I55C+I5V, G182*+N79A+I5V, V115I+A161L+Q1*, V115I+A161L+L2*, V115I+A161L+A43C, V115I+A161L+I55C, V115I+A161L+N79A, V115I+A161L+I5V, V115I+Q1*+L2*, V115I+Q1*+A43C, V115I+Q1*+I55C, V115I+Q1*+N79A, V115I+Q1*+I5V, V115I+L2*+A43C, V115I+L2*+I55C, V115I+L2*+N79A, V115I+L2*+I5V, V115I+A43C+I55C, V115I+A43C+N79A, V115I+A43C+I5V, V115I+I55C+N79A, V115I+I55C+I5V, V115I+N79A+I5V, A161L+Q1*+L2*, A161L+Q1*+A43C, A161L+Q1*+I55C, A161L+Q1*+N79A, A161L+Q1*+I5V, A161L+L2*+A43C, A161L+L2*+I55C, A161L+L2*+N79A, A161L+L2*+I5V, A161L+A43C+I55C, A161L+A43C+N79A, A161L+A43C+I5V, A161L+I55C+N79A, A161L+I55C+I5V, A161L+N79A+I5V, Q1*+L2*+A43C, Q1*+L2*+I55C, Q1*+L2*+N79A, Q1*+L2*+I5V, Q1*+A43C+I55C, Q1*+A43C+N79A, Q1*+A43C+I5V, Q1*+I55C+N79A, Q1*+I55C+I5V, Q1*+N79A+I5V, L2*+A43C+I55C, L2*+A43C+N79A, L2*+A43C+I5V, L2*+I55C+N79A, L2*+I55C+I5V, L2*+N79A+I5V, A43C+I55C+N79A, A43C+I55C+I5V, A43C+N79A+I5V, I55C+N79A+I5V or composed of them.

[0092] On the other hand, this variant includes SEQ ID NO: Substitution of mature peptides of 2: R181P+G182*+V115I+A161L, R181P+G182*+V115I+Q1*, R181P+G182*+V115I+L2*, R181P+G182*+V115I+A43C, R181P+G182*+V115I+I55C, R181P+G182*+V115I+N79A, R181P+G182*+V115I+I5V, R181P+G182*+A161L+Q1*, R181P+G182*+A161L+L2*, R181P+G182*+A161L+A43C, R181P +G182*+A161L+I55C, R181P+G182*+A161L+N79A, R181P+G182*+A161L+I 5V, R181P+G182*+Q1*+L2*, R181P+G182*+Q1*+A43C, R181P+G182*+Q1*+ I55C, R181P+G182*+Q1*+N79A, R181P+G182*+Q1*+I5V, R181P+G182*+L2 *+A43C, R181P+G182*+L2*+I55C, R181P+G182*+L2*+N79A, R181P+G182* +L2*+I5V, R181P+G182*+A43C+I55C, R181P+G182*+A43C+N79A, R181P+G 182*+A43C+I5V, R181P+G182*+I55C+N79A, R181P+G182*+I55C+I5V, R18 1P+G182*+N79A+I5V, R181P+V115I+A161L+Q1*, R181P+V115I+A161L+L2 *, R181P+V115I+A161L+A43C, R181P+V115I+A161L+I55C, R181P+V115I+ A161L+N79A, R181P+V115I+A161L+I5V, R181P+V115I+Q1*+L2*, R181P+V 115I+Q1*+A43C, R181P+V115I+Q1*+I55C, R181P+V115I+Q1*+N79A, R181 P+V115I+Q1*+I5V, R181P+V115I+L2*+A43C, R181P+V115I+L2*+I55C, R1 81P+V115I+L2*+N79A, R181P+V115I+L2*+I5V, R181P+V115I+A43C+I55C,R181P+V115I+A43C+N79A、R181P+V115I+A43C+I5V、R181P+V115I+I55C+N 79A、R181P+V115I+I55C+I5V、R181P+V115I+N79A+I5V、R181P+A161L+Q1*+ L2*、R181P+A161L+Q1*+A43C、R181P+A161L+Q1*+I55C、R181P+A161L+Q1*+ N79A、R181P+A161L+Q1*+I5V、R181P+A161L+L2*+A43C、R181P+A161L+L2*+ I55C, R181P+A161L+L2*+N79A, R181P+A161L+L2*+I5V, R181P+A161L+A43C+I55C, R181P+A161L+A43C+N79A, R181P+A161L+A43C+I5V, R181P+A161L+ I55C+N79A, R181P+A161L+I55C+I5V, R181P+A161L+N79A+I5V, R181P+Q1*+L2*+A43C, R181P+Q1*+L2*+I55C, R181P+Q1*+L2*+N79A, R181P+Q1*+L2*+I 5V、R181P+Q1*+A43C+I55C、R181P+Q1*+A43C+N79A、R181P+Q1*+A43C+I5V 、R181P+Q1*+I55C+N79A、R181P+Q1*+I55C+I5V、R181P+Q1*+N79A+I5V、R18 1P+L2*+A43C+I55C, R181P+L2*+A43C+N79A, R181P+L2*+A43C+I5V, R181P+L2*+I55C+N79A, R181P+L2*+I55C+I5V, R181P+L2*+N79A+I5V, R181P+A43C +I55C+N79A, R181P+A43C+I55C+I5V, R181P+A43C+N79A+I5V, R181P+I55C+N79A+I5V, G182*+V115I+A161L+Q1*, G182*+V115I+A161L+L2*, G182*+V1 15I+A161L+A43C, G182*+V115I+A161L+I55C, G182*+V115I+A161L+N79A, G182*+V115I+A161L+I5V, G182*+V115I+Q1*+L2*, G182*+V115I+Q1*+A43CG182*+V115I+Q1*+I55C, G182*+V115I+Q1*+N79A, G182*+V115I+Q1*+I5V, G182*+V115I+L2*+A43C, G182*+V115I+L2*+I55C, G182*+V115I+L2*+N79A G182*+V115I+L2*+I5V, G182*+V115I+A43C+I55C, G182*+V115I+A43C+N79A, G182*+V115I+A43C+I5V, G182*+V115I+I55C+N79A, G182*+V115I+I55C+ I5V, G182*+V115I+N79A+I5V, G182*+A161L+Q1*+L2*, G182*+A161L+Q1*+A43C, G182*+A161L+Q1*+I55C, G182*+A161L+Q1*+N79A, G182*+A161L+Q1*+I 5V, G182*+A161L+L2*+A43C, G182*+A161L+L2*+I55C, G182*+A161L+L2*+N79A, G182*+A161L+L2*+I5V, G182*+A161L+A43C+I55C, G182*+A161L+A43C+ N79A, G182*+A161L+A43C+I5V, G182*+A161L+I55C+N79A, G182*+A161L+I55C+I5V, G182*+A161L+N79A+I5V, G182*+Q1*+L2*+A43C, G182*+Q1*+L2*+I5 5C, G182*+Q1*+L2*+N79A, G182*+Q1*+L2*+I5V, G182*+Q1*+A43C+I55C, G182*+Q1*+A43C+N79A, G182*+Q1*+A43C+I5V, G182*+Q1*+I55C+N79A, G182*+ Q1*+I55C+I5V, G182*+Q1*+N79A+I5V, G182*+L2*+A43C+I55C, G182*+L2*+A43C+N79A, G182*+L2*+A43C+I5V, G182*+L2*+I55C+N79A, G182*+L2*+I55C +I5V, G182*+L2*+N79A+I5V, G182*+A43C+I55C+N79A, G182*+A43C+I55C+I5V, G182*+A43C+N79A+I5V, G182*+I55C+N79A+I5V, V115I+A161L+Q1*+L2*V115I+A161L+Q1*+A43C, V115I+A161L+Q1*+I55C, V115I+A161L+Q1*+N79A, V115I+A161L+Q1*+I5V, V115I+A161L+L2*+A43C, V115I+A161L+L2*+I55C V115I+A161L+L2*+N79A, V115I+A161L+L2*+I5V, V115I+A161L+A43C+I55C, V115I+A161L+A43C+N79A, V115I+A161L+A43C+I5V, V115I+A161L+I55C+ N79A, V115I+A161L+I55C+I5V, V115I+A161L+N79A+I5V, V115I+Q1*+L2*+A43C, V115I+Q1*+L2*+I55C, V115I+Q1*+L2*+N79A, V115I+Q1*+L2*+I5V, V1 15I+Q1*+A43C+I55C, V115I+Q1*+A43C+N79A, V115I+Q1*+A43C+I5V, V115I+Q1*+I55C+N79A, V115I+Q1*+I55C+I5V, V115I+Q1*+N79A+I5V, V115I+L2*+ A43C+I55C, V115I+L2*+A43C+N79A, V115I+L2*+A43C+I5V, V115I+L2*+I55C+N79A, V115I+L2*+I55C+I5V, V115I+L2*+N79A+I5V, V115I+A43C+I55C+N 79A, V115I+A43C+I55C+I5V, V115I+A43C+N79A+I5V, V115I+I55C+N79A+I5V, A161L+Q1*+L2*+A43C, A161L+Q1*+L2*+I55C, A161L+Q1*+L2*+N79A, A16 1L+Q1*+L2*+I5V, A161L+Q1*+A43C+I55C, A161L+Q1*+A43C+N79A, A161L+Q1*+A43C+I5V, A161L+Q1*+I55C+N79A, A161L+Q1*+I55C+I5V, A161L+Q1*+N 79A+I5V, A161L+L2*+A43C+I55C, A161L+L2*+A43C+N79A, A161L+L2*+A43C+I5V, A161L+L2*+I55C+N79A, A161L+L2*+I55C+I5V, A161L+L2*+N79A+I5VA161L + A43C + I55C + N79A, A161L + A43C + I55C + I5V, A161L + A43C + N79A + I5V, A161L + I55C + N79A + I5V, Q1* + L2* + A43C + I55C, Q1* + L2* + A43C + N79A, Q1* + L2* + A43C + I5V, Q1* + L2* + I55C + N79A, Q1* + L2* + I55C + I5V, Q1* + L2* + N79A + I5V, Q1* + A43C + I55C + N79A, Q1* + A43C + I55C + I5V, Q1* + A43C + N79A + I5V, Q1* + I55C + N79A + I5V, L2* + A43C + I55C + N79A, L2* + A43C + I55C + I5V, L2* + A43C + N79A + I5V, L2* + I55C + N79A + I5V, A43C + I55C + N79A + I5V or composed of them.

[0093] On the other hand, this variant includes SEQ ID NO: Substitution of mature polypeptides of 2: R181P+G182*+V115I+A161L+Q1*, R181P+G182*+V115I+A161L+L2*, R181P+G182*+V115I+A161L+A43C, R181P+G182*+V115I+A161L+I55C, R181P+G182*+V115I+A161L+N79A, R181P+G182*+V115I+A161L+I5V, R181P+G182*+V115I+Q1*+L2*, R181P+G182*+V115I+Q1*+A43C, R181P+G 182*+V115I+Q1*+I55C, R181P+G182*+V115I+Q1*+N79A, R181P+G182*+V 115I+Q1*+I5V, R181P+G182*+V115I+L2*+A43C, R181P+G182*+V115I+L2 *+I55C, R181P+G182*+V115I+L2*+N79A, R181P+G182*+V115I+L2*+I5V, R181P+G182*+V115I+A43C+I55C, R181P+G182*+V115I+A43C+N79A, R181P +G182*+V115I+A43C+I5V, R181P+G182*+V115I+I55C+N79A, R181P+G182 *+V115I+I55C+I5V, R181P+G182*+V115I+N79A+I5V, R181P+G182*+A161 L+Q1*+L2*, R181P+G182*+A161L+Q1*+A43C, R181P+G182*+A161L+Q1*+I 55C, R181P+G182*+A161L+Q1*+N79A, R181P+G182*+A161L+Q1*+I5V, R181 P+G182*+A161L+L2*+A43C, R181P+G182*+A161L+L2*+I55C, R181P+G182 *+A161L+L2*+N79A, R181P+G182*+A161L+L2*+I5V, R181P+G182*+A161L +A43C+I55C、R181P+G182*+A161L+A43C+N79A、R181P+G182*+A161L+A43 C+I5V, R181P+G182*+A161L+I55C+N79A, R181P+G182*+A161L+I55C+I5V,R181P+G182*+A161L+N79A+I5V, R181P+G182*+Q1*+L2*+A43C, R181P+G182*+Q1*+L2*+I55C, R181P+G182*+Q1*+L2*+N79A, R181P+G182*+Q1*+L2*+I5 V、R181P+G182*+Q1*+A43C+I55C、R181P+G182*+Q1*+A43C+N79A、R181P+G1 82*+Q1*+A43C+I5V、R181P+G182*+Q1*+I55C+N79A、R181P+G182*+Q1*+I55 C+I5V, R181P+G182*+Q1*+N79A+I5V, R181P+G182*+L2*+A43C+I55C, R181P+G182*+L2*+A43C+N79A, R181P+G182*+L2*+A43C+I5V, R181P+G182*+L2*+ I55C+N79A, R181P+G182*+L2*+I55C+I5V, R181P+G182*+L2*+N79A+I5V, R181P+G182*+A43C+I55C+N79A, R181P+G182*+A43C+I55C+I5V, R181P+G182* +A43C+N79A+I5V, R181P+G182*+I55C+N79A+I5V, R181P+V115I+A161L+Q1*+L2*, R181P+V115I+A161L+Q1*+A43C, R181P+V115I+A161L+Q1*+I55C, R18 1P+V115I+A161L+Q1*+N79A、R181P+V115I+A161L+Q1*+I5V、R181P+V115I+ A161L+L2*+A43C、R181P+V115I+A161L+L2*+I55C、R181P+V115I+A161L+L2 *+N79A、R181P+V115I+A161L+L2*+I5V、R181P+V115I+A161L+A43C+I55C、R181P+V115I+A161L+A43C+N79A、R181P+V115I+A161L+A43C+I5V、R181P+V1 15I+A161L+I55C+N79A、R181P+V115I+A161L+I55C+I5V、R181P+V115I+A161L+N79A+I5V、R181P+V115I+Q1*+L2*+A43C、R181P+V115I+Q1*+L2*+I55C、R181P+V115I+Q1*+L2*+N79A, R181P+V115I+Q1*+L2*+I5V, R181P+V115I+Q1*+A43C+I55C, R181P+V115I+Q1*+A43C+N79A, R181P+V115I+Q1*+A43C+I5 V, R181P+V115I+Q1*+I55C+N79A, R181P+V115I+Q1*+I55C+I5V, R181P+V115I+Q1*+N79A+I5V, R181P+V115I+L2*+A43C+I55C, R181P+V115I+L2*+A43C +N79A、R181P+V115I+L2*+A43C+I5V、R181P+V115I+L2*+I55C+N79A、R181P+V115I+L2*+I55C+I5V、R181P+V115I+L2*+N79A+I5V、R181P+V115I+A43C+ I55C+N79A, R181P+V115I+A43C+I55C+I5V, R181P+V115I+A43C+N79A+I5V, R181P+V115I+I55C+N79A+I5V, R181P+A161L+Q1*+L2*+A43C, R181P+A161L+ Q1*+L2*+I55C、R181P+A161L+Q1*+L2*+N79A、R181P+A161L+Q1*+L2*+I5V、 R181P+A161L+Q1*+A43C+I55C、R181P+A161L+Q1*+A43C+N79A、R181P+A161 L+Q1*+A43C+I5V、R181P+A161L+Q1*+I55C+N79A、R181P+A161L+Q1*+I55C+ I5V、R181P+A161L+Q1*+N79A+I5V、R181P+A161L+L2*+A43C+I55C、R181P+A 161L+L2*+A43C+N79A、R181P+A161L+L2*+A43C+I5V、R181P+A161L+L2*+I55C+N79A、R181P+A161L+L2*+I55C+I5V、R181P+A161L+L2*+N79A+I5V、R181 P+A161L+A43C+I55C+N79A、R181P+A161L+A43C+I55C+I5V、R181P+A161L+A 43C+N79A+I5V、R181P+A161L+I55C+N79A+I5V、R181P+Q1*+L2*+A43C+I55C、R181P+Q1*+L2*+A43C+N79A, R181P+Q1*+L2*+A43C+I5V, R181P+Q1*+L2*+I55C+N79A, R181P+Q1*+L2*+I55C+I5V, R181P+Q1*+L2*+N79A+I5V, R181P+Q 1*+A43C+I55C+N79A、R181P+Q1*+A43C+I55C+I5V、R181P+Q1*+A43C+N79A+I5V、R181P+Q1*+I55C+N79A+I5V、R181P+L2*+A43C+I55C+N79A、R181P+L2* +A43C+I55C+I5V, R181P+L2*+A43C+N79A+I5V, R181P+L2*+I55C+N79A+I5V, R181P+A43C+I55C+N79A+I5V, G182*+V115I+A161L+Q1*+L2*, G182*+V115 I+A161L+Q1*+A43C, G182*+V115I+A161L+Q1*+I55C, G182*+V115I+A161L+Q1*+N79A, G182*+V115I+A161L+Q1*+I5V, G182*+V115I+A161L+L2*+A43C G182*+V115I+A161L+L2*+I55C, G182*+V115I+A161L+L2*+N79A, G182*+V115I+A161L+L2*+I5V, G182*+V115I+A161L+A43C+I55C, G182*+V115I+A161 L+A43C+N79A、G182*+V115I+A161L+A43C+I5V、G182*+V115I+A161L+I55C+ N79A、G182*+V115I+A161L+I55C+I5V、G182*+V115I+A161L+N79A+I5V、G18 2*+V115I+Q1*+L2*+A43C, G182*+V115I+Q1*+L2*+I55C, G182*+V115I+Q1*+L2*+N79A, G182*+V115I+Q1*+L2*+I5V, G182*+V115I+Q1*+A43C+I55C, G1 82*+V115I+Q1*+A43C+N79A、G182*+V115I+Q1*+A43C+I5V、G182*+V115I+Q 1*+I55C+N79A、G182*+V115I+Q1*+I55C+I5V、G182*+V115I+Q1*+N79A+I5V、G182*+V115I+L2*+A43C+I55C, G182*+V115I+L2*+A43C+N79A, G182*+V115I+L2*+A43C+I5V, G182*+V115I+L2*+I55C+N79A, G182*+V115I+L2*+I5 5C+I5V, G182*+V115I+L2*+N79A+I5V, G182*+V115I+A43C+I55C+N79A, G182*+V115I+A43C+I55C+I5V, G182*+V115I+A43C+N79A+I5V, G182*+V115 I+I55C+N79A+I5V, G182*+A161L+Q1*+L2*+A43C, G182*+A161L+Q1*+L2*+I55C, G182*+A161L+Q1*+L2*+N79A, G182*+A161L+Q1*+L2*+I5V, G182*+ A161L+Q1*+A43C+I55C、G182*+A161L+Q1*+A43C+N79A、G182*+A161L+Q1* +A43C+I5V、G182*+A161L+Q1*+I55C+N79A、G182*+A161L+Q1*+I55C+I5V、

[0094] G182*+A161L+Q1*+N79A+I5V, G182*+A161L+L2*+A43C+I55C, G182*+A161L+L2*+A43C+N79A, G182*+A161L+L2*+A43C+I5V, G182*+A161L+L2*+I55C+N 79A, G182*+A161L+L2*+I55C+I5V, G182*+A161L+L2*+N79A+I5V, G182*+A161L+A43C+I55C+N79A, G182*+A161L+A43C+I55C+I5V, G182*+A161L+A43C+N 79A+I5V, G182*+A161L+I55C+N 79A+I5V, G182*+Q1*+L2*+A43C+I55C, G182*+Q1*+L2*+A43C+N 79A, G182*+Q1*+L2*+A43C+I5V, G182*+Q1*+L2*+I55C+N 79A, G182*+Q1*+L2*+I55C+I5V, G182*+Q1*+L2*+N79A+I5V, G182*+Q1*+A43C+I55C+N79A, G182*+Q1*+A43C+I55C+I5V, G182*+Q1*+A43C+N79A+I5V, G1 82*+Q1*+I55C+N79A+I5V、G182*+L2*+A43C+I55C+N79A、G182*+L2*+A43C+I55C+I5V、G182*+L2*+A43C+N79A+I5V、G182*+L2*+I55C+N79A+I5V、G182* +A43C+I55C+N79A+I5V、V115I+A161L+Q1*+L2*+A43C、V115I+A161L+Q1*+L2*+I55C、V115I+A161L+Q1*+L2*+N79A、V115I+A161L+Q1*+L2*+I5V、V115I+ A161L+Q1*+A43C+I55C、V115I+A161L+Q1*+A43C+N79A、V115I+A161L+Q1*+ A43C+I5V、V115I+A161L+Q1*+I55C+N79A、V115I+A161L+Q1*+I55C+I5V、V11 5I+A161L+Q1*+N79A+I5V、V115I+A161L+L2*+A43C+I55C、V115I+A161L+L2*+A43C+N79A、V115I+A161L+L2*+A43C+I5V、V115I+A161L+L2*+I55C+N79A、V115I+A161L+L2*+I55C+I5V, V115I+A161L+L2*+N79A+I5V, V115I+A161L+A43C+I55C+N79A, V115I+A161L+A43C+I55C+I5V, V115I+A161L+A43C+N79 A+I5V, V115I+A161L+I55C+N79A+I5V, V115I+Q1*+L2*+A43C+I55C, V115I+Q1*+L2*+A43C+N79A, V115I+Q1*+L2*+A43C+I5V, V115I+Q1*+L2*+I55C+N 79A, V115I+Q1*+L2*+I55C+I5V, V115I+Q1*+L2*+N79A+I5V, V115I+Q1*+A43C+I55C+N79A, V115I+Q1*+A43C+I55C+I5V, V115I+Q1*+A43C+N79A+I5V V115I+Q1*+I55C+N79A+I5V, V115I+L2*+A43C+I55C+N79A, V115I+L2*+A43C+I55C+I5V, V115I+L2*+A43C+N79A+I5V, V115I+L2*+I55C+N79A+I5V, V11 5I+A43C+I55C+N79A+I5V, A161L+Q1*+L2*+A43C+I55C, A161L+Q1*+L2*+A43C+N79A, A161L+Q1*+L2*+A43C+I5V, A161L+Q1*+L2*+I55C+N79A, A161L+ Q1*+L2*+I55C+I5V, A161L+Q1*+L2*+N79A+I5V, A161L+Q1*+A43C+I55C+N79A, A161L+Q1*+A43C+I55C+I5V, A161L+Q1*+A43C+N79A+I5V, A161L+Q1*+I 55C+N79A+I5V, A161L+L2*+A43C+I55C+N79A, A161L+L2*+A43C+I55C+I5V, A161L+L2*+A43C+N79A+I5V, A161L+L2*+I55C+N79A+I5V, A161L+A43C+I5 5C+N79A+I5V, Q1*+L2*+A43C+I55C+N79A, Q1*+L2*+A43C+I55C+I5V, Q1*+L2*+A43C+N79A+I5V, Q1*+L2*+I55C+N79A+I5V, Q1*+A43C+I55C+N79A+I5VL2*+A43C+I55C+N79A+I5V or composed of these.

[0095] On the other hand, this variant includes SEQ ID NO: Substitution of mature polypeptides of 2: R181P+G182*+V115I+A161L+Q1*+L2*, R181P+G182*+V115I+A161L+Q1*+A43C, R181P+G182*+V115I+A161L+Q1*+I55C, R181P+G182*+V115I+A161L+Q1*+N79A, R181P+G182*+V115I+A161L+Q1*+I5V, R181P+G182*+V115I+A161L+L2*+A43C, R181P+G182*+V115I+A161L+L2*+I55C, R181P+ G182*+V115I+A161L+L2*+N79A, R181P+G182*+V115I+A161L+L2*+I5V, R 181P+G182*+V115I+A161L+A43C+I55C, R181P+G182*+V115I+A161L+A43C +N79A、R181P+G182*+V115I+A161L+A43C+I5V、R181P+G182*+V115I+A161 L+I55C+N79A, R181P+G182*+V115I+A161L+I55C+I5V, R181P+G182*+V115 I+A161L+N79A+I5V, R181P+G182*+V115I+Q1*+L2*+A43C, R181P+G182*+ V115I+Q1*+L2*+I55C, R181P+G182*+V115I+Q1*+L2*+N79A, R181P+G182* +V115I+Q1*+L2*+I5V、R181P+G182*+V115I+Q1*+A43C+I55C、R181P+G182 *+V115I+Q1*+A43C+N79A, R181P+G182*+V115I+Q1*+A43C+I5V, R181P+G1 82*+V115I+Q1*+I55C+N79A, R181P+G182*+V115I+Q1*+I55C+I5V, R181P+ G182*+V115I+Q1*+N79A+I5V, R181P+G182*+V115I+L2*+A43C+I55C, R181 P+G182*+V115I+L2*+A43C+N79A, R181P+G182*+V115I+L2*+A43C+I5V, R1 81P+G182*+V115I+L2*+I55C+N79A, R181P+G182*+V115I+L2*+I55C+I5V,R181P+G182*+V115I+L2*+N79A+I5V, 181P+G182*+V115I+A43C+I55C+N79A, R181P+G182*+V115I+A43C+I55C+I5V, 181P+G182*+V115I+A43C+N79A+I5 V, R181P+G182*+V115I+I55C+N79A+I5V, R181P+G182*+A161L+Q1*+L2*+A43C, R181P+G182*+A161L+Q1*+L2*+I55C, R181P+G182*+A161L+Q1*+L2*+N7 9A, R181P+G182*+A161L+Q1*+L2*+I5V, R181P+G182*+A161L+Q1*+A43C+I55C, R181P+G182*+A161L+Q1*+A43C+N79A, R181P+G182*+A161L+Q1*+A43C+ I5V, R181P+G182*+A161L+Q1*+I55C+N79A, R181P+G182*+A161L+Q1*+I55C+I5V, R181P+G182*+A161L+Q1*+N79A+I5V, R181P+G182*+A161L+L2*+A43C+ I55C, R181P+G182*+A161L+L2*+A43C+N79A, R181P+G182*+A161L+L2*+A43C+I5V, 181P+G182*+A161L+L2*+I55C+N79A, R181P+G182*+A161L+L2*+I55 C+I5V, R181P+G182*+A161L+L2*+N79A+I5V, 181P+G182*+A161L+A43C+I55C+N79A, R181P+G182*+A161L+A43C+I55C+I5V, R181P+G182*+A161L+A43C+ N79A+I5V, R181P+G182*+A161L+I55C+N79A+I5V, R181P+G182*+Q1*+L2*+A43C+I55C, R181P+G182*+Q1*+L2*+A43C+N79A, R181P+G182*+Q1*+L2*+A43 C+I5V, R181P+G182*+Q1*+L2*+I55C+N79A, R181P+G182*+Q1*+L2*+I55C+I5V, R181P+G182*+Q1*+L2*+N79A+I5V, R181P+G182*+Q1*+A43C+I55C+N79AR181P+G182*+Q1*+A43C+I55C+I5V, R181P+G182*+Q1*+A43C+N79A+I5V, R181P+G182*+Q1*+I55C+N79A+I5V, R181P+G182*+L2*+A43C+I55C+N79A, R1 81P+G182*+L2*+A43C+I55C+I5V、R181P+G182*+L2*+A43C+N79A+I5V、R181P+G182*+L2*+I55C+N79A+I5V、R181P+G182*+A43C+I55C+N79A+I5V、R181P +V115I+A161L+Q1*+L2*+A43C、R181P+V115I+A161L+Q1*+L2*+I55C、R181P +V115I+A161L+Q1*+L2*+N79A、R181P+V115I+A161L+Q1*+L2*+I5V、R181P+ V115I+A161L+Q1*+A43C+I55C、R181P+V115I+A161L+Q1*+A43C+N79A、R181 P+V115I+A161L+Q1*+A43C+I5V、R181P+V115I+A161L+Q1*+I55C+N79A、R18 1P+V115I+A161L+Q1*+I55C+I5V, R181P+V115I+A161L+Q1*+N79A+I5V, R181P+V115I+A161L+L2*+A43C+I55C, R181P+V115I+A161L+L2*+A43C+N79A R181P+V115I+A161L+L2*+A43C+I5V, R181P+V115I+A161L+L2*+I55C+N79A, R181P+V115I+A161L+L2*+I55C+I5V, R181P+V115I+A161L+L2*+N79A+I5V 、181P+V115I+A161L+A43C+I55C+N79A、R181P+V115I+A161L+A43C+I55C+I 5V、R181P+V115I+A161L+A43C+N79A+I5V、R181P+V115I+A161L+I55C+N79A +I5V, R181P+V115I+Q1*+L2*+A43C+I55C, R181P+V115I+Q1*+L2*+A43C+N79A, R181P+V115I+Q1*+L2*+A43C+I5V, R181P+V115I+Q1*+L2*+I55C+N79AR181P+V115I+Q1*+L2*+I55C+I5V, R181P+V115I+Q1*+L2*+N79A+I5V, R181P+V115I+Q1*+A43C+I55C+N79A, R181P+V115I+Q1*+A43C+I55C+I5V, R18 1P+V115I+Q1*+A43C+N79A+I5V、R181P+V115I+Q1*+I55C+N79A+I5V、R181P+V115I+L2*+A43C+I55C+N79A、R181P+V115I+L2*+A43C+I55C+I5V、R181P +V115I+L2*+A43C+N79A+I5V、R181P+V115I+L2*+I55C+N79A+I5V、R181P+V115I+A43C+I55C+N79A+I5V、R181P+A161L+Q1*+L2*+A43C+I55C、R181P+A 161L+Q1*+L2*+A43C+N79A、R181P+A161L+Q1*+L2*+A43C+I5V、R181P+A161L+Q1*+L2*+I55C+N79A、R181P+A161L+Q1*+L2*+I55C+I5V、R181P+A161L+ Q1*+L2*+N79A+I5V, R181P+A161L+Q1*+A43C+I55C+N79A, R181P+A161L+Q1*+A43C+I55C+I5V, R181P+A161L+Q1*+A43C+N79A+I5V, R181P+A161L+Q1* +I55C+N79A+I5V、R181P+A161L+L2*+A43C+I55C+N79A、R181P+A161L+L2*+A43C+I55C+I5V、R181P+A161L+L2*+A43C+N79A+I5V、R181P+A161L+L2*+I 55C+N79A+I5V, R181P+A161L+A43C+I55C+N79A+I5V, R181P+Q1*+L2*+A43C+I55C+N79A, R181P+Q1*+L2*+A43C+I55C+I5V, R181P+Q1*+L2*+A43C+N79 A+I5V, R181P+Q1*+L2*+I55C+N79A+I5V, R181P+Q1*+A43C+I55C+N79A+I5V, R181P+L2*+A43C+I55C+N79A+I5V, G182*+V115I+A161L+Q1*+L2*+A43CG182*+V115I+A161L+Q1*+L2*+I55C, G182*+V115I+A161L+Q1*+L2*+N79A, G182*+V115I+A161L+Q1*+L2*+I5V, G182*+V115I+A161L+Q1*+A43C+I55C 、G182*+V115I+A161L+Q1*+A43C+N79A、G182*+V115I+A161L+Q1*+A43C+I 5V、G182*+V115I+A161L+Q1*+I55C+N79A、G182*+V115I+A161L+Q1*+I55C+ I5V, G182*+V115I+A161L+Q1*+N79A+I5V, G182*+V115I+A161L+L2*+A43C+I55C, G182*+V115I+A161L+L2*+A43C+N79A, G182*+V115I+A161L+L2*+A4 3C+I5V, G182*+V115I+A161L+L2*+I55C+N79A, G182*+V115I+A161L+L2*+I55C+I5V, G182*+V115I+A161L+L2*+N79A+I5V, 182*+V115I+A161L+A43C+I 55C+N79A, G182*+V115I+A161L+A43C+I55C+I5V, G182*+V115I+A161L+A43C+N79A+I5V, G182*+V115I+A161L+I55C+N79A+I5V, G182*+V115I+Q1*+L2 *+A43C+I55C、G182*+V115I+Q1*+L2*+A43C+N79A、G182*+V115I+Q1*+L2*+A43C+I5V、G182*+V115I+Q1*+L2*+I55C+N79A、G182*+V115I+Q1*+L2*+I55 C+I5V, G182*+V115I+Q1*+L2*+N79A+I5V, G182*+V115I+Q1*+A43C+I55C+N79A, G182*+V115I+Q1*+A43C+I55C+I5V, G182*+V115I+Q1*+A43C+N79A+I 5V, G182*+V115I+Q1*+I55C+N79A+I5V, G182*+V115I+L2*+A43C+I55C+N79A, G182*+V115I+L2*+A43C+I55C+I5V, G182*+V115I+L2*+A43C+N79A+I5VG182*+V115I+L2*+I55C+N79A+I5V, G182*+V115I+A43C+I55C+N79A+I5V, G182*+A161L+Q1*+L2*+A43C+I55C, G182*+A161L+Q1*+L2*+A43C+N79A, G 182*+A161L+Q1*+L2*+A43C+I5V, G182*+A161L+Q1*+L2*+I55C+N79A, G182*+A161L+Q1*+L2*+I55C+I5V, G182*+A161L+Q1*+L2*+N79A+I5V, G182*+A 161L+Q1*+A43C+I55C+N79A、G182*+A161L+Q1*+A43C+I55C+I5V、G182*+A 161L+Q1*+A43C+N79A+I5V、G182*+A161L+Q1*+I55C+N79A+I5V、G182*+A1 61L+L2*+A43C+I55C+N79A, G182*+A161L+L2*+A43C+I55C+I5V, G182*+A161L+L2*+A43C+N79A+I5V, G182*+A161L+L2*+I55C+N79A+I5V, G182*+A161 L+A43C+I55C+N79A+I5V, G182*+Q1*+L2*+A43C+I55C+N79A, G182*+Q1*+L2*+A43C+I55C+I5V, G182*+Q1*+L2*+A43C+N79A+I5V, G182*+Q1*+L2*+I5 5C+N79A+I5V, G182*+Q1*+A43C+I55C+N79A+I5V, G182*+L2*+A43C+I55C+N79A+I5V, V115I+A161L+Q1*+L2*+A43C+I55C, V115I+A161L+Q1*+L2*+A43 C+N79A, V115I+A161L+Q1*+L2*+A43C+I5V, V115I+A161L+Q1*+L2*+I55C+N79A, V115I+A161L+Q1*+L2*+I55C+I5V, V115I+A161L+Q1*+L2*+N79A+I5 V、V115I+A161L+Q1*+A43C+I55C+N79A、V115I+A161L+Q1*+A43C+I55C+I5 V、V115I+A161L+Q1*+A43C+N79A+I5V、V115I+A161L+Q1*+I55C+N79A+I5V、V115I + A161L + L2* + A43C + I55C + N79A, V115I + A161L + L2* + A43C + I55C + I5V, V115I + A161L + L2* + A43C + N79A + I5V, V115I + A161L + L2* + I55C + N79A + I5V, V115I + A161L + A43C + I55C + N79A + I5V, V115I + Q1* + L2* + A43C + I55C + N79A, V115I + Q1* + L2* + A43C + I55C + I5V, V115I + Q1* + L2* + A43C + N79A + I5V, V115I + Q1* + L2* + I55C + N79A + I5V, V115I + Q1* + A43C + I55C + N79A + I5V, V115I + L2* + A43C + I55C + N79A + I5V, A161L + Q1* + L2* + A43C + I55C + N79A, A161L + Q1* + L2* + A43C + I55C + I5V, A161L + Q1* + L2* + A43C + N79A + I5V, A161L + Q1* + L2* + I55C + N79A + I5V, A161L + Q1* + A43C + I55C + N79A + I5V, A161L + L2* + A43C + I55C + N79A + I5V, Q1* + L2* + A43C + I55C + N79A + I5V or composed thereof.

[0096] On the other hand, this variant includes SEQ ID NO: Substitution of mature peptides of 2: R181P+G182*+V115I+A161L+Q1*+L2*+A43C, R181P+G182*+V115I+A161L+Q1*+L2*+I55C, R181P+G182*+V115I+A161L+Q1*+L2*+N79A, R181P+G182*+V115I+A161L+Q1*+L2*+I5V, R181P+G182*+V115I+A161L+Q1*+A43C+I55C, R181P+G182*+V115I+A161L+Q1*+A43C+N79A, R181P+G182* +V115I+A161L+Q1*+A43C+I5V、R181P+G182*+V115I+A161L+Q1*+I55C+N 79A, R181P+G182*+V115I+A161L+Q1*+I55C+I5V, R181P+G182*+V115I+A1 61L+Q1*+N79A+I5V, R181P+G182*+V115I+A161L+L2*+A43C+I55C, R181P+ G182*+V115I+A161L+L2*+A43C+N79A, R181P+G182*+V115I+A161L+L2*+A 43C+I5V, R181P+G182*+V115I+A161L+L2*+I55C+N79A, R181P+G182*+V1 15I+A161L+L2*+I55C+I5V, R181P+G182*+V115I+A161L+L2*+N79A+I5V, R 181P+G182*+V115I+A161L+A43C+I55C+N79A, R181P+G182*+V115I+A161L +A43C+I55C+I5V, R181P+G182*+V115I+A161L+A43C+N79A+I5V, R181P+G1 82*+V115I+A161L+I55C+N79A+I5V, R181P+G182*+V115I+Q1*+L2*+A43C +I55C、R181P+G182*+V115I+Q1*+L2*+A43C+N79A、R181P+G182*+V115I+Q 1*+L2*+A43C+I5V, R181P+G182*+V115I+Q1*+L2*+I55C+N79A, R181P+G18 2*+V115I+Q1*+L2*+I55C+I5V, R181P+G182*+V115I+Q1*+L2*+N79A+I5V,R181P+G182*+V115I+Q1*+A43C+I55C+N79A、R181P+G182*+V115I+Q1*+A 43C+I55C+I5V、R181P+G182*+V115I+Q1*+A43C+N79A+I5V、R181P+G182*+ V115I+Q1*+I55C+N79A+I5V, R181P+G182*+V115I+L2*+A43C+I55C+N79A, R181P+G182*+V115I+L2*+A43C+I55C+I5V, R181P+G182*+V115I+L2*+A4 3C+N79A+I5V, R181P+G182*+V115I+L2*+I55C+N79A+I5V, R181P+G182*+V115I+A43C+I55C+N79A+I5V, R181P+G182*+A161L+Q1*+L2*+A43C+I55C R181P+G182*+A161L+Q1*+L2*+A43C+N79A, R181P+G182*+A161L+Q1*+L2*+A43C+I5V, R181P+G182*+A161L+Q1*+L2*+I55C+N79A, R181P+G182*+A16 1L+Q1*+L2*+I55C+I5V, R181P+G182*+A161L+Q1*+L2*+N79A+I5V, R181P+G182*+A161L+Q1*+A43C+I55C+N79A, R181P+G182*+A161L+Q1*+A43C+I5 5C+I5V, R181P+G182*+A161L+Q1*+A43C+N79A+I5V, R181P+G182*+A161L+Q1*+I55C+N79A+I5V, R181P+G182*+A161L+L2*+A43C+I55C+N79A, R181P +G182*+A161L+L2*+A43C+I55C+I5V、R181P+G182*+A161L+L2*+A43C+N79A+I5V、R181P+G182*+A161L+L2*+I55C+N79A+I5V、R181P+G182*+A161L+ A43C+I55C+N79A+I5V, R181P+G182*+Q1*+L2*+A43C+I55C+N79A, R181P+G182*+Q1*+L2*+A43C+I55C+I5V, R181P+G182*+Q1*+L2*+A43C+N79A+I5VR181P+G182*+Q1*+L2*+I55C+N79A+I5V, R181P+G182*+Q1*+A43C+I55C+N79A+I5V, R181P+G182*+L2*+A43C+I55C+N79A+I5V, R181P+V115I+A161L+Q 1*+L2*+A43C+I55C、R181P+V115I+A161L+Q1*+L2*+A43C+N79A、R181P+V115I+A161L+Q1*+L2*+A43C+I5V、R181P+V115I+A161L+Q1*+L2*+I55C+N79A R181P+V115I+A161L+Q1*+L2*+I55C+I5V, R181P+V115I+A161L+Q1*+L2*+N79A+I5V, R181P+V115I+A161L+Q1*+A43C+I55C+N79A, R181P+V115I+A16 1L+Q1*+A43C+I55C+I5V, R181P+V115I+A161L+Q1*+I55C+N79A+I5V, R181P+V115I+A161L+L2*+A43C+I55C+N79A, R181P+V115I+A161L+L2*+A43C+I55 C+I5V, R181P+V115I+A161L+L2*+A43C+N79A+I5V, R181P+V115I+A161L+L2*+I55C+N79A+I5V, R181P+V115I+A161L+A43C+I55C+N79A+I5V, R181P+V1 15I+Q1*+L2*+A43C+I55C+N79A、R181P+V115I+Q1*+L2*+A43C+I55C+I5V、R181P+V115I+Q1*+L2*+A43C+N79A+I5V、R181P+V115I+Q1*+L2*+I55C+N79A +I5V, R181P+V115I+Q1*+A43C+I55C+N79A+I5V, R181P+V115I+L2*+A43C+I55C+N79A+I5V, R181P+A161L+Q1*+L2*+A43C+I55C+N79A, R181P+A161L+Q 1*+L2*+A43C+I55C+I5V、R181P+A161L+Q1*+L2*+A43C+N79A+I5V、R181P+A161L+Q1*+L2*+I55C+N79A+I5V、R181P+A161L+Q1*+A43C+I55C+N79A+I5V、R181P+A161L+L2*+A43C+I55C+N79A+I5V, R181P+Q1*+L2*+A43C+I55C+N79A+I5V, G182*+V115I+A161L+Q1*+L2*+A43C+I55C, G182*+V115I+A161L+Q 1*+L2*+A43C+N79A, G182*+V115I+A161L+Q1*+L2*+A43C+I5V, G182*+V115I+A161L+Q1*+L2*+I55C+N79A, G182*+V115I+A161L+Q1*+L2*+I55C+I5V G182*+V115I+A161L+Q1*+L2*+N79A+I5V, G182*+V115I+A161L+Q1*+A43C+I55C+N79A, G182*+V115I+A161L+Q1*+A43C+I55C+I5V, G182*+V115I+A16 1L+Q1*+A43C+N79A+I5V, G182*+V115I+A161L+Q1*+I55C+N79A+I5V, G182*+V115I+A161L+L2*+A43C+I55C+N79A, G182*+V115I+A161L+L2*+A43C+I55 C+I5V, G182*+V115I+A161L+L2*+A43C+N79A+I5V, G182*+V115I+A161L+L2*+I55C+N79A+I5V, G182*+V115I+A161L+A43C+I55C+N79A+I5V, G182*+V1 15I+Q1*+L2*+A43C+I55C+N79A、G182*+V115I+Q1*+L2*+A43C+I55C+I5V、G182*+V115I+Q1*+L2*+A43C+N79A+I5V、G182*+V115I+Q1*+L2*+I55C+N79A +I5V, G182*+V115I+Q1*+A43C+I55C+N79A+I5V, G182*+V115I+L2*+A43C+I55C+N79A+I5V, G182*+A161L+Q1*+L2*+A43C+I55C+N79A, G182*+A161L+Q 1*+L2*+A43C+I55C+I5V, G182*+A161L+Q1*+L2*+A43C+N79A+I5V, G182*+A161L+Q1*+L2*+I55C+N79A+I5V, G182*+A161L+Q1*+A43C+I55C+N79A+I5VG182*+A161L+L2*+A43C+I55C+N79A+I5V, G182*+Q1*+L2*+A43C+I55C+N79A+I5V, V115I+A161L+Q1*+L2*+A43C+I55C+N79A, V115I+A161L+Q1*+L2*+A43C+I55C+I5V, V115I+A161L+Q1*+L2*+A43C+N79A+I5V, V115I+A161L+Q1*+L2*+I55C+N79A+I5V, V115I+A161L+Q1*+A43C+I55C+N79A+I5V, V115I+A161L+L2*+A43C+I55C+N79A+I5V, V115I+Q1*+L2*+A43C+I55C+N79A+I5V, A161L+Q1*+L2*+A43C+I55C+N79A+I5V or composed of them.

[0097] On the other hand, this variant includes SEQ ID NO: Substitution of mature peptides of 2: R181P+G182*+V115I+A161L+Q1*+L2*+A43C+I55C, R181P+G182*+V115I+A161L+Q1*+L2*+A43C+N79A, R181P+G182*+V115I+A161L+Q1*+L2*+A43C+I5V, R181P+G182*+V115I+A161L+Q1*+L2*+I55C+N79A, R181P+G182*+V115I+A161L+Q1*+L2*+I55C+I5V, R181P+G182*+V115I+A161L +Q1*+L2*+N79A+I5V, R181P+G182*+V115I+A161L+Q1*+A43C+I55C+N79A , R181P+G182*+V115I+A161L+Q1*+A43C+I55C+I5V, R181P+G182*+V115I +A161L+Q1*+A43C+N79A+I5V、R181P+G182*+V115I+A161L+Q1*+I55C+N7 9A+I5V, R181P+G182*+V115I+A161L+L2*+A43C+I55C+N79A, R181P+G182* +V115I+A161L+L2*+A43C+I55C+I5V、R181P+G182*+V115I+A161L+L2*+A 43C+N79A+I5V, R181P+G182*+V115I+A161L+L2*+I55C+N79A+I5V, R181P +G182*+V115I+A161L+A43C+I55C+N79A+I5V、R181P+G182*+V115I+Q1*+ L2*+A43C+I55C+N79A, R181P+G182*+V115I+Q1*+L2*+A43C+I55C+I5V, R1 81P+G182*+V115I+Q1*+L2*+A43C+N79A+I5V, R181P+G182*+V115I+Q1*+ L2*+I55C+N79A+I5V, R181P+G182*+V115I+Q1*+A43C+I55C+N79A+I5V, R 181P+G182*+V115I+L2*+A43C+I55C+N79A+I5V, R181P+G182*+A161L+Q1 *+L2*+A43C+I55C+N79A, R181P+G182*+A161L+Q1*+L2*+A43C+I55C+I5V,R181P + G182* + A161L + Q1* + L2* + A43C + N79A + I5V, R181P + G182* + A161L + Q1* + L2* + I55C + N79A + I5V, R181P + G182* + A161L + Q1* + A43C + I55C + N79A + I5V, R181P + G182* + A161L + L2* + A43C + I55C + N79A + I5V, R181P + G182* + Q1* + L2* + A43C + I55C + N79A + I5V, R181P + V115I + A161L + Q1* + L2* + A43C + I55C + N79A, R181P + V115I + A161L + Q1* + L2* + A43C + I55C + I5V, R181P + V115I + A161L + Q1* + L2* + A43C + N79A + I5V, R181P + V115I + A161L + Q1* + L2* + I55C + N79A + I5V, R181P + V115I + A161L + Q1* + A43C + I55C + N79A + I5V, R181P + V115I + A161L + L2* + A43C + I55C + N79A + I5V, R181P + V115I + Q1* + L2* + A43C + I55C + N79A + I5V, R181P + A161L + Q1* + L2* + A43C + I55C + N79A + I5V, G182* + V115I + A161L + Q1* + L2* + A43C + I55C + N79A, G182* + V115I + A161L + Q1* + L2* + A43C + I55C + I5V, G182* + V115I + A161L + Q1* + L2* + A43C + N79A + I5V, G182* + V115I + A161L + Q1* + L2* + I55C + N79A + I5V, G182* + V115I + A161L + Q1* + A43C + I55C + N79A + I5V, G182* + V115I + A161L + L2* + A43C + I55C + N79A + I5V, G182* + V115I + Q1* + L2* + A43C + I55C + N79A + I5V, G182* + A161L + Q1* + L2* + A43C + I55C + N79A + I5V, V115I + A161L + Q1* + L2* + A43C + I55C + N79A + I5V or composed of them.,

[0098] On the other hand, this variant includes the mature peptide of SEQ ID NO: 2, specifically R181P+G182*+V115I+A161L+Q1*+L2*+A43C+I55C+N79A, R181P+G182*+V115I+A161L+Q1*+L2*+A43C+I55C+I5V, R181P+G182*+V115I+A161L+Q1*+L2*+A43C+N79A+I5V, R181P+G182*+V115I+A161L+Q1*+A43C+I55C+N79A+I5V, and R181P+G182*+V115I+A161L+Q1*+A43C+I55C+N79A+ I5V, R181P+G182*+V115I+A161L+L2*+A43C+I55C+N79A+I5V, R181P+G 182*+V115I+Q1*+L2*+A43C+I55C+N79A+I5V, R181P+G182*+A161L+Q1* +L2*+A43C+I55C+N79A+I5V、R181P+V115I+A161L+Q1*+L2*+A43C+I55C +N79A+I5V, G182*+V115I+A161L+Q1*+L2*+A43C+I55C+N79A+I5V or their combinations.

[0099] On the other hand, the variant includes or consists of the mature polypeptide of SEQ ID NO: 2, substituted with R181P+G182*+V115I+A161L+Q1*+L2*+A43C+I55C+N79A+I5V.

[0100] These variants may further include one or more additional changes in one or more other locations (e.g., several).

[0101] These amino acid changes can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions typically of 1–30 amino acids; small amino- or carboxyl-terminal extensions, such as methionine residues at the amino terminus; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function, such as polyhistidine tracts, antigenic epitopes, or binding domains.

[0102] Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific reactivity are known in the art and are described, for example, by H. Neurath and R.R. Hill, 1979, in *The Proteins*, Academic Press, New York. Common substitutes are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0103] Alternatively, amino acid alterations have the property of changing the physicochemical properties of peptides. For example, amino acid alterations can improve the thermal stability of peptides, change substrate specificity, change the optimal pH, etc.

[0104] Essential amino acids in peptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the resulting mutant molecule is tested for keratinase activity to identify amino acid residues essential for the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem., 271: 4699-4708. Alternatively, mutations in amino acids at hypothetical contact sites can be combined with physical analysis of the structure, such as by techniques like NMR, crystallography, electron diffraction, or photoaffinity labeling, to determine the enzyme's active site or other biological interactions. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J.Mol.Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. Essential amino acids can also be identified by comparison with related peptides.

[0105] In one aspect, the variant may consist of or include at least 172 amino acid residues (e.g., amino acids 17 to 188 of SEQ ID NO: 2). In another aspect, the variant comprises 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% of the number of amino acids in SEQ ID NO: 2, but less than 100%. In another aspect, the variant comprises at least 172 amino acid residues of the number of amino acids in SEQ ID NO: 2 (e.g., amino acids 17 to 188 of SEQ ID NO: 2) and 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%.

[0106] In some aspects, the present invention relates to variants including an N-terminal extension. This extension may constitute a position corresponding to amino acid -17 to -1 or a truncated portion thereof, as specified in SEQ ID NO: 2. In some aspects, the present invention relates to a variant wherein the N-terminal extension is selected from: (a) AAVDSNHTPAVPELVAR; (b) AVDSNHTPAVPELVAR; (c) VDSNHTPAVPELVAR; (d) DSNHTPAVPELVAR; (e) SNHTPAVPELVAR; (f) NHTPAVPELVAR; (g) HTPAVPELVAR; (h) TPAVPELVAR; (i) PAVPELVAR; (j) AVPELVAR; (k); (l) PELVAR; (m) PELVAR; (n) ELVAR; (o) LVAR; (p) VAR; (q) AR; or (r) R. Variants that include deletions (e.g., Q1*, L2*, or Q1*+L2*) at one or more (e.g., several) positions of amino acids 1 and / or 2 corresponding to SEQ ID NO: 2 may have different processing of the propeptide region. In some aspects of the invention, this can produce variants that include both deletions (e.g., Q1*, L2*, or Q1*+L2*) and N-terminal extensions at one or more (e.g., several) positions corresponding to amino acids 1 and / or 2 of SEQ ID NO: 2, the N-terminal extensions being selected from: (a) AAVDSNHTPAVPELVAR; (b) AVDSNHTPAVPELVAR; (c) VDSNHTPAVPELVAR; (d) DSNHTPAVPELVAR; (e) SNHTPAVPELVAR; (f) NHTPAVPELVAR; (g) HTPAVPELVAR; (h) TPAVPELVAR; (i) PAVPELVAR; (j) AVPELVAR; (k); (l) PELVAR; (m) PELVAR; (n) ELVAR; (o) LVAR; (p) VAR; (q) AR; or (r) R.

[0107] On the one hand, this variant exhibits improved specific activity compared to the parent enzyme. Specific activity can be determined by hydrolyzing BETEB as described in Example 3, and / or by the increase in pH and / or OD change after hydrolyzing PET as described in Example 4.

[0108] On the one hand, this variant has improved substrate binding compared to the parent enzyme.

[0109] On one hand, this variant exhibits improved substrate cleavage compared to the parent enzyme. Substrate cleavage can be determined by hydrolysis of BETEB as described in Example 3, and / or by hydrolysis of PET followed by an increase in pH and / or OD changes as described in Example 4.

[0110] In one respect, this variant exhibits improved substrate specificity compared to the parent enzyme. Substrate specificity can be determined by hydrolyzing BETEB as described in Example 3, and / or by the increase in pH and / or OD change after hydrolyzing PET as described in Example 4.

[0111] In one respect, the variant exhibits improved thermal stability compared to the parent enzyme. Thermal stability can be determined by differential scanning calorimetry (DSC) or, as described in Example 3, by determining residual activity after incubation at a specified temperature, as measured by hydrolysis of BETEB, and / or, as described in Example 4, by the increase in pH and / or OD changes via hydrolysis of PET.

[0112] Thermal stability determined by differential scanning calorimetry (DSC) was performed using a VP-capillary differential scanning calorimeter (MicroCal Inc., Piscateway, NJ, USA). The denaturation temperature Td (°C) was considered the apex of the denaturation peak (predominantly endothermic) in the thermochromatogram (Cp vs. T), obtained after heating the enzyme solution (approximately 0.5 mg / mL) in buffer (50 mM Tris; 100 mM NaCl pH 9) at a constant programmed heating rate of 200 K / hr. Sample and reference solutions (approximately 0.2 mL) were loaded into the calorimeter from storage conditions at 10°C (reference: buffer without enzyme) and pre-equilibrated at 20°C for 20 min, followed by DSC scans from 20°C to 100°C. Denaturation temperatures were determined with an accuracy of + / -1°C.

[0113] On the one hand, this variant exhibits a reduced tendency to pill compared to the parent enzyme. The tendency to pill can be determined by performing a pilling record test, as described in Example 4.

[0114] Parental keratinase

[0115] The parental asparaginase can be (a) a polypeptide having at least 60% sequence identity with the mature polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes with the coding sequence of the mature polypeptide of SEQ ID NO: 1 or its full-length complement under low stringency conditions; or (c) a polypeptide encoded by a polynucleotide that has at least 60% sequence identity with the coding sequence of the mature polypeptide of SEQ ID NO: 1.

[0116] In one respect, the parent has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO: 2, which has keratinase activity. In another respect, the amino acid sequence of the parent differs from the mature polypeptide of SEQ ID NO: 2 by up to 20 amino acids, such as 1-15, 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0117] In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO: 2. In another aspect, the parent comprises or consists of the mature polypeptide of SEQ ID NO: 2. In another aspect, the parent comprises or consists of amino acids 1 to 194 of SEQ ID NO: 2.

[0118] In another aspect, the parent is a fragment of the mature polypeptide of SEQ ID NO: 2, the fragment comprising or including at least 172 amino acid residues (e.g., amino acids 17 to 188 of SEQ ID NO: 2). In another aspect, the fragment comprises 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% of the number of amino acids in SEQ ID NO: 2, but less than 100%. In another aspect, the fragment comprises at least 172 amino acid residues of the number of amino acids in SEQ ID NO: 2 (e.g., amino acids 17 to 188 of SEQ ID NO: 2) and 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%.

[0119] On the other hand, the parent is an allelic variant of the mature polypeptide of SEQ ID NO: 2.

[0120] On the other hand, the parent is encoded by a polynucleotide that hybridizes with the mature polypeptide coding sequence of (i) SEQ ID NO: 1 or its full-length complement under very low stringency, low stringency, medium stringency, medium-high stringency, high stringency or very high stringency conditions (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York).

[0121] Nucleic acid probes can be designed using the polynucleotide or a subsequence of SEQ ID NO: 1, together with the polypeptide or fragment of SEQ ID NO: 2, to identify and clone parental DNA encoding strains from different genera or species according to methods well known in the art. Specifically, such probes can be hybridized with genomic DNA or cDNA of cells of interest according to standard DNA blotting procedures to identify and isolate the corresponding genes therein. Such probes can be significantly shorter than the complete sequence, but should be at least 15, for example at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, for example at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, or at least 900 nucleotides. Both DNA and RNA probes can be used. Typically, the probes are labeled (e.g., with...). 32 P, 3 H, 35 (S, biotin, or avidin) to detect the corresponding gene. This invention covers such probes.

[0122] Genomic DNA or cDNA libraries prepared from other strains of this type can be screened for DNA that hybridizes to and encodes the parental DNA as described above. Genomic DNA or other DNA from these other strains can be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the library or separated DNA can be transferred and immobilized on nitrocellulose or other suitable vector materials. The vector material is used for DNA blotting to identify clones or DNA that hybridize to SEQ ID NO: 1 or its subsequences.

[0123] For the purposes of this invention, hybridization indicates that a polynucleotide hybridizes with a labeled nucleic acid probe under very low to very high stringency conditions, the probe corresponding to (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1; (iii) its full-length complementary sequence; or (iv) its subsequence. Molecules hybridizing with the nucleic acid probe under these conditions can be detected using, for example, X-ray film or any other detection method known in the art.

[0124] In one respect, the nucleic acid probe is the coding sequence of the mature polypeptide of SEQ ID NO: 1. In another respect, the nucleic acid probe is nucleotides 106 to 687 of SEQ ID NO: 1. In yet another respect, the nucleic acid probe is a polynucleotide encoding a polypeptide of SEQ ID NO: 2, its mature polypeptide, or a fragment thereof. In yet another respect, the nucleic acid probe is SEQ ID NO: 1.

[0125] On the other hand, the parent is encoded by a polynucleotide that has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide encoding sequence of SEQ ID NO: 1.

[0126] The polypeptide can be a hybrid polypeptide, in which a region of one polypeptide is fused to the N-terminus or C-terminus of a region of another polypeptide.

[0127] The parent peptide can be a fusion peptide or a cleavable fusion peptide, wherein another peptide is fused at the N-terminus or C-terminus of the peptide of the present invention. Fusion peptides are generated by fusing a polynucleotide encoding another peptide into the polynucleotide of the present invention. Techniques for generating fusion peptides are known in the art and include linking the coding sequences of the peptides such that they are in a frame and that the expression of the fusion peptide is under the control of one or more promoters and terminators. Fusion peptides can also be constructed using integrin technology, wherein the fusion peptide is generated post-translational (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

[0128] Fusion peptides may further include a cleavage site between the two peptides. Upon secretion of the fusion protein, this site is activated, thereby releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

[0129] The parent can be obtained from any genus of microorganisms. For the purposes of this invention, the term "obtained from" as used herein in conjunction with a given source should mean that the parent encoded by the polynucleotide is produced by that source or by a strain in which a polynucleotide from that source has been inserted. In one aspect, the parent is extracellularly secreted.

[0130] The parent can be a bacterial keratinase. For example, the parent can be a Gram-positive bacterial polypeptide, such as a keratinase from Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces; or a Gram-negative bacterial polypeptide, such as Campylobacter or Escherichia coli. Keratinases from the genera *Coli*, *Flavobacterium*, *Fusobacterium*, *Helicobacter*, *Ilyobacter*, *Neisseria*, *Pseudomonas*, *Salmonella*, or *Ureaplasma*.

[0131] On one hand, the parent is a alkalophilic Bacillus, amyloliquefaciens, brevis, circulans, clausii, coagulating Bacillus, firmus, lautus, lentus, licheniformis, megaterium, pumilus, stearothermophilus, subtilis, or thuringiensis keratinase.

[0132] In another respect, the parent is a keratinase of Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus lactis, or Streptococcus equi subsp. Zooepidemicus.

[0133] On the other hand, this parent does not produce keratinase from Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans.

[0134] The parent can be a bacterial keratinase. For example, the parent can be a yeast keratinase, such as a keratinase from the genera *Candida*, *Kluyveromyces*, *Pichia*, *Saccharomyces*, *Schizosaccharomyces*, or *Yarrowia*.Or a filamentous fungus with keratinase, such as *Acremonium*, *Agaricus*, *Alternaria*, *Aspergillus*, *Aureobasidium*, *Botryospaeria*, *Ceriporiopsis*, *Chaetomidium*, *Chrysosporium*, *Claviceps*, *Cochliobolus*, *Coprinopsis*, or *Coptis*. *Otermes*, *Corynascus*, *Cryphonectria*, *Cryptococcus*, *Diplodia*, *Exidia*, *Filibasidium*, *Fusarium*, *Gibberella*, *Holomastigotoides*, *Humicola*, *Irpex*, *Lentinula*, *Leptospaeria*, *Pyrethrum* Genus: Magnaporthe, Melanocarpus, Meriplus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudot *Rhizomucor*, *Schizophyllum*, *Scytalidium*, *Talaromyces*, *Thermoascus*, *Thielavia*, *Tolypocladium*, *Trichoderma*, *Trichophaea*, *Verticillium*, *Volvariella*, or *Xylaria* cutinase.

[0135] On the other hand, the parent is a cutinase from Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis.

[0136] On the other hand, the parent species are *Acremonium cellulolyticus*, *Aspergillus aculeatus*, *Aspergillus awamori*, *Aspergillus foetidus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Chrysosporium inops*, *Chrysosporium keratinophilum*, *Chrysosporium lucknowense*, *Chrysosporium merdarium*, *Chrysosporium pannicola*, and *Chrysosporium queenslandense*. Queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulfideum Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium sulphureumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thievia achromatica, Thievia albomyces, Thievia albopilosa, Thievia australeinsis, Thievia fimeti, Thievia microsporum microspora, Thievora ovispora, Thievora peruviana, Thievora setosa, Thievora spededonium, Thievora subthermophila, Thievora terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride keratinase.

[0137] On the other hand, the parent is a specific putrefactive fungal keratinase, or the keratinase of SEQ ID NO: 2, or its mature polypeptide. Alternatively, the parent can be a strain of *Rhizoctonia*, such as *Rhizoctonia solani*, or a strain of *Alternaria*, such as *Alternaria brassicicola* (WO94 / 03578). The keratinase can also be a variant of the parental keratinase, such as those described in WO 00 / 34450 or WO 01 / 92502.

[0138] It will be understood that, for the species mentioned above, this invention covers both perfect and imperfect states, as well as other taxonomic equivalents, such as asexual forms, regardless of their known species names. Those skilled in the art will readily identify the appropriate equivalents.

[0139] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Culture Collection (DSMZ), the Netherlands Culture Collection (CentraalbureauVoor Schimmelcultures, CBS), and the Northern Research Center (NRRL) of the Patent Culture Collection of the Agricultural Research Service.

[0140] The parent can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.), using the probes mentioned above. Techniques for directly isolating microorganisms and DNA from their natural environment are well known in the art. The polynucleotide encoding the parent can then be obtained by similarly screening a library of genomic DNA or cDNA from another microorganism or a mixed DNA sample. Once the polynucleotide encoding the parent is detected with one or more probes, it can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[0141] Preparation of variants

[0142] The present invention also relates to methods for obtaining variants having keratinase activity, the methods comprising: (a) introducing a change into a parental keratinase at one or more (e.g., several) positions corresponding to positions 181, 182, 115, 161, 1, 2, 43, 55, 79, or 5 of a mature polypeptide of SEQ ID NO: 2, wherein the change is a substitution for positions 181, 115, 161, 43, 55, 79, and 5, and a deletion for positions 1, 2, and 182, wherein the variant has at least 75%, 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 the mature polypeptide of SEQ ID NO: 2, and wherein the variant has keratinase activity; and (b) recovering the variant.

[0143] These 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.

[0144] Site-directed mutagenesis is a technique that introduces one or more (e.g., several) mutations at one or more designated sites in a polynucleotide encoding the parent.

[0145] Site-directed mutagenesis can be achieved in vitro using PCR involving primers containing oligonucleotides with the desired mutation. Site-directed mutagenesis can also be performed in vitro via cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid containing a polynucleotide encoding the parent and subsequent ligation of the mutated oligonucleotide into the polynucleotide. Typically, the restriction enzyme used to digest the plasmid is the same as that used to digest the oligonucleotide to allow the sticky ends of the plasmid and the insert to ligate to each other. See, for example, Scherer and Davis, 1979, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

[0146] Site-directed mutagenesis can also be achieved in vivo using methods known in the art. See, for example, US 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15-16.

[0147] Any site-directed mutagenesis procedure can be used in this invention. Many commercially available kits are available for preparing variants.

[0148] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode polypeptides of interest. Gene synthesis can be performed using a variety of techniques, such as the multi-channel microchip-based technique described by Tian et al. (2004, Nature 432: 1050-1054), and similar techniques involving the synthesis and assembly of oligonucleotides on optically programmable microfluidic chips.

[0149] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or truncation, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; US 5223409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0150] The activity of cloned, mutagenic peptides expressed by host cells can be detected by combining mutagenesis / reorganization methods with high-throughput automated screening methods (Ness et al., 1999, Nature Biotechnology 17:893-896). The mutagenic DNA molecules encoding the active peptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0151] Semi-synthetic gene construction is achieved through a combination of various methods, including synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction typically involves combining the process of synthesizing polynucleotide fragments with PCR technology. Therefore, specific regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenesis primers, and still others can undergo error-prone or non-error-prone PCR amplification. The polynucleotide subsequence can then be shuffled.

[0152] Polynucleotides

[0153] The present invention also relates to polynucleotides encoding variants of the invention.

[0154] Nucleic acid constructs

[0155] The present invention also relates to nucleic acid constructs comprising polynucleotides operably linked to one or more control sequences encoding variants of the invention, the one or more control sequences guiding the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0156] The polynucleotide can be manipulated in a variety of ways to provide expression of the variant. Depending on the expression vector, manipulation of the polynucleotide before its insertion into the vector may be desired or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0157] The control sequence can be a promoter, which is a polynucleotide recognized by the host cell for the expression of that polynucleotide. The promoter contains a transcriptional control sequence that mediates the expression of that variant. The promoter can be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant, truncated, and heterozygous promoters, and can be derived from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0158] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of this invention in bacterial host cells are promoters obtained from the following genes: Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis α-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus thermophilus maltose amylase gene (amyM), Bacillus subtilis fructan sucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), Escherichia coli lac operon, Escherichia coli trc promoter (Egon et al., 1988, Gene 69: 301-315), *Streptomyces cerevisiae* agar hydrolase gene (dagA), and prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), and the tac promoter (DeBoer et al., 1983, Proc. Natl. 80: 21-25). Other promoters are described in Gilbert et al., 1980, Scientific American 242: 74-94, “Useful proteins from recombinant bacteria”; and in Sambrook et al., 1989, see above. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0159] Examples of suitable promoters for guiding the transcription of the nucleic acid constructs of this invention in filamentous fungal host cells are promoters obtained from the genes of: Aspergillus nidulans acetamase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucosylamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 96 / 00787). 00 / 56900), Rhizomucormiehei cutinase, Rhizomucormiehei aspartic protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiose hydrolase I, Trichoderma reesei cellobiose hydrolase II, Trichoderma reesei endodextranase I, Trichoderma reesei endodextranase II, Trichoderma reesei endodextranase III, Trichoderma reesei endodextranase IV, Trichoderma reesei endodextranase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, ... Trichoderma β-xylosidase, and the NA2-tpi promoter (a modified promoter derived from the Aspergillus neutral α-amylase gene, wherein the untranslated leader sequence is replaced by the untranslated leader sequence of the Aspergillus triose phosphate isomerase gene; non-restrictive examples include a modified promoter derived from the Aspergillus niger neutral α-amylase gene, wherein the untranslated leader sequence is replaced by the untranslated leader sequence of the Aspergillus niger or Aspergillus oryzae triose phosphate isomerase gene); and its mutant promoters, truncated promoters, and heterozygous promoters.

[0160] In the yeast host, useful promoters are derived from the following genes: *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), *Saccharomyces cerevisiae* triose phosphate isomerase (TPI), *Saccharomyces cerevisiae* metallothionein (CUP1), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Romanos et al., 1992, *Yeast* 8: 423-488, describe other useful promoters in the yeast host cell.

[0161] The control sequence can also be a transcription terminator recognized by the host cell to terminate transcription. This terminator sequence is operatively linked to the 3' end of the polynucleotide encoding that variant. Any terminator that is functional in the host cell can be used.

[0162] Preferred terminators for bacterial host cells were obtained from the genes of Bacillus clausti alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0163] The preferred terminator for filamentous fungal host cells is derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0164] Preferred terminators for yeast host cells are derived from the genes of *Saccharomyces cerevisiae* enolase, *Saccharomyces cerevisiae* cytochrome C (CYC1), and *Saccharomyces cerevisiae* glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described above by Romanos et al., 1992.

[0165] The control sequence can also be a stabilizer region of mRNA downstream of the promoter and upstream of the gene coding sequence, which increases the expression of the gene.

[0166] Examples of suitable mRNA stable regions were obtained from the following: Bacillus thuringiensis cryIIIA gene (WO94 / 25612) and Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

[0167] The control sequence can also be a leader sequence, a non-translated mRNA region that is important for translation in the host cell. The leader sequence is operatively linked to the 5' end of the polynucleotide encoding that variant. Any leader sequence that is functional in the host cell can be used.

[0168] The preferred leader sequence for use in filamentous fungal host cells was obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0169] The leader sequence suitable for yeast host cells is obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0170] The control sequence can also be a polyadenylation sequence, i.e., a sequence operatively linked to the 3' end of the variant-coding sequence and recognized by the host cell during transcription as a signal to add polyadenylate residues to the transcribed mRNA. Any polyadenylation sequence that functions in the host cell can be used.

[0171] Preferred polyadenylated sequences for use in filamentous fungal host cells are derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0172] The polyadenylation sequence useful for yeast host cells is described in Guo and Sherman, 1995, Molecular Cellular Biology, 15: 5983-5990.

[0173] The control sequence can also be a signal peptide coding region, encoding a signal peptide linked to the N-terminus of the variant and guiding the variant into the cell's secretory pathway. The 5' end of the polynucleotide coding sequence may inherently contain a signal peptide coding sequence naturally linked within the translation reading frame to a segment encoding the variant's coding sequence. Alternatively, the 5' end of the coding sequence may include a signal peptide coding sequence that is exogenous to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, an exogenous signal peptide coding sequence may simply replace the native signal peptide coding sequence to increase the variant's secretion. However, any signal peptide coding sequence that guides the expressed variant into the host cell's secretory pathway can be used.

[0174] Effective signal peptide coding sequences for bacterial host cells are obtained from the following genes: maltose amylase produced by Bacillus NCIB 11837, subtilisin from Bacillus licheniformis, β-lactamase from Bacillus licheniformis, α-amylase from Bacillus thermophilus, neutral proteases (nprT, nprS, nprM) from Bacillus thermophilus, and prsA from Bacillus subtilis. Additional signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

[0175] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the following genes: Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Aspergillus oryzae cellulase, Aspergillus oryzae endoglucanase V, Aspergillus pubescens cutinase, and Rhizopus oryzae aspartic protease.

[0176] Signal peptides useful to yeast host cells are obtained from genes of *Saccharomyces cerevisiae* α-factor and *Saccharomyces cerevisiae* invertase. Romanos et al., 1992, ibid., described other useful signal peptide coding sequences.

[0177] The control sequence can also be a propeptide-coding sequence encoding a propeptide located at the N-terminus of the variant. The resulting polypeptide is called a proenzyme or propeptide progenitor (or, in some cases, a zymogen). The propeptide progenitor is usually inactive and can be converted into an active polypeptide by catalytic cleavage or autocatalytic cleavage of the propeptide progenitor. The propeptide-coding sequence can be obtained from the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Thermophilus laccase (WO 95 / 33836), Mucor mannii aspartic protease, and Saccharomyces cerevisiae α-factor.

[0178] In the presence of both the signal peptide sequence and the propeptide sequence, the propeptide sequence is positioned immediately adjacent to the N-terminus of the variant, and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0179] Also desirable is the addition of regulatory sequences that modulate the expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operon systems. In yeast, the ADH2 or GAL1 systems can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter, and the *Aspergillus oryzae* glucosylamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the variant will be operatively linked to the regulatory sequence.

[0180] expression carrier

[0181] The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcription and translation termination signals. Different nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction enzyme sites to allow insertion or substitution of the polynucleotide encoding the variant at these sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located within the vector, such that the coding sequence is operatively linked to the suitable control sequence for expression.

[0182] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that facilitates recombinant DNA procedures and induces the expression of polynucleotides. The choice of vector typically depends on its compatibility with the host cell to which it will be introduced. The vector can be a linear or closed circular plasmid.

[0183] The vector can be a self-replicating vector, that is, a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any elements necessary to ensure self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome and replicates along with one or more chromosomes in which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids (which together contain the total DNA from the genome to be introduced into the host cell), or transposons can be used.

[0184] The vector preferably contains one or more selective markers that allow for convenient selection of cells such as transformed cells, transfected cells, and transduced cells. A selective marker is a gene whose product provides resistance to biocides or viruses, heavy metal resistance, or auxotrophic prototrophs, etc.

[0185] Examples of bacterial selective markers include the dal gene in *Bacillus licheniformis* or *Bacillus subtilis*, or markers that confer antibiotic resistance (e.g., resistance to ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline). Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (glufosinate-amylase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (adenosyl sulfate transtransferase), and trpC (o-aminobenzoic acid synthase), along with their equivalents. Preferred markers for use in *Aspergillus* cells are the amdS and pyrG genes from *Aspergillus nidus* or *Aspergillus oryzae*, and the bar gene from *Streptomyces hygroscopicus*.

[0186] The vector preferably contains one or more elements that allow the vector to integrate into the host cell's genome or to replicate autonomously in the cell independently of the genome.

[0187] For integration into the host cell genome, the vector can rely on a polynucleotide sequence encoding the variant or any other element of the vector for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector can contain additional polynucleotides to guide integration into one or more precise locations on one or more chromosomes within the host cell genome via homologous recombination. To increase the likelihood of integration at precise locations, these integrating elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have high sequence identity with the corresponding target sequence to enhance the likelihood of homologous recombination. These integrating elements can be any sequence homologous to the target sequence within the host cell genome. Furthermore, these integrating elements can be non-coding or coding polynucleotides. On the other hand, the vector can integrate into the host cell genome via non-homologous recombination.

[0188] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicon that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replicator" refer to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0189] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in Escherichia coli, and the origins of replication of plasmids pUB110, pE194, pTA1060, and pAMß1, which allow replication in Bacillus.

[0190] Examples of replication origins used in yeast host cells are 2-micron replication origins ARS1, ARS4, combinations of ARS1 and CEN3, and combinations of ARS4 and CEN6.

[0191] Examples of origins of replication used in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acid Research 15: 9163-9175; WO 00 / 24883). The isolation of the AMA1 gene and the construction of plasmids or vectors containing this gene can be performed according to the methods disclosed in WO 00 / 24883.

[0192] More than one copy of the polynucleotide of the present invention can be inserted into a host cell to increase the generation of variants. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene along with the polynucleotide, wherein cells containing the amplified copy of the selectable marker gene, and thus additional copies of the polynucleotide, can be selected by culturing cells in the presence of a suitable selectivity reagent.

[0193] The procedures for connecting the above-described elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, ibid.).

[0194] host cells

[0195] This invention also relates to recombinant host cells comprising a polynucleotide operably linked to one or more control sequences encoding a variant of the invention, the one or more control sequences directing the generation of the variant. A construct or vector comprising the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrase or as an autonomously replicating extrachromosomal vector, as previously described. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations occurring during replication. The selection of the host cell will depend largely on the gene encoding the variant and its origin.

[0196] The host cell can be any cell that is useful in the recombinant-generated variants, such as prokaryotic or eukaryotic cells.

[0197] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Bacillus aeruginosa, Lactobacillus, Lactococcus, Marine Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Coliform, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0198] The bacterial host cell can be any Bacillus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus croceus, Bacillus coagulans, Bacillus sturdier, Bacillus splendidus, Bacillus stagnation, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, and Bacillus thuringiensis.

[0199] The bacterial host cell can also be any streptococcal cell, including but not limited to Streptococcus equi, Streptococcus pyogenes, Streptococcus mammae, and Streptococcus equi subsp. veterinaryis.

[0200] The bacterial host cell can also be any Streptomyces cell, including but not limited to chromogenic Streptomyces, insecticidal Streptomyces, blue Streptomyces, gray Streptomyces, and pale blue Streptomyces cells.

[0201] DNA can be introduced into Bacillus cells via the following methods: protoplast transformation (see, for example, Chang and Cohen, 1979, Molecular Genetics and Genomics, 168: 111-115); competent cell transformation (see, for example, Young and Spizizen, 1961, Journal of Bacteriol., 81: 823-829; or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology, 56: 209-221); and electroporation (see, for example, Shigekawa and Dower, 1988, Biotechniques, 6: ). 742-751), or conjugation (see, for example, Koehler and Thorne, 1987, Journal of Bacteriology 169: 5271-5278). DNA can be introduced into E. coli cells via protoplast transformation (see, for example, Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, for example, Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). DNA can be introduced into Streptomyces cells through protoplast transformation, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, for example, Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or transduction (see, for example, Burke et al., 2001, Proceedings of the National Academy of Sciences of the United States of America 98: 6289-6294). DNA can be introduced into Pseudomonas cells by electroporation (see, for example, Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, for example, Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57).Introducing DNA into Streptococcus cells can be achieved through: native competent cells (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplast transformation (see, for example, Catt and Jollick, 1991, Microbios 68: 189-207), electroporation (see, for example, Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, for example, Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0202] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0203] The host cell can be a fungal cell. As used herein, “fungus” includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, along with Oomycota and all mitotic fungi (as defined by Hawksworth et al. in Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0204] The host cell of this fungus can be a yeast cell. As used herein, "yeast" includes *Entomophyceae* (Endosporales), *Basidiomycetes*, and yeasts belonging to the class Deuteromycetes (Bacillus). Since the classification of yeast may change in the future, for the purposes of this invention, yeast should be defined as described in *Biology and Activities of Yeast* (edited by Skinner, Passmore, and Davenport, Soc. App. Bacteriol. Symposium, No. 9, 1980).

[0205] Yeast host cells can be cells from the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia pastoris*, *Saccharomyces*, *Saccharomyces*, or *Yarrowia*, such as *Kluyveromyces lactis*, *Kluyveromyces*, *Saccharomyces cerevisiae*, *Saccharomyces sacchariformis*, *Saccharomyces davidiana*, *Saccharomyces douglas*, *Kluyveromyces*, *Nordiya*, *Ovoyces*, or *Yarrowia lipolytica*.

[0206] Fungal host cells can be filamentous fungal cells. "Filamentous fungi" includes all filamentous forms of the phylum Eumycota and subphyla of Oomycetes (as defined by Hawksworth et al., 1995, see above). Filamentous fungi are typically characterized by a hyphal wall composed of chitosan, cellulose, glucan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal extension, and carbon metabolism is obligate aerobic. In contrast, yeast (such as Saccharomyces cerevisiae) grows vegetatively through budding of single-celled cells, and carbon metabolism can be fermentative.

[0207] The host cells of filamentous fungi can be cells from genera such as *Apertoire*, *Aspergillus*, *Bjerkandera*, *Pseudomonas*, *Aureospora*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Filibasidium*, *Fusarium*, *Pyrophyllus*, *Pleurotus*, *Mucor*, *Pyrophyllus*, *Neurophyllus*, *Penicillium*, *Penicillium*, *Phlebia*, *Pleurotus*, *Schizophyllum*, *Ascomycetes*, *Fusarium*, *Pleurotus ... or *Trichoderma*.

[0208] For example, the host cells of filamentous fungi can be *Aspergillus amblymorii*, *Aspergillus sulphureus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus oryzae*, *Ceriporiopsis carnegiea*, *Ceriporiopsis pannocinta*, *Ceriporiopsis rivulosa*, *Ceriporiopsis subrufa*, *Ceriporiopsis inops*, *Chrysosporium inops*, *Chrysosporium lucknowense*, *Chrysosporium merdarium*, *Chrysosporium merdarium*, and *Chrysosporium quercetinum*. Queenslandicum), Tropical golden spores, Brown golden spores, Grey-capped coprinus, Hairy turkey, Fusarium spores, Gracilaria, Fusarium kuwai, Yellow Fusarium, Gracilaria, Gracilaria, Fusarium heterospore, Fusarium albinosum, Fusarium oxysporum, Fusarium multibranchum, Fusarium pink, Fusarium elderberry, Fusarium scabiosaefolium, Fusarium sulfideum, Fusarium rotundum, Fusarium spheroides, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Fusarium moniliforme, Mucor, Mucor, Neurospora crassa, Penicillium purpureoides, Protozoa chrysophyllus, Leptozoa radiata, Pleurotus ostreatus, Clostridium perfringens, Trametes versicolor, Trichoderma harzianum, Trichoderma cornigeri, Trichoderma longifolia, Trichoderma reesei, or cells of Trichoderma viride.

[0209] Fungal cells can be transformed through a process involving protoplast formation, protoplast transformation, and cell wall regeneration in a manner known per se. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988, Bio / Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96 / 00787. Yeast can be transformed using procedures described in the following literature: Becker and Guarente, in Abelson, JN and Simon, MI eds., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences 75: 1920.

[0210] Production methods

[0211] The present invention also relates to methods for generating variants, the methods comprising: (a) culturing host cells of the present invention under conditions suitable for expressing the variant; and (b) recovering the variant.

[0212] These host cells are cultured in a nutrient medium suitable for producing the variant using methods known in the art. For example, the cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-feed fermentation, or solid-state fermentation) in a suitable medium and under conditions that allow for the expression and / or isolation of the variant in a laboratory or industrial fermenter. The culture occurs using procedures known in the art in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the U.S. Center for Type Culture Collection). If the variant is secreted into the nutrient medium, it can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0213] The variant can be detected using methods specific to these variants known in the art. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, enzyme assays can be used to determine the activity of the variant. Keratinase activity can be determined as hydrolytic activity against BETEB substrates, as described in Example 3.

[0214] The variant can be recovered using methods known in the art. For example, the variant can be recovered from the nutrient medium through a variety of routine procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0215] Variants can be purified to obtain substantially pure variants by a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric point focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, edited by Janson and Ryden, VCH Publishers, New York, 1989).

[0216] In an alternative, instead of recycling the variant, the host cell of the present invention expressing the variant is used as a source of the variant.

[0217] Composition

[0218] In some aspects, the present invention also relates to compositions comprising one or more (e.g., several) variations.

[0219] use

[0220] The keratinase variant of the present invention can be used, for example, to enzymatically hydrolyze cyclic oligomers of poly(ethylene terephthalate), such as cyclic tri(ethylene terephthalate), abbreviated as c3ET. Such cyclic oligomers can be removed from polyester-containing fabrics or yarns by treating them with the keratinase variant, optionally followed by rinsing the fabric or yarn with an aqueous solution having a pH in the range of pH 7 to pH 11. Treatment of the polyester can conveniently be carried out above the glass transition temperature of c3ET (about 55°C) and below the glass transition temperature of the polyester (about 70°C). Therefore, the treatment can suitably be carried out at 50°C-100°C, 50°C-95°C, 50°C-90°C, 50°C-85°C, 50°C-80°C, or 60°C-75°C. This method can be performed in a manner similar to WO 97 / 27237.

[0221] This keratinase variant can be used to treat textiles / fabrics composed of or including polyesters, such as PET (a polymer of ethylene glycol and terephthalic acid), P3GT (a polymer of 1,3-propylene glycol and terephthalic acid), or any blend thereof. Such polyester blends may include, for example, cotton (polyester / cotton blend) and / or other suitable fibers. This treatment can provide benefits to textiles / fabrics composed of or including polyesters, such as reduced pilling tendency.

[0222] This keratinase variant can be used to improve the performance of functional finishing agents on PET-containing yarns or fabrics by treating them with the keratinase variant followed by treatment with processing agents such as softeners, anti-wrinkle resins, antistatic agents, stain-resistant agents, or agents designed to reduce wrinkle-free, sustained-pressure, or flame-retardant effects. Treatment with the keratinase variant increases the number of functional groups on the surface, which can be used to adhere functional finishing agents. Examples of finishing agents are described in "SENSHOKU SIAGEKAKO BENRAN" published by Nihon SeniSentaa KK on October 15, 1998.

[0223] This keratase variant can also be used to degrade and recycle polyesters such as poly(propylene caproate) (PCL), polyethylene glycol terephthalate (PET), polylactic acid, polybutene succinate, and poly(hydroxybutyrate) hydroxyvalerate, for example, films and bottles, as described in JP-A5-344897.

[0224] In some aspects, the present invention relates to a method for modifying polyesters, the method comprising using one or more variants (e.g., several variants).

[0225] In some aspects, the present invention relates to a method for hydrolyzing cyclic oligomers of polyethylene terephthalate, the method comprising using one or more variants (e.g., several variants).

[0226] In some aspects, the present invention relates to a method for modifying polyester / cotton blended fabrics, the method comprising using one or more of keratinase and cellulase.

[0227] In some aspects, the present invention relates to a method for reducing the pilling tendency of fabrics comprising or composed of polyester, the method comprising using one or more (e.g., several) variants. Improvement in pilling resistance can be determined by using a Martindale pilling tester (Swiss Standard SN 198525), as described in the following paragraph “Materials and Methods”.

[0228] On the other hand, the present invention also relates to the following embodiments:

[0229] 1. A variant having the keratinase activity of a parent keratinase, the variant comprising alterations at one or more (e.g., several) positions corresponding to positions 181, 182, 115, 161, 1, 2, 43, 55, 79, or 5 of SEQ ID NO: 2, wherein the alteration is a substitution for positions 181, 115, 161, 43, 55, 79, and 5, and a deletion for positions 1, 2, and 182, and wherein the variant has at least 75% but less than 100% sequence identity with the mature polypeptide of SEQ ID NO: 2.

[0230] 2. The variant as described in embodiment 1, wherein the parental keratinase is selected from the group consisting of the following items:

[0231] a. A polypeptide having at least 75% sequence identity with the mature polypeptide of SEQ ID NO: 2;

[0232] b. A polypeptide encoded by a polynucleotide that hybridizes with the coding sequence of the mature polypeptide of SEQ ID NO:1 or its full-length complement under low stringency conditions;

[0233] c. A polypeptide encoded by a polynucleotide that has at least 75% identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1; and

[0234] d. Fragment of the mature polypeptide of SEQ ID NO: 2.

[0235] 3. A variant of any one of embodiments 1-2, wherein the variant comprises 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%, of the number of amino acids of SEQ ID NO: 2.

[0236] 4. A variant of any one of the embodiments 1-3, wherein the number of changes is 1-20, for example 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 changes.

[0237] 5. A variant of any one of embodiments 1-4, the variant comprising one or more (e.g., several) changes selected from the group consisting of: R181P, G182*, V115I, A161L, Q1*, L2*, A43C, I55C, N79A, and I5V.

[0238] 6. A variation of any one of embodiments 1-5, wherein the variation includes or comprises changes selected from or constitutes thereof, the group consisting of the following:

[0239] a. V115I +R181P +G182*

[0240] b. A161L +R181P +G182*

[0241] c. Q1* +L2* +I5V +A43C +I55C +N79A +V115I +R181P +G182*

[0242] d. Q1* +L2* +A43C +I55C +N79A +V115I +R181P +G182*

[0243] e. I5V +A43C +I55C +N79A +V115I

[0244] f. A43C +I55C +N79A

[0245] g. I5V +A43C +I55C +N79A +V115I +R181P +G182*

[0246] h. Q1* +L2*

[0247] i. I5V

[0248] j. A43C +I55C

[0249] k. N79A

[0250] l. V115I

[0251] m. A161L

[0252] n.R181P +G182*.

[0253] 7. A variant as described in any one of embodiments 1-6, the variant further comprising an N-terminal extension.

[0254] 8. The variant as described in embodiment 7, wherein the N-terminal extension is selected from:

[0255] a. AAVDSNHTPAVPELVAR

[0256] b. AVDSNHTPAVPELVAR

[0257] c. VDSNHTPAVPELVAR

[0258] d. DSNHTPAVPELVAR

[0259] e. SNHTPAVPELVAR

[0260] f. NHTPAVPELVAR

[0261] g. HTPAVPELVAR

[0262] h. TPAVPELVAR

[0263] i. PAVPELVAR

[0264] j. AVPELVAR

[0265] k. VPELVAR

[0266] l. PELVAR

[0267] m. ELVAR

[0268] n. LVAR

[0269] o. VAR

[0270] p. AR

[0271] qR.

[0272] 9. A variant as described in any one of embodiments 1-8, wherein the variant has improved properties relative to the parent, wherein such improved properties are selected from the group consisting of: specific activity, substrate binding, substrate cleavage, substrate specificity, thermal stability, and reduced pilling tendency.

[0273] 10. A composition comprising any of the variants described in any of embodiments 1-9.

[0274] 11. A method for modifying polyester, the method comprising using a variant as described in any one of embodiments 1-9.

[0275] 12. A method for hydrolyzing cyclic oligomers of polyethylene terephthalate, the method comprising using a variant as described in any one of embodiments 1-9.

[0276] 13. A method for reducing the pilling tendency of fabrics comprising or composed of polyester using a variant as described in any one of embodiments 1-9.

[0277] 14. A polynucleotide encoding a variant as described in any one of embodiments 1-9.

[0278] 15. A nucleic acid construct comprising the polynucleotides as described in embodiment 14.

[0279] 16. An expression vector comprising the polynucleotide as described in embodiment 14.

[0280] 17. A host cell comprising the polynucleotides as described in embodiment 14.

[0281] 18. A method for producing a keratinase variant, the method comprising:

[0282] a. Culture host cells as described in embodiment 17 under conditions suitable for expressing the variant; and

[0283] b. Recycle the variant.

[0284] 19. A method for obtaining a keratinase variant, the method comprising introducing a modification into a parental keratinase at one or more positions corresponding to positions 181, 182, 115, 161, 1, 2, 43, 55, 79, 5, and 71 of a mature polypeptide corresponding to SEQ ID NO: 2, wherein the modification is a substitution at positions 181, 115, 161, 43, 55, 79, and 5 and a deletion at positions 1, 2, and 182, and the variant possesses keratinase activity. The variant is then recovered.

[0285] The invention is further described through the following examples, but should not be construed as limiting the scope of the invention.

[0286] Materials and Methods

[0287] Unless otherwise specified, these materials are reagent grade.

[0288] Table 1: Changes in the amino acid positions corresponding to SEQ ID No: 2 in the variants.

[0289]

[0290] Example 1: Cloning and Expression

[0291] Variants were generated by site-directed mutagenesis using forward primers containing the mutation. The PCR reaction mixture contained 50 ng template DNA; 10 pmol mutagenic primers; 300 μmol dNTPs; 1X 5X HF Phusion buffer; 0.5 mM MgCl2; 1 unit of Phusion polymerase; and up to 25 μL MilliQ water. PCR cycles were performed at 98°C / 2:00 min; 18x (98°C / 1:00 min, 62°C / 1:00 min, 72°C / (2*plasmid length, in kb) min); 72°C / 20:00 min; and stored at 4°C. Annealing temperatures were varied according to the Tm of the mutagenic primers used. Digestion with DpnI was performed at 37°C for 6 hours in a PCR instrument using 0.5 μL of DpnI enzyme (added directly to the PCR reaction mixture).

[0292] 5.0 μL of the reaction mixture was directly transformed into chemically competent Escherichia coli DH5α cells. 2–6 colonies were inoculated for plasmid isolation and plasmid DNA sequencing to confirm the inclusion of the desired mutation.

[0293] The plasmid DNA with the identified promising mutations was then transformed into Aspergillus oryzae, and the resulting clones were screened for protein expression at a small scale (2 mL in a 24-well plate).

[0294] Small-scale expression:

[0295] The spore suspension was prepared by autoclaving 0.2 g agar (RM026, HiMedia), 0.05 g Tween 20 (P9416, Sigma), and water to a final volume of 100 mL and then dispensing into 1 mL Nunc tubes.

[0296] YPM medium (10 g yeast extract (RM027, HiMedia); 20 g peptone (RM001, HiMedia); and water added to 1000 mL; maltose (RM3050, HiMedia) from 20% stock solution added after autoclaving to a final concentration of 2%).

[0297] Spores from transformed colonies were transferred from agar plates and inoculated into 2 mL of YPM medium in 12- or 24-well plates using an inoculation loop and spore suspension. These plates were incubated at 34°C under humid conditions for 3 days without shaking. The mycelial layer that formed on the surface of the medium was removed, and the medium was analyzed by SDS-PAGE and viability assays.

[0298] A 40 μL aliquot was mixed with 10 μL of SDS-PAGE, heated at 100 °C for 10 min, and then loaded into a 12% agarose gel, followed by Coomassie Brilliant Blue staining. Colonies with the best expression were streaked onto COVE-N agar slants for shake-flask fermentation.

[0299] Shake flask fermentation :

[0300] G2-Gly medium (18 g yeast extract (RM027, HiMedia); 24.0 g glycerol (87%, 10409405001730, Merck); 1.0 mL Dowfax 63N10 (Novozymes); and replenished to 1000 mL of ion-exchanged water).

[0301] MDU-2BP medium (45.0 g maltose RM3050, HiMedia); 1.0 g magnesium sulfate (61777005001730, Merck); 1.0 g sodium chloride (1.93206.0521, Merck); 2.0 g potassium sulfate (61777405001730, Merck); 12.0 g potassium dihydrogen phosphate (1.93205.0521, Merck); 7.0 g yeast extract (RM027, HiMedia); 0.1 mL Dowfax 63N10 (Novozymes); 0.5 mL AMG Spormetal (KU6) (see below); and replenished to 1000 mL of ion-exchanged water.

[0302] AMG Spormetal (KU6) (6.8 g zinc chloride (61752905001046, Merck); 2.5 g copper sulfate (61775905001730, Merck); 0.13 g anhydrous nickel chloride (8067220100, Merck); 13.9 g ferric sulfate (61751005001730, Merck); 8.45 g manganese sulfate (61754805001730, Merck); 3 g citric acid (60024205001730, Merck); and replenished to 1000 mL of ion-exchanged water).

[0303] Approximately 5 mL of G2-Gly medium from a baffle flask (200 mL culture base in a 500 mL flask) was poured into a sporulation slant of a given variant. Spores were scraped from the slant using an inoculation loop and resuspended in the medium, which was then poured back into the flask containing the remaining medium. These flasks were covered with a thick layer of Mira-like cloth and paper and incubated at 34°C for 24 hours at 180 rpm. After overnight growth, 2–5 mL of the G2-Gly culture was inoculated into 300 mL of MDU-2BP medium in a 1 L baffle flask. Before inoculation, 50% urea was added to the heat-pressed MDU-2BP medium to a final concentration of 0.5%. These flasks were incubated at 34°C and 180 rpm for 72 hours. After 72 hours of growth, the supernatant was analyzed for expression on 12% SDS-PAGE. Once SDS-PAGE shows the expression of the desired protein, the fermentation broth is delivered for purification.

[0304] Example 2: Purification

[0305] The fermentation broth was filtered through a Buchner funnel (145 mm) fitted with a glass fiber membrane sandwich. The membrane sandwich consisted of GFD / A / C / B and F membranes, with membrane D at the top of the Buchner funnel and membrane F at the bottom. The culture was then forced through a GE QuixStand membrane fixed at a transmembrane pressure maintained at 5 psi. ® The instrument features a 0.2 μm hollow fiber filter membrane.

[0306] Sterile filtered culture medium was loaded into HEA-HyperCel resin containing a mixed state. ® (HEA is hexylamine) on the first column. The resin was packaged in a glass column (Kronlab) with a column diameter of 15 mm and a bed height of 200 mm. BioRad Duoflow pathfinder20 was used. ® The purification process is automated using the sequence of steps outlined below. All volumes, except for the sample volume, are 8–10 times the column volume.

[0307]

[0308] The elution fractions from the first column were combined and their conductivity adjusted to 4 mS / cm by dilution with 50 mM sodium acetate buffer (pH 4.5), and then loaded into UnoS cation exchange resin packaged in glass columns (Kron Labs). ® On the second column, the glass column has a column diameter of 15 mm and a bed height of 200 mm. Using BioRad Duoflow pathfinder20. ® The purification process is automated using the sequence of steps outlined below. All volumes, except for the sample volume, are 8–10 times the column volume.

[0309]

[0310] The eluted fractions are combined, parsed on an SDS-PAGE for recording, and then... 280 Measure, package, and deliver.

[0311] Large-scale purification

[0312] The culture medium was centrifuged (20000x g, 20 min), and the supernatant was carefully decanted from the precipitate and filtered through a Nalgene 0.2 μm filter.

[0313] Add 5M NaCl solution to the 0.2 μm filtrate to a final concentration of 1M NaCl. Apply this mixture to a decylamine-agarose column (from Upfront Chromatography) equilibrated in 40 mM H3BO3 / NaOH, 1M NaCl (pH 9.0), and then wash with 5x column volume equilibration buffer and elute gradually with a mixture of 70% (50 mM H3BO3 / NaOH, pH 9.0) and 30% isopropanol.

[0314] The elution peak from the decylamine agarose step was applied to an SP-crosslinked dextran FF column (from GE Healthcare) equilibrated in 20 mM acetic acid / NaOH (pH 5.0). After thorough washing of the column with equilibration buffer, keratinase was eluted with a linear gradient of equilibration buffer, 20 mM acetic acid / NaOH, and 1.0 M NaCl (pH 5.0) for more than 3x column volumes. The main peak from the SP-crosslinked dextran FF column containing keratinase was analyzed by SDS-PAGE, and fractions were pooled as purified preparations when only one band was visible on a Coomassie-stained SDS-PAGE gel.

[0315] Example 3: Keratinase Activity

[0316] To calculate the residual activity of the parent and variant enzymes, the hydrolytic activity against the BETEB substrate (12.5 mg / mL BETEB; 0.1% Triton X-100, H2O) was determined at two temperatures: 85 °C was considered to be 100% activity and 90 °C was considered to show reduced activity.

[0317] For each test temperature, two controls were included (“substrate blank” and “enzyme blank”) and tested under conditions similar to those of the enzyme sample. The enzyme sample was prepared by mixing 25 μL of culture supernatant (sub) from Example 1 or purified enzyme (pure) from Example 2 with 100 μL of substrate, and then adding to 1 mL of 40 mM Britton-Robinson buffer pH 7.0 (Britton-Robinson buffer is an equimolar mixture of boric acid, orthophosphate, and acetic acid. The pH is adjusted using a 5x molar solution of NaOH). The “substrate blank” contained 100 μL of substrate and was added to 1 mL of 40 mM Britton-Robinson buffer pH 7.0. The “enzyme blank” contained 25 μL of culture supernatant, 100 μL of 0.1% Triton X-100, and was added to 1 mL of 40 mM Britton-Robinson buffer pH 7.0. (The text abruptly ends here, so the translation stops as well.) After incubating at 85°C or 90°C for 20 minutes, the reaction was immediately stopped by placing the sample on ice for 1 to 5 minutes. The sample was then centrifuged at 13,000 rpm for 1 minute, and the absorbance of the supernatant was measured at 254 nm.

[0318] The improved residual activity of the variant compared to the parent enzyme is expressed as a relative %RA greater than 100. The relative %RA is calculated as follows: Relative %RA = (Variant %RA) / (SEQ ID NO: 2 %RA) * 100

[0319] Table 2: Residual activity (RA) of keratinase variants relative to SEQ ID No: 2

[0320]

[0321] Example 4: Biopolishing with keratinase in a laundry index meter

[0322] Biopolishing with keratinase was performed on the SDL-Atlas LP2 Laundry Index (LOM) at both small-scale (SSLOM) and full-scale (FSLOM) settings.

[0323] Cut the fabric into rectangular pieces / small samples of 5 x 10 cm (approximately 1 g, for SSLOM) and 14 x 14 cm (approximately 4-5 g, for FSLOM). Overlock the fabric by sewing. Condition the pieces for 24 hours at 65% + / - 5% relative humidity and 20°C + / - 1°C before numbering them, weigh and record the weight using an analytical balance (for samples under 100 g) or a precision balance (for samples over 100 g).

[0324] A conditioned fragment was placed in each beaker along with 10 small acid-resistant steel balls (M6M-SR-A4-80) for mechanical assistance. Buffer (Britton-Robinson buffer, pH = 8) and enzyme solution were added at a liquid-to-fabric v / w ratio of 10:1, based on calculations of the actual fabric weight, as indicated in these tables. The OD absorbance at 254 nm and the initial pH of the solution were measured at time 0 h.

[0325] Each beaker is fitted with a lid lined with two neoprin gaskets and tightly closed with a metal clamping device. These beakers are loaded into a LOM preheated to 70°C. In the vertical position, in each of the four drum positions, five beakers are received and secured using a metal frame. The LOM lid is then closed and the beakers are rinsed.

[0326] Two hours later, the fabrics were transferred to a passivation solution (2 g / L sodium carbonate) and kept at 95°C for 10 minutes, followed by rinsing twice in 1 L of hot water and then twice in 1 L of cold water. The fabrics were then tumble-dried (AEG, LAVATHERM 37700, Germany) for 1 hour, after which they were conditioned as described above before evaluation.

[0327] The treatment bath from each beaker was centrifuged at 13,000 rpm for 1 minute, and the pH and OD absorbance at 254 nm were determined. Pilling records and weight loss of the fabric were evaluated.

[0328] According to the product manual, use BCA TMProtein Assay Kit (BCA) TM The Protein Assay Kit (product number 23225, available commercially from Thermo Fisher Scientific Inc.) measures enzyme proteins.

[0329] OD absorbance and pH measurement

[0330] Keratinase activity was studied by hydrolyzing PET or BETEB in Eppendorf tubes. The hydrolysis products are terephthalates and their esters, which have characteristic absorption peaks around 254 nm (UV). The absorbance at 254 nm (OD) was [not specified in the original text]. 254 This reflects the enzyme's hydrolytic activity toward polyester. Increased enzyme activity toward PET or BETEB leads to OD. 254 The increase in OD. 254 Read the absorbance using a SpectraMax M2 microplate reader (Molecular Devices, LLC.). If the absorbance exceeds the effective range of the microplate reader by 1.5, dilute the solution. Dilution x15 means the solution has been diluted 15 times.

[0331] The hydrolysis product, terephthalate, is acidic and will lower the pH of the solution. Therefore, enzyme activity can be measured by measuring the pH change before and after the reaction.

[0332] Balling record test

[0333] Fabrics (both treated and untreated) that had been pre-conditioned for at least 24 hours under standard climatic conditions (65% humidity, 20°C) were tested for pilling records using a Nu-Martindale tester (James H. Hill Heal Co. Ltd, England), with the same type of untreated fabric used as the wear test fabric. After 2000 revolutions, a standard pilling test (Swiss Standard (SN) 198525) was performed, using a scale of 1-5 with the following meanings: 1 indicating poor pilling resistance and 5 indicating excellent pilling resistance. Therefore, the higher the Martindale pilling record score, the more effective the biopolishing treatment.

[0334] Record 5: No pilling

[0335] Record 4: Slight pilling

[0336] Record 3: Moderate pilling

[0337] Record 2: Obvious pilling

[0338] Record 1: Severe pilling

[0339] Allow 1 / 2 and 1 / 4 records

[0340] Three separate readings were taken for each sample by different people, and the average of these three readings was used as the final result of the pilling record.

[0341] Table 3: 100% Stable PET Woven Fabrics at SSLOM 1 Bioblasting in China

[0342]

[0343] 1 SSLOM was performed on 100% stable PET woven fabric (cationically dyeable, CHN-2011-00461) at 70°C and pH 8.0.

[0344] 2 The concentration is expressed as mg of enzyme protein / g of fabric.

[0345] 3 The change in the balling record is approximately 0.2.

[0346] Table 4: 100% Stable PET Woven Fabrics at SSLOM 1 Bioblasting in China

[0347]

[0348]

[0349] 1 SSLOM was performed on 100% stable PET woven fabric (cationically dyeable, CHN-2011-00461) at 70°C and pH 8.0.

[0350] 2 The concentration is expressed as mg of enzyme protein / g of fabric.

[0351] 3 The change in the balling record is approximately 0.2.

[0352] Table 5: 100% Stable PET Woven Fabrics at FSLOM 1 Biological grinding

[0353]

[0354]

[0355] Table 6: 100% Stable PET Woven Fabrics at FSLOM 1 Biological grinding

[0356]

[0357]

[0358]

[0359] 1 FSLOM was performed on 100% stable PET woven fabric (cationically dyeable, CHN-2011-00461) at 70°C and pH 8.0.

[0360] 2 The concentration is expressed as mg of enzyme protein / g of fabric.

[0361] 3 The change in the balling record is approximately 0.2.

[0362] Table 7: Different PET-blended cotton fabrics in FSLOM 1 Biological grinding

[0363]

[0364] 1 FSLOM was performed using different PET-blended cotton fabrics at 70°C and pH 8.0.

[0365] 2 The concentration is expressed as mg of enzyme protein / g of fabric.

[0366] The invention described and claimed herein is not limited to the specific aspects disclosed herein, as these aspects are intended to illustrate several aspects of the invention. Any equivalent aspects are contemplated to be within the scope of the invention. In fact, various modifications to the invention, other than those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the disclosure including the definition shall prevail.

[0367]

Claims

1. A variant having the keratinase activity of a parent keratinase, the variant comprising alterations at one or more (e.g., several) positions corresponding to positions 181, 182, 115, 161, 1, 2, 43, 55, 79, or 5 of SEQ ID NO: 2, wherein the alteration is a substitution for positions 181, 115, 161, 43, 55, 79, and 5, and a deletion for positions 1, 2, and 182, and wherein the variant has at least 75% but less than 100% sequence identity with the mature polypeptide of SEQ ID NO:

2.

2. The variant of claim 1, wherein the parental keratinase is selected from the group consisting of the following: a. A polypeptide having at least 75% sequence identity with the mature polypeptide of SEQ ID NO: 2; b. A polypeptide encoded by a polynucleotide that hybridizes with the mature polypeptide coding sequence of SEQ ID NO: 1 or its full-length complement under low stringency conditions; c. A polypeptide encoded by a polynucleotide that has at least 75% identity with the sequence encoding the mature polypeptide of SEQ ID NO: 1; and d. Fragment of the mature polypeptide of SEQ ID NO:

2.

3. The variant of any one of claims 1-2, wherein the variant comprises 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%, of the number of amino acids of SEQ ID NO:

2.

4. The variant of any one of claims 1-3, wherein the number of changes is 1-20, for example 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 changes.

5. A variation of any one of claims 1-4, wherein the variation comprises one or more (e.g., several) changes selected from the group consisting of: R181P, G182*, V115I, A161L, Q1*, L2*, A43C, I55C, N79A, and I5V.

6. A variation of any one of claims 1-5, wherein the variation comprises or consists of changes selected from or constitutes the group consisting of: a. V115I +R181P +G182* b. A161L +R181P +G182* c. Q1* +L2* +I5V +A43C +I55C +N79A +V115I +R181P +G182* d. Q1* +L2* +A43C +I55C +N79A +V115I +R181P +G182* e. I5V +A43C +I55C +N79A +V115I f. A43C +I55C +N79A g. I5V +A43C +I55C +N79A +V115I +R181P +G182* h. Q1* +L2* i. I5V j. A43C +I55C k. N79A l. V115I m. A161L n.R181P +G182*.

7. The variant of any one of claims 1-6, further comprising an N-terminal extension.

8. The variant of claim 7, wherein the N-terminal extension is selected from: a. AAVDSNHTPAVPELVAR b. AVDSNHTPAVPELVAR c. VDSNHTPAVPELVAR d. DSNHTPAVPELVAR e. SNHTPAVPELVAR f. NHTPAVPELVAR g. HTPAVPELVAR h. TPAVPELVAR i. PAVPELVAR j. AVPELVAR k. VPELVAR l. PELVAR m. ELVAR n. LVAR o. VAR p. AR qR.

9. A variant as claimed in any one of claims 1-8, wherein the variant has improved properties relative to the parent, wherein such improved properties are selected from the group consisting of the following: Specific activity, substrate binding, substrate cleavage, substrate specificity, thermal stability, and reduced tendency to pill.

10. A composition comprising a variant as claimed in any one of claims 1-9.

11. A method for modifying polyester, the method comprising using a variant as described in any one of claims 1-9.

12. A method for hydrolyzing cyclic oligomers of polyethylene terephthalate, the method comprising using a variant as described in any one of claims 1-9.

13. A method for reducing the pilling tendency of fabrics comprising or composed of polyester using the variants of any one of claims 1-9.

14. A polynucleotide encoding a variant as described in any one of claims 1-9.

15. A nucleic acid construct comprising the polynucleotide as described in claim 14.

16. An expression vector comprising the polynucleotide as described in claim 14.

17. A host cell comprising the polynucleotide as described in claim 14.

18. A method for producing a keratinase variant, the method comprising: a. Culture the host cells as described in claim 17 under conditions suitable for expressing the variant; and b. Recycle the variant.

19. A method for obtaining a keratinase variant, the method comprising introducing a modification into a parental keratinase at one or more positions corresponding to positions 181, 182, 115, 161, 1, 2, 43, 55, 79, 5, and 71 of a mature polypeptide corresponding to SEQ ID NO: 2, wherein the modification is a substitution at positions 181, 115, 161, 43, 55, 79, and 5 and a deletion at positions 1, 2, and 182, and the variant possesses keratinase activity. The variant is then recovered.

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