Protease variants and uses thereof

Abstract

Disclosed herein are one or more subtilisin variants, nucleic acids encoding the same, and compositions and methods related to production and use thereof, the one or more subtilisin variants include one or more subtilisin variants having improved stability and / or soil removability as compared to one or more reference subtilisins.

Description

[0001] This application is a divisional application of Chinese application No. 201780046448.9, whose application date is May 31, 2017 and whose invention name is “Protease variants and uses thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application is related to and claims the benefit of priority to U.S. Provisional Patent Application Serial No. 62 / 343,618, filed on May 31, 2016, which is incorporated herein by reference in its entirety.

[0004] References to sequence listings submitted electronically

[0005] The contents of the sequence listing submitted electronically with the application as an ASCII text file (name: NB40785WOPCT_ST25; size: 882KB; creation date: May 26, 2017) form part of the present application and are incorporated herein by reference in their entirety.

[0006] Disclosed herein are one or more subtilisin protease variants, nucleic acids encoding the same, and compositions and methods related to the production and use thereof, including one or more subtilisin protease variants having improved stability and / or stain removal compared to one or more reference subtilisin proteases.

[0007] Serine proteases are enzymes with active site serines that initiate hydrolysis of protein peptide bonds (EC numbering 3.4.21). Serine proteases comprise a wide variety of enzymes with broad specificity and biological functions, which are further divided into chymotrypsin-like (trypsin-like) and subtilisin-like based on the structures of these enzymes. The prototype subtilisin (EC numbering 3.4.21.62) was initially obtained from Bacillus subtilis. Subtilisin and its homologues are members of the S8 peptidase family of the MEROPS classification scheme. Members of family S8 have a catalytic triad in the order of Asp, His and Ser in their amino acid sequence. Although many variant proteases that can be used for cleaning applications have been developed, there is still a need for improved protease variants.

[0008] One embodiment provides one or more subtilisin protease variants comprising an amino acid sequence comprising two, three, or four or more variants at positions corresponding to SEQ ID NO: 2, wherein the positions are selected from: (i) 1, 3, 9, 10, 15, 22, 24, 26, 27, 28, 29, 30, 31, 35, 37, 40, 43, 45, 48, 52, 68, 71, 76, 77, 78, 86, 87, 95, 96, 98, 99, 100, 101, 102, 103, 105, 108, 111, 114, 115, 117, 119, 123, 125, 126, 127, 128, 129, 130, 136, 143, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 47, 151, 155, 158, 160, 161, 165, 184, 187, 193, 202, 203, 204, 210, 211, 216, 217, 234, 238, 239, 242, 243, 250, 255, 258, 259, 260, 264 and 274; (ii) 1, 3, 9, 10, 15, 22, 24, 26, 28, 29, 30, 31, 35, 37, 40, 45, 48, 52, 68, 71, 76, 77, 78, 86, 87, 95, 96, 98, 99, 100, 101, 102, 105, 108, 111, 114, 115, 117, 119, 123, 125, 126, 127, 128, 129, 130, 136, 143, 146, 147, 151, 155, 158, 160, 161, 165, 184, 187, 193, 202, 203, 204, 210, 211, 216, 217, 234, 238, 239, 242, 243, 250, 255, 258, 259, 260, 264 and 274; (iii) 1, 3, 9, 10, 24, 26, 29, 30, 31, 35, 37, 40, 45, 48, 52, 68, 71, 77, 78, 86, 87, 95, 96, 98, 99, 100, 101, 102, 105, 108, 111, 115, 117, 119, 123, 127, 128, 129, 130, 136, 143, 146, 147, 151, 155, 158, 160, 161, 165, 184, 187, 193, 202, 203, 204, 210, 211, 216, 217, 234, 238, 239, 242, 243, 250, 258, 259, 260, 264, and 274;(iv) 3, 9, 10, 35, 68, 77, 78, 86, 87, 99, 101, 115, 123, 127, 128, 165, 184, 187, 202, 203, 210, 217, 242, 250, 258, and 264; or (v) 3, 68, 77, 78, 123, 127, 128, 165, 184, 202, 210, 217, and 258; provided that two, three, or four or more of the variations One or more of the above are artificial mutations or substitutions; wherein the variant has at least 85% but less than 100% amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3, and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13, and motif 14; and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2. ;

[0009] Yet another embodiment provides one or more subtilisin variants comprising an amino acid sequence comprising two, three or four or more amino acid substitutions at two, three or four or more positions corresponding to SEQ ID NO: 2, wherein the substitutions are selected from: (i) 1Q, 3Q / V, 9E, 10M, 15I, 22Y, 24N / Q, 26Q, 27R, 28A, 29S, 30T, 31I, 35A, 37T, 40E, 43A, 45I / Q / R, 46Y, 47N / Q, ​​48R, 49S, 50T, 51I, 52A, 53T, 54E, 55I / Q / R, 56Y, 57N / Q, ​​58R, 59S, 60T, 61I, 62I / Q / R, 63I / Q / R, 64Y, 65I / Q / R, 66Y, 67R, 68A, 69S, 71 8I / T / V, 52R, 68S, 71A, 76K, 77D / E / H / N / Q / S, 78I, 86H / N / R, 87S / T, 95A / F / Q, 96S, 98 H / R, 99A / E / Q / S, 100N, 101A, 102D / T, 103I, 105N, 108H / K, 111Q, 114N, 115F, 117S, 1 19Q, 123I, 125N, 126Q, 127E / F / Q / S / T / V, 128P / R / S, 129H / R, 130S, 136N / R, 143Q / V, 146A / S, 147L, 151G, 155E / G / N, 158D / N / Q, 160C / D / K, 161D / Q, 165A / E / Q / S, 184E / Q, 187E / P, 193D / Q, 202V, 203E / N / Q, 204I, 210E / H / I / P, 211S, 216Q, 217S, 234I / W, 238 Q / R, 239K / S / T, 242G / N, 243I, 250G, 255R, 258D / E / P, 259E / P, 260W, 264H / Q / S and 274A;(ii) 1Q, 3Q / V, 9E, 10M, 15I, 22Y, 24N / Q, 26Q, 28A, 29S, 30T, 31I, 35A, 37T, 40E, 45I / Q / R, 48I / T / V, 52R, 68S, 71A, 76K, 77D / E / H / N / S, 78I, 86H / N / R, 87S / T, 95A / F / Q, 96S, 98K / R, 99A / E / Q / S, 100N, 101A, 102D / T, 105N, 108H / K, 111Q, 114N, 115F, 117S, 119Q, 123I, 125N, 126Q, 127E / F / Q / S / T / V, 128P / R / S, 129H / R, 130S, 136N, 143Q / V, 146A / S, 147L, 151G, 155E / G / N, 158D / N / Q, 160D / K, 161D / Q, 165A / E / Q / S, 184E / Q, 187E / P, 193D / Q, 194E, 202V, 203E / N / Q, 204I, 210I / P, 211S, 216Q, 217S, 234I / W, 238Q / R, 239K / S / T, 242G / N, 243I, 250G, 255R, 258D / P, 259E / P, 260W, 264H / Q / S and 274A; (iii) 1Q, 3V, 9E, 10M, 24Q, 26Q, 29S, 30T, 31I, 35A, 37T, 40E, 45I / Q / R, 48T / V, 52R, 68S, 71A, 77E / N, 78I, 86H / R, 87S, 95A / F, 96S, 98R, 99A / E / Q / S, 100N, 101A, 102D / T, 105N, 108H, 111Q, 115F, 117S, 119Q, 123I, 127E / Q / S / T / V, 128P / R, 129H / R, 130S, 136N, 143Q / V, 146A, 147L, 151G, 155G / N, 158D / N / Q, 160D / K, 161D / Q, 165A / E / Q, 184E / Q, 187E / P, 193Q, 202V, 203E / N / Q, 204I, 210I / P, 211S, 216Q, 217S, 234W, 238Q, 239S, 242G / N, 243I, 250G, 258D / P, 259E / P, 260W, 264H / Q / S and 274A;(iv) 3V, 9E, 10M, 35A, 68S, 77N, 78I, 86H, 87S, 99Q / S, 101A, 115F, 123I, 127S / T, 128P / R, 165E / Q, 184Q, 187E, 202V, 203E, 210P, 217S, 242G, 250G, 258D / P, and 264H; or (v) 3V, 68S, 77N, 78I, 123I, 127S, 128P, 165Q, 184Q, 202V, 210P, 217S, and 258D / P; provided that one or more of the two, three or four or more substitutions are artificial substitutions; wherein the variant has at least 85% but less than 100% amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14; and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2. ;

[0010] In one embodiment, the AprL-clade enzyme comprises motif A, motif B, or a combination thereof. In other embodiments, the AprL-clade enzyme comprises motif A and motif B. In some embodiments, one or more subtilisin variants described herein comprise motif A, motif B, or a combination thereof. In still other embodiments, one or more subtilisin variants described herein comprise motif A and motif B. In another embodiment, motif A is selected from motif 1, motif 2, motif 3, and motif 4. In still other embodiments, motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13, and motif 14.

[0011] Some embodiments relate to compositions comprising one or more subtilisin variants described herein. Additional embodiments relate to a method of cleaning comprising contacting a surface or item in need of cleaning with one or more subtilisin variants described herein or one or more compositions described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1AA-1D A CLUSTAL W sequence alignment of AprL, Blid02339 and other AprL-clade enzymes is provided.

[0013] Figure 2 A phylogenetic tree of subtilisins including AprL, Blid02339, other members of the AprL-clade, and other subtilisins including variants described herein is provided.

[0014] Unless otherwise specified herein, one or more subtilisin variants described herein can be prepared and used by conventional techniques commonly used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, recombinant DNA fields and industrial enzyme use and development. Undefined terms and abbreviations should conform to the conventional meanings used in the art. Unless otherwise defined herein, all scientific and technological terms used herein have the same meaning as those of ordinary skill in the art generally understand. Any definition provided herein will be interpreted as a whole in the context of the specification. Unless otherwise clearly indicated by the context, as used herein, the singular "one / a kind" and "the / said" include plural. Unless otherwise specified, the nucleic acid sequence is written from left to right in the 5' to 3' direction; and the amino acid sequence is written from left to right in the amino to carboxyl direction. Each numerical range used herein will include each narrower numerical range falling within such a wider numerical range, as if such a narrower numerical range is all clearly written here.

[0015] The term "about" as used herein in conjunction with a numerical value refers to a range of + / -0.5 of the numerical value, unless the term is otherwise specifically defined in the context. For example, the phrase "a pH value of about 6" means a pH value of from 5.5 to 6.5, unless the pH value is otherwise specifically defined.

[0016] The naming of the amino acid substitution or variation of one or more subtilisin variants described herein uses one or more of the following: position; position: amino acid substitution or one or more variations; or one or more starting amino acids: position: one or more amino acid substitutions. Reference to "position" (i.e., 5, 8, 17, 22, etc.) covers any starting amino acid that may be present at such positions, and any variation from the starting amino acid or the substitution that may be present at such positions. Reference to "position: one or more amino acid substitutions and / or one or more variations" (i.e., 1S / T / G, 3G, 17T, etc.) covers any starting amino acid that may be present at the position and one or more amino acids that may be different from the starting amino acid and / or may replace the starting amino acid. In the case where the substituted amino acid is the same as the starting amino acid, the amino acid substitution excludes the starting amino acid. Reference to the starting amino acid or the substituted amino acid or the amino acid variation can be further represented as several starting, substituted or mutated amino acids separated by a slash (" / "). For example, D275S / K means that position 275 is substituted with serine (S) or lysine (K), and P / S197K means that the starting amino acid proline (P) or serine (S) at position 197 is substituted with lysine (K).

[0017] The positions of the amino acid residues in a given amino acid sequence are numbered by corresponding to the amino acid sequence of SEQ ID NO:2. That is, the amino acid sequence of the AprL-clade enzyme shown in SEQ ID NO:2 is used as a reference sequence. For example, the amino acid sequence of the AprL-clade subtilisin or one or more subtilisin variants described herein is aligned with the amino acid sequence of SEQ ID NO:2 using an alignment algorithm as described herein, and each amino acid residue in a given amino acid sequence aligned (preferably, optimally aligned) with the amino acid residues in SEQ ID NO:2 is conveniently numbered by reference to the position of the corresponding amino acid residue represented by a number. When compared to a query sequence, a sequence alignment algorithm such as described herein will identify the position at which insertions or deletions occur in the subject sequence.

[0018] With respect to polypeptide amino acid sequences, the term "variant" refers to a polypeptide amino acid sequence that is different from a specified wild-type, parent or reference polypeptide in that it includes one or more artificial amino acid substitutions, insertions or deletions. Similarly, with respect to polynucleotide nucleic acid sequences, the term "variant" refers to a polynucleotide nucleic acid sequence that is different from a specified wild-type, parent or reference polynucleotide in that it includes one or more artificial nucleic acid substitutions, insertions or deletions. The identity of the wild-type, parent or reference polypeptide nucleic acid sequence or polynucleotide nucleic acid sequence will be apparent from the context.

[0019] When used, for example, in the phrase "two, three, or four or more amino acid variations relative to SEQ ID NO: 2" or "two, three, or four or more variations at positions corresponding to SEQ ID NO: 2," the term "one or more variations" encompasses every amino acid that is different from the amino acid present at the corresponding position in SEQ ID NO: 2. For example, the sequence of a variant of interest is aligned with SEQ ID NO: 2, and each position in the variant is compared to SEQ ID NO: 2 to identify an amino acid at each position that is different from the amino acid present at the corresponding position in SEQ ID NO: 2, and each amino acid that is different from the corresponding amino acid in SEQ ID NO: 2 is a variation.

[0020] The terms "protease" and "proteinase" refer to enzymes with the ability to decompose proteins and peptides. Protease has the ability to "proteolysis" by hydrolysis of peptide bonds, which connect amino acids together in the peptide or polypeptide chain that forms a protein. This activity of protease as a protein digestive enzyme is referred to as "proteolytic activity". There are many well-known procedures for measuring proteolytic activity. For example, proteolytic activity can be determined by comparative tests that analyze the ability of each protease to hydrolyze a suitable substrate. Exemplary substrates that can be used to analyze protease or proteolytic activity include but are not limited to dimethyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625) and bovine keratin (ICN Biomedical 902111). Colorimetric assays using these substrates are well known in the art (see, for example, WO 99 / 34011 and US 6,376,450). The pNA peptide base assay (see, e.g., Del Mar et al., Anal Biochem, 99:316-320, 1979) can also be used to determine the active enzyme concentration. The assay measures the rate at which p-nitroaniline is released when the enzyme hydrolyzes a soluble synthetic substrate such as succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline (suc-AAPF-pNA). The rate of generation of the yellow color from the hydrolysis reaction is measured at 410 nm on a spectrophotometer and is proportional to the active enzyme concentration. In addition, absorbance measurements at 280 nanometers (nm) can be used to determine the total protein concentration in a purified protein sample. The activity of substrate / protein concentration gives the enzyme specific activity.

[0021] The phrase "one or more compositions that are substantially free of boron" or "one or more detergents that are substantially free of boron" refers to one or more compositions or detergents, respectively, that contain trace amounts of boron (e.g., less than about 1000 ppm (1 mg / kg or L (equal to 1 ppm)), less than about 100 ppm, less than about 50 ppm, less than about 10 ppm, or less than about 5 ppm, or less than about 1 ppm), which may come from other composition or detergent ingredients.

[0022] As used herein, "Bacillus" includes all species within the genus "Bacillus" as known to those skilled in the art, including, but not limited to, Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkaliphilus, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus gibsonii, and Bacillus thuringiensis. It should be recognized that the genus Bacillus is constantly undergoing taxonomic reorganization. Therefore, the genus is intended to include species that have been reclassified, including, but not limited to, such organisms as B. stearothermophilus (now called "Geobacillus stearothermophilus") or B. polymyxa (now "Paenibacillus polymyxa"). The production of resistant endospores under stressful environmental conditions is considered a defining characteristic of the genus Bacillus, although this feature also applies to the more recently named genera Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

[0023] As used herein, the term "mutation" refers to an artificial change in a reference amino acid or nucleic acid sequence. The term is intended to encompass artificial substitutions, insertions, and deletions.

[0024] As used herein, the term "vector" refers to a nucleic acid construct for introducing or transferring one or more nucleic acids into a target cell or target tissue. Typically, a vector is used to introduce exogenous DNA into a cell or tissue. Vectors include plasmids, cloning vectors, phages, viruses (e.g., viral vectors), cosmids, expression vectors, shuttle vectors, etc. Typically, vectors include an origin of replication, a multiple cloning site, and a selectable marker. Typically, the process of inserting a vector into a target cell is referred to as transformation. In certain embodiments, the present invention includes a vector comprising a DNA sequence of a coding serine protease polypeptide (e.g., a precursor or mature serine protease polypeptide) that is effectively connected to a suitable leader sequence (e.g., secretion, a signal peptide sequence, etc.), the vector being able to achieve expression of the DNA sequence in a suitable host, and folding and translocation of the recombinant polypeptide chain.

[0025] As used herein, in the context of introducing a nucleic acid sequence into a cell, the term "introducing" refers to any method suitable for transferring a nucleic acid sequence into a cell. Such methods of introduction include, but are not limited to, protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. Transformation refers to a genetic change in a cell caused by uptake, optional genomic incorporation, and expression of genetic material (e.g., DNA).

[0026] "Expression cassette" or "expression vector" refers to a nucleic acid construct or vector (e.g., exogenous nucleic acid or transgene) produced recombinantly or synthetically for expressing a target nucleic acid in a target cell. Typically, the target nucleic acid expresses a target protein. Typically, the expression vector or expression cassette comprises a promoter nucleotide sequence that drives or promotes the expression of the exogenous nucleic acid. Typically, the expression vector or expression cassette also includes other specific nucleic acid elements that allow transcription of specific nucleic acids in the target cell. The recombinant expression cassette can be integrated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Some expression vectors have the ability to integrate and express heterologous DNA fragments in a host cell or a host cell genome. Many prokaryotic and eukaryotic expression vectors are commercially available. It is within the knowledge of those skilled in the art to select a suitable expression vector for expressing a protein from the nucleic acid sequence incorporated into the expression vector.

[0027] As used herein, a nucleic acid is "effectively connected" to another nucleic acid sequence when the nucleic acid is placed in a functional relationship with the other nucleic acid sequence. For example, if a promoter affects the transcription of a coding sequence, a promoter or enhancer is effectively connected to a nucleotide coding sequence. If a ribosome binding site is positioned so as to promote the translation of a coding sequence, the ribosome binding site can be effectively connected to a coding sequence. Typically, "effectively connected" DNA sequences are contiguous. However, enhancers do not need to be contiguous. Connection is achieved by connecting at a convenient restriction site. If such sites do not exist, synthetic oligonucleotide adapters or joints can be used according to conventional practice.

[0028] As used herein, the term "gene" refers to a polynucleotide (e.g., DNA segment) that encodes a polypeptide and includes regions before and after the coding region. In some cases, a gene includes intervening sequences (introns) between individual coding segments (exons).

[0029] As used herein, when used with respect to a cell, "recombinant" typically indicates that the cell has been modified by the introduction of an exogenous nucleic acid sequence, or that the cell is derived from a cell so modified. For example, a recombinant cell may contain genes that do not exist in the same form in a cell in a natural (non-recombinant) form, or a recombinant cell may contain native genes (found in the native form of the cell) that have been modified and reintroduced into the cell. A recombinant cell may contain nucleic acids endogenous to the cell that have been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene substitution, site-specific mutations, and related techniques known to those of ordinary skill in the art. Recombinant DNA technology includes techniques for producing recombinant DNA in vitro and transferring the recombinant DNA into cells where it can be expressed or propagated to produce recombinant polypeptides. "Recombination" (recombination and recombination) of polynucleotides or nucleic acids generally refers to assembling or combining two or more nucleic acids or polynucleotide chains or fragments to produce new polynucleotides or nucleic acids.

[0030] If a nucleic acid or polynucleotide can be transcribed and / or translated to produce a polypeptide or fragment thereof in its native state or when manipulated by methods known to those skilled in the art, then the nucleic acid or polynucleotide is said to "encode" the polypeptide. The antisense strand of such a nucleic acid is also said to encode the sequence.

[0031] The terms "host strain" and "host cell" refer to a suitable host for an expression vector comprising a DNA sequence of interest.

[0032] "Protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. The terms "protein" and "polypeptide" are used interchangeably herein. Single-letter and 3-letter codes for amino acids defined in accordance with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) are used throughout this disclosure. The single letter X refers to any one of the twenty amino acids. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

[0033] "Leader sequence" or "leader peptide sequence" refers to an amino acid sequence between the signal peptide sequence and the mature protease sequence that is necessary for the correct folding and secretion of the protease; they are sometimes referred to as intramolecular chaperones. Cleavage of the leader sequence or leader peptide sequence produces a mature active protease. Bacterial serine proteases are usually expressed as proenzymes.

[0034] The terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that can participate in the secretion or directional transport of a mature or precursor form of a protein. Typically, the signal sequence is located at the N-terminus of the precursor or mature protein sequence. The signal sequence can be endogenous or exogenous. Signal sequences are generally not present in mature proteins. Typically, after protein transport, the signal sequence is cut from the protein by a signal peptidase.

[0035] The term "mature" form of a protein, polypeptide or peptide refers to the functional form of the protein, polypeptide or peptide without the signal peptide sequence and the leader peptide sequence.

[0036] The term "precursor" form of a protein or peptide refers to a mature form of the protein having a leader sequence operatively linked to the amino or carboxyl terminus of the protein. The precursor may also have a "signal" sequence operatively linked to the amino terminus of the leader sequence. The precursor may also have additional polypeptides involved in post-translational activity (e.g., polypeptides that are cleaved from it to leave the mature form of the protein or peptide).

[0037] With respect to amino acid sequences or nucleic acid sequences, the term "wild-type" means that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance (e.g., a protein or polynucleotide sequence) found in nature. In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., a recombinant polynucleotide and protein sequence produced in a laboratory, or a modification of a wild-type sequence).

[0038] As used herein, with respect to amino acid residue positions, "corresponding to" or "corresponds to" refers to the amino acid residue at the recited position in a protein or peptide, or an amino acid residue that is similar, homologous, or identical to the recited residue in a protein or peptide. As used herein, a "corresponding region" generally refers to an analogous position in a related protein or reference protein.

[0039] The terms "derived from" and "obtained from" refer not only to proteins produced or producible by strains of the organism in question, but also to proteins encoded by DNA sequences isolated from such strains and produced in host organisms containing such DNA sequences. In addition, the term refers to proteins encoded by synthetic and / or cDNA-derived DNA sequences and having the identifying characteristics of the protein in question. For example, "proteases derived from Bacillus" refer to those enzymes having proteolytic activity naturally produced by Bacillus, as well as serine proteases, such as those produced by Bacillus sources but produced by other host cells transformed with nucleic acids encoding serine proteases using genetic engineering techniques.

[0040] The term "identity" in the context of two polynucleotide or polypeptide sequences refers to the nucleic acids or amino acids in the two sequences that are the same when aligned for maximum correspondence as measured using a sequence comparison or analysis algorithm described below or known in the art.

[0041] "% identity" or "percent identity" or "PID" refers to protein sequence identity. Percent identity can be determined using standard techniques known in the art. The percentage of amino acid identity shared by a target sequence can be determined by aligning the sequences to directly compare the sequence information, for example by using programs such as BLAST, MUSCLE, or CLUSTAL. The BLAST algorithm is described in, for example, Altschul et al., J Mol Biol [Journal of Molecular Biology], 215:403-410 (1990) and Karlin et al., Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 90:5873-5787, 1993). The percentage (%) amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues in the "reference" sequence (including any gaps created by the program for optimal / maximal alignment). The BLAST algorithm refers to the "reference" sequence as the "query" sequence.

[0042] As used herein, "homologous proteins" or "homologous proteases" refer to proteins with different similarities in primary, secondary and / or tertiary structures. When comparing proteins, protein homology can refer to the similarity of linear amino acid sequences. Homology can be determined by amino acid sequence alignment, for example, using programs such as BLAST, MUSCLE or CLUSTAL. Homology searches for protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST using a threshold (E value cutoff) of 0.001. (Altschul et al., "Gapped BLAST and PSI BLAST a new generation of protein database search programs", Nucleic Acids Res, Group 1; 25(17):3389-402 (1997)). The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most related sequences based on biological similarity, but is not recommended for query sequences of less than 20 residues (Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997 and Schaffer et al., Nucleic Acids Res. 29:2994-3005, 2001). Exemplary default BLAST parameters for nucleic acid sequence searches include: Neighborhood Wordsize Threshold=11; E-value Cutoff=10; Scoring Matrix=NUC.3.1 (Match=1, Mismatch=-3); GapOpening=5; and Gap Extension=2. Exemplary default BLAST parameters for amino acid sequence searches include: Wordsize=3; E-value Cutoff=10; Scoring Matrix=BLOSUM62; GapOpening=11; and Gap Extension=1. Using this information, protein sequences can be grouped and / or a phylogenetic tree can be constructed therefrom. Amino acid sequences can be input in programs such as Vector NTI Advance suites, and a guide tree (Saitou and Nei, Mol Biol Evol [Molecular Biology and Evolution], 4:406-425, 1987) can be created using the Neighbor Joining (NJ) method. Tree structure can be calculated using the Kimura correction for sequence distance and ignoring the position with a vacancy. Programs such as AlignX can display the distance value calculated in brackets after the molecular name displayed on the phylogenetic tree.

[0043] Understanding the homology between molecules can reveal the evolutionary history of the molecules as well as information about their functions; if a newly sequenced protein is homologous to an already characterized protein, there is a strong indication of the biochemical function of the new protein. The most basic relationship between two entities is homology; if two molecules originate from a common ancestor, they are said to be homologous. Homologous molecules or homologs can be divided into two categories: paralogs and orthologs. Paralogs are homologs that exist within one species. Paralogs often differ in their detailed biochemical functions. Orthologs are homologs that exist in different species and have very similar or identical functions. A protein superfamily is the largest grouping (branch) of proteins from which a common ancestor can be inferred. Usually this common ancestor is based on sequence alignments and mechanical similarities. Typically, a superfamily contains several protein families that show sequence similarity within the family. Based on the MEROPS protease classification system, the term "protein clan" is often used for the protease superfamily.

[0044] The CLUSTAL W algorithm is another example of a sequence alignment algorithm (see Thompson et al., Nucleic Acids Res 22:4673-4680, 1994). The default parameters for the CLUSTAL W algorithm include: Gap Open Penalty = 10.0; Gap Extension Penalty = 0.05; Protein Weight Matrix = BLOSUM Series; DNA Weight Matrix = IUB; Delayed Divergent Sequence % = 40; Gap Separation Distance = 8; DNA Transition Weight = 0.50; List Hydrophilic Residues = GPSNDQEKR; Use Negativity Matrix = Off; Switch Special Residue Penalty = On; Switch Hydrophilic Penalty = On; and Switch End Gap Separation Penalty = Off. In the CLUSTAL algorithm, deletions occurring at either end are included. For example, a variant having five amino acid deletions at either end of a 500 amino acid polypeptide (or within a polypeptide) has a percent sequence identity of 99% (495 / 500 identical residues x 100) relative to a "reference" polypeptide. Such variants would be encompassed by variants having "at least 99% sequence identity" to the polypeptide.

[0045] When nucleic acid or polynucleotide is separated from other components (including but not limited to, for example, other proteins, nucleic acids, cells, etc.) at least in part or completely, the nucleic acid or polynucleotide is "separated". Similarly, when polypeptide, protein or peptide is separated from other components (including but not limited to, for example, other proteins, nucleic acids, cells, etc.) at least in part or completely, the polypeptide, protein or peptide is "separated". In mole, the species separated in the composition is more abundant than other species. For example, the species separated can include at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% (in mole) of all macromolecular species present. Preferably, the species of interest is purified to basic homogeneity (that is, the contaminant species can not be detected in the composition by conventional detection methods). Purity and homogeneity can be determined using a variety of techniques well known in the art such as agarose or polyacrylamide gel electrophoresis of nucleic acid or protein samples, respectively, followed by visualization after staining. If necessary, high resolution techniques such as high performance liquid chromatography (HPLC) or similar methods can be used to purify the material.

[0046] The term "purified" as applied to nucleic acids or polypeptides generally refers to a nucleic acid or polypeptide that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms discrete bands in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that produces essentially one band in an electrophoretic gel is "purified." A purified nucleic acid or polypeptide is at least about 50% pure, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percentages by weight on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of that molecule after application of a purification or enrichment technique. The term "enriched" refers to the presence in a composition of a compound, polypeptide, cell, nucleic acid, amino acid or other specific substance or component at a relative or absolute concentration greater than that of the starting composition.

[0047] As used herein, the term "functional assay" refers to an assay that provides an indication of protein activity. In some embodiments, the term refers to an assay system in which a protein is analyzed for its ability to function in its usual capacity. For example, in the case of a protease, a functional assay involves determining the effectiveness of the protease in hydrolyzing a protein substrate.

[0048] The term "cleaning activity" refers to the cleaning performance achieved by a serine protease polypeptide or a reference subtilisin under the conditions mainly during the disclosed proteolysis, hydrolysis, cleaning or other processes. In certain embodiments, the cleaning performance of a serine protease or a reference subtilisin can be determined by using various assays for cleaning one or more enzyme-sensitive stains (e.g., stains caused by food, grass, blood, ink, milk, oil and / or egg protein) on an article or surface. The cleaning performance of one or more subtilisin variants as described herein or a reference subtilisin can be determined by subjecting the stain on an article or surface to one or more standard wash conditions and by using various chromatograms, spectrophotometers or other quantitative methods to evaluate the degree of stain removal. Exemplary cleaning assays and methods are known in the art, and include but are not limited to those described in WO 99 / 34011 and US 6,605,458, and those cleaning assays and methods included in the examples provided below.

[0049] The term "cleaning effective amount" of one or more subtilisin variants or reference subtilisin described herein refers to the amount of a protease that achieves a desired level of enzymatic activity in a particular cleaning composition. Such an effective amount can be readily determined by one of ordinary skill in the art and is based on a number of factors, such as the specific protease used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, tablet, stick) composition is desired, etc.

[0050] The term "cleaning auxiliary material" refers to any liquid, solid or gaseous material included in the cleaning compositions except one or more subtilisin variants as described herein. In certain embodiments, the cleaning compositions of the present disclosure include one or more cleaning auxiliary materials. Typically, depending on the specific type and form (e.g., liquid, granule, powder, rod-shaped, paste, spray, tablet, gel, foam or other compositions) of the cleaning compositions, each cleaning auxiliary material is selected. Preferably, each cleaning auxiliary material is compatible with the protease used in the compositions.

[0051] Cleaning compositions and cleaning formulations include any composition suitable for cleaning, bleaching, disinfecting and / or sterilizing any object, article and / or surface. Such compositions and formulations include, but are not limited to, for example, liquid and / or solid compositions, including cleaning or detergent compositions (e.g., liquid, tablet, gel, stick, granular and / or solid laundry cleaning or detergent compositions) and fine fabric detergent compositions; hard surface cleaning compositions and formulations, such as for glass, wood, ceramic and metal countertops and windows; carpet cleaners; stove cleaners; fabric fresheners; fabric softeners; and textiles, clothing synergistic cleaning or detergent compositions, clothing additive cleaning compositions and clothing pre-spotter cleaning compositions; dishwashing compositions, including hand-washing or manual dishwashing compositions (e.g., "hand-washing" or "manual" dishwashing detergents) and automatic dishwashing compositions (e.g., "automatic dishwashing detergents"). The present invention can also use single dosage unit forms, including but not limited to pills, tablets, gelca or other single dosage units such as pre-measured powders or liquids.

[0052] Unless otherwise specified, cleaning compositions or cleaning formulations as used herein include general-purpose or heavy-duty detergents in granular or powder form, especially cleaning detergents; general-purpose detergents in liquid, granular, gel, solid, tablet, paste or unit dose form, especially so-called heavy-duty liquid (HDL) detergents or heavy-duty dry cleaning (HDD) detergent types; liquid fine fabric detergents; hand or manual dishwashing detergents, including those of the high-sudsing type; hand or manual dishwashing detergents, automatic dishwashing detergents, or tableware or tableware detergents, including various tablet, powder, solid, granular, liquid, gel and rinse aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand wash types, cleaning bars, mouthwashes, denture cleaners, car wash shampoos, carpet shampoos, bathroom cleaners; hair shampoos and / or hair dyes for humans and other animals; body washes and bubble baths and metal cleaners; and cleaning aids, such as bleach additives and "stain bars" or pre-treatment types. In some embodiments, particulate compositions are in a "compacted" form; in some embodiments, liquid compositions are in a "concentrated" form.

[0053] As used herein, "fabric cleaning compositions" include hand and machine laundry detergent compositions, including laundry additive compositions and compositions suitable for soaking and / or pretreatment of stained fabrics (e.g., clothes, linens, and other textile materials).

[0054] As used herein, "non-fabric cleaning compositions" include non-textile (i.e., non-fabric) surface cleaning compositions, including, but not limited to, for example, hand washing or manual or automatic dishwashing detergent compositions, oral cleaning compositions, denture cleaning compositions, contact lens cleaning compositions, wound debridement compositions, and personal cleansing compositions.

[0055] As used herein, the term "detergent composition" or "detergent formulation" is used with respect to compositions intended for use in washing media for cleaning contaminated or dirty objects (including specific fabrics and / or non-fabric objects or articles). In certain embodiments, detergents of the present disclosure include one or more subtilisin variants as described herein, and additionally include one or more surfactants, one or more transferases, hydrolases, oxidoreductases, builders (e.g., builder salts), bleaching agents, bleach activators, bluing agents, fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants and / or solubilizing agents. In some cases, the builder salt is a mixture of silicate and phosphate, preferably with more silicate (e.g., sodium metasilicate) than phosphate (e.g., sodium tripolyphosphate). Some embodiments relate to cleaning compositions or detergent compositions, do not include any phosphate (e.g., phosphate or phosphate builder).

[0056] As used herein, the term "bleaching" refers to treating a material (e.g., fabric, clothing, pulp, etc.) or a surface for a sufficient period of time and / or treating a material (e.g., fabric, clothing, pulp, etc.) or a surface under appropriate pH and / or temperature conditions to achieve whitening (i.e., whitening) and / or cleaning of the material. Examples of chemicals suitable for bleaching include, but are not limited to, for example, ClO2, H2O2, peracids, NO2, etc.

[0057] As used herein, " washing performance " of protease (for example, one or more subtilisin variants as described herein or its recombinant polypeptide or active fragment) refers to the contribution that one or more subtilisin variants as described herein provide other cleaning performance to washing compared with the detergent that does not add one or more subtilisin variants as described herein to composition.Compare washing performance under relevant washing conditions.In some test systems, other relevant factors, for example detergent composition, foam concentration (sud concentration), water hardness, washing mechanics, time, pH and / or temperature can be controlled in such a way as follows: imitate one or more conditions typical for household applications in certain market segments (for example hand-washing or manual dishwashing, automatic dishwashing, dishwashing cleaning, tableware cleaning, fabric cleaning, etc.).

[0058] The term "relevant washing conditions" as used herein denotes the conditions actually used in households in the hand dishwashing, automatic dishwashing or laundry detergent segments, in particular washing temperature, time, washing mechanics, foam concentration, detergent type and water hardness.

[0059] As used herein, the term "disinfecting" refers to removing contaminants from a surface, as well as inhibiting or killing microorganisms on the surface of an item.

[0060] The "compact" form of the cleaning composition herein is best reflected by density, and in terms of the composition, by the amount of inorganic filler salts. Inorganic filler salts are conventional ingredients of detergent compositions in powder form. In conventional detergent compositions, filler salts are present in considerable amounts, typically from about 17% to about 35% (by weight) of the total composition. In contrast, in compact compositions, filler salts are present in an amount of no more than about 15% of the total composition. In certain embodiments, filler salts are present in an amount of no more than about 10% or more preferably about 5% of the composition (by weight). In certain embodiments, the inorganic filler salts are selected from alkali salts and alkaline earth metal salts of sulfates and chlorides. In certain embodiments, the filler salt is sodium sulfate.

[0061] One or more subtilisin protease variants that can be used for cleaning applications and cleaning methods and multiple industrial applications are disclosed herein. One or more separated, recombinant, substantially pure or non-naturally occurring subtilisin protease variants are disclosed herein. In certain embodiments, one or more subtilisin protease variants as described herein can be used for cleaning applications, and can be incorporated into the cleaning compositions that can be used for cleaning articles or surface methods that are needed therefor.

[0062] One embodiment provides one or more subtilisin protease variants comprising two, three, or four or more variations relative to SEQ ID NO:2 at positions selected from the group consisting of: (i) 1, 3, 9, 10, 15, 17, 19, 22, 24, 25, 26, 27, 28, 30, 35, 37, 38, 43, 45, 48, 68, 71, 74, 76, 77, 78, 86, 87, 91, 95, 96, 98, 99, 100, 102, 103, 108, 111, 114, 115, 120, 123, 125, 126, 127, 128, 129, 130, 136, 143, 146, 147, 151, 155, 160, 165, 183, 184, 187, 193, 202, 203, 210, 217, 234, 238, 239, 240, 242, 247, 250, 251, 255, 258, 259, 260 and 274; (ii) 1, 3, 9, 15, 22, 24, 28, 30, 35, 37, 45, 48, 68, 71, 74, 76, 77, 78, 86, 87, 95, 96, 98, 99, 100, 108, 114, 115, 120, 123, 125, 126, 127, 128, 129, 130, 14 3, 146, 147, 151, 155, 165, 183, 184, 187, 193, 202, 203, 210, 217, 234, 238, 239, 240, 242, 247, 250, 255, 258, 259, 260 and 274; (iii) 1, 3, 15, 35, 68, 71, 77, 86, 87, 95, 99, 115, 123, 127, 128, 147, 155, 165, 184, 202, 210, 217, 234, 239, 242, 250, 258 and 274; or (iv) 3, 6 8, 77, 86, 99, 115, 123, 127, 128, 165, 184, 202, 210, 217 and 258; provided that one or more of the two, three or four or more variations is a non-naturally occurring amino acid; wherein the variant has 85% or more amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14;and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2. Another embodiment relates to one or more subtilisin protease variants comprising an amino acid sequence comprising two, three, or four or more variants at positions corresponding to SEQ ID NO: 2, wherein the positions are selected from: (i) 1, 3, 9, 10, 15, 22, 24, 26, 27, 28, 29, 30, 31, 35, 37, 40, 43, 45, 48, 52, 68, 71, 76, 77, 78, 86, 87, 95, 96, 98, 99, 100, 101, 102, 103, 105, 108, 111, 114, 115, 117, 119, 123, 125, 126, 127, 128, 129, 130, 136, 143, 146, 147, 148, 150 47, 151, 155, 158, 160, 161, 165, 184, 187, 193, 202, 203, 204, 210, 211, 216, 217, 234, 238, 239, 242, 243, 250, 255, 258, 259, 260, 264 and 274; (ii) 1, 3, 9, 10, 15, 22, 24, 26, 28, 29, 30, 31, 35, 37, 40, 45, 48, 52, 68, 71, 76, 77, 78, 86, 87, 95, 96, 98, 99, 100, 101, 102, 105, 108, 111, 114, 115, 117, 119, 123, 125, 126, 127, 128, 129, 130, 136, 143, 146, 147, 151, 155, 158, 160, 161, 165, 184, 187, 193, 202, 203, 204, 210, 211, 216, 217, 234, 238, 239, 242, 243, 250, 255, 258, 259, 260, 264 and 274; (iii) 1, 3, 9, 10, 24, 26, 29, 30, 31, 35, 37, 40, 45, 48, 52, 68, 71, 77, 78, 86, 87, 95, 96, 98, 99, 100, 101, 102, 105, 108, 111, 115, 117, 119, 123, 127, 128, 129, 130, 136, 143, 146, 147, 151, 155, 158, 160, 161, 165, 184, 187, 193, 202, 203, 204, 210, 211, 216, 217, 234, 238, 239, 242, 243, 250, 258, 259, 260, 264, and 274;(iv) 3, 9, 10, 35, 68, 77, 78, 86, 87, 99, 101, 115, 123, 127, 128, 165, 184, 187, 202, 203, 210, 217, 242, 250, 258, and 264; or (v) 3, 68, 77, 78, 123, 127, 128, 165, 184, 202, 210, 217, and 258; provided that two, three, or four or more of the variations One or more of the above are artificial mutations or substitutions; wherein the variant has at least 85% but less than 100% amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3, and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13, and motif 14; and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2. ;

[0063] Still another embodiment provides one or more subtilisins comprising two, three, or four or more variations relative to SEQ ID NO:2 selected from the group consisting of: (i) 1Q, 3Q / V, 9E / T, 10M, 15I / V, 17H, 19E, 22Y, 24N, 25D, 26R, 27R, 28A, 30T, 35A, 37T, 38G, 43A, 45I / R, 48I, 68S, 71A, 74G, 76K, 77D / H / N / Q / S, 78I, 86H / N / R, 87S / T, 91G, 95A / Q, 96S, 98H / K / R, 99L / S, 100N / R, 102R, 103I, 108H / K, 111Q, 114N, 115F, 120 A, 123I, 125N, 126Q, 127F / T, 128P / S, 129T, 130S, 136A / R, 143V, 146A / S, 147L, 151G, 155A / E / G, 160C / R, 165A / E / Q / S, 183A / G, 184Q, 187P, 1 93D,202V,203E / N,210E / H / P,217S,234I / W,238R / T,239K / S / T,240N,242G,247W,250G,251E,255R,258E / H / P,259P,260W and 274A / F; (ii)1Q, 3Q / V, 9E, 15I, 22Y, 24N, 28A, 30T, 35A, 37T, 45I, 48I, 68S, 71A, 74G, 76K, 77D / H / N / S, 78I, 86H / N / R, 87S / T, 95A / Q, 96S, 98K, 99L / S, 100N, 10 8H / K, 114N, 115F, 120A, 123I, 125N, 126Q, 127F / T, 128P / S, 129T, 130S , 143V, 146A / S, 147L, 151G, 155A / E / G, 165A / E / Q / S, 183G, 184Q, 187P, 193D, 202V, 203E / N, 210P, 217S, 234I / W, 238R / T, 239K / S / T, 240N, 242G, 247W, 250G, 255R, 258P, 259P, 260W and 274A; (iii) 1Q, 3V, 15I, 35A, 68S, 71A, 77N, 86H, 87S, 95A, 99S, 115F, 123I, 127T, 128P / S, 147L, 155G, 165Q / S, 184Q, 202V, 210P, 217S, 234W, 239S, 242G, 250G, 258P and 274A;or (iv) 3V, 68S, 77N, 86H, 99S, 115F, 123I, 127T, 128P, 165Q, 184Q, 202V, 210P, 217S and 258P; provided that one or more of the two, three or four or more variations is a non-naturally occurring amino acid; wherein the variant has 85% or more amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14; and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2.;

[0064] Yet another embodiment provides one or more subtilisin protease variants comprising an amino acid sequence comprising two, three, or four or more amino acid substitutions selected from the group consisting of: (i) A / G1, T3, P9, L / Q10, K15, H / Q17, Q19, K22, A24, N25, V26, K27, G / V28, I / V30, I35, A / S / T37, S38, N / K43, V45, A48, A68, V71, L74, N76, S / T77, I / T78, N / S86, V87, A / I91, L95, N96 , S98, G99, S100, S / T102, Y103, S108, E111, T114, A / Q / T115, V120, M123, L125, G126, G127, A / P / T128, S129, G / V130, Q136, A / S143 , A / I / V146, V147, A / S151, S155, N / S / Y160, G165, G / K / N / S183, N / Q184, S187, A / P / S193, K194, A / S / V202, G / S203, S / T210, S211, N2 17. L234, P238, A / N / T239, A242, D / N / T247, S250, R / S251, N / Y255, D / S258, S259, F260 and Q274; (ii) A / G1, T3, P9, K15, K22, A24, G / V 28. I / V30, I35, A / S / T37, V45, A48, A68, V71, L74, N76, S / T77, I / T78, N / S86, V87, L95, N96, S98, G99, S100, S108, T114, A / Q / T115, V120, M123, L125, G126, G127, A / P / T128, S129, G / V130, A / S143, A / I / V146, V147, A / S151, S155, G165, K / S183, N184, S187, A / P / S1 93, K194, A / S / V202, G / S203, S / T210, S211, N217, L234, P238, A / N / T239, A242, D / N / T247, S250, N / Y255, D / S258, S259, F260 and Q274;or (iii) A / G1, T3, K15, I35, A68, V71, S / T77, N / S86, V87, L95, G99, A / Q / T115, M123, G127, A / P / T128, V147, S155, G165, N184, A / S / V202, S / T210, N217, L234, A / N / T239, A242, S250, D / S258, and Q274; or (iv) T3, A68, S / T77, N / S86, G99, A / Q / T115, M123, G127, A / P / T128, G16 5, N184, A / S / V202, S / T210, N217 and D / S258; provided that one or more of the two, three or four or more substitutions are non-naturally occurring amino acids; wherein the variant has 85% or more amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14; and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:2. Still another embodiment provides one or more subtilisin variants comprising an amino acid sequence comprising two, three, or four or more amino acid substitutions selected from the group consisting of: (i) A / G1, T3, P9, L10, K15, H / Q17, Q19, K22, A24, N25, V26, K27, G / V28, I / V30, I35, A / S / T37, S38, N / K43, V45, A48, A68, V71, L74, N76, T77, T78, N / S86, V87, A91, L95, N96, S98, G99, S100, S / T102, Y103, S108, E111, T114, Q / T115, V120, M123, L125, G126, G127, A / P128, S129, G / V13 0. Q136, A143, A / I / V146, V147, A / S151, S155, N / S160, G165, K / S183, N18 4. S187, A / S193, K194, A / V202, G / S203, S / T210, S211, N217, L234, P238, N / T239, A242, N / T247, S250, R / S251, N / Y255, D / S258, S259, F260 and Q274;(ii)A / G1, T3, P9, K15, K22, A24, G / V28, I / V30, I35, A / S / T37, V45, A48, A68, V71, L74, N76, T77, T78, N / S86, V87, L95, N96, S98, G99, S100, S108, T114, Q / T115, V120, M123, L125, G126, G127, A / P128, S129, G / V130, A143, A / I / V14 6. V147, A / S151, S155, G165, K / S183, N184, S187, A / S193, K194, A / V202, G / S203, S / T210, S211, N217, L234, P238, N / T239, A242, N / T247, S250, N / Y255, D / S258, S259, F260 and Q274; (iii) A / G1, T3, K15, I35, A68, V71, T77, N / S86, V8 7. L95, G99, Q / T115, M123, G127, A / P128, V147, S155, G165, N184, A / V202, S / T210, N217, L234, N / T239, A242, S250, D / S258, and Q274; or (iv) T3, A68, T77, N / S86, G99, Q / T115, M123, G127, A / P128, G165, N184, A / V202, S / T210, N217, and D / S258; provided that one or more of the two, three or four or more substitutions is a non-naturally occurring amino acid; wherein the variant has 85% or more amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14; and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2. ;

[0065] Another embodiment provides one or more subtilisin variants comprising an amino acid sequence comprising two, three, or four or more amino acid substitutions selected from the group consisting of: (i) 1Q, 3Q / V, 9E / T, 10M, 15I / V, 17H, 19E, 22Y, 24N, 25D, 26R, 27R, 28A, 30T, 35A, 37T, 38G, 43A, 45I / R, 48I, 68S, 71A, 74G, 76K, 77D / H / N / Q / S, 78I, 86H / N / R / S, 87S / T, 91G, 95A / Q, 96S, 98H / K / R, 99L / S, 100N / R, 102R, 103R, 3I, 108H / K, 111Q, 114N, 115F, 120A, 123I, 125N, 126Q, 127F / T, 128P / S, 12 9T, 130S, 136A / R, 143V, 146A / S, 147L, 151G, 155A / E / G, 160C / R, 165A / E / Q / S, 183A / G, 184Q, 187P, 193D, 194E, 202V, 203E / N, 210E / H / P, 211N, 217S, 234I / W, 238R / T, 239K / S / T, 242G, 247W, 250G, 251E, 255R, 258E / H / P, 259P , 260W and 274A / F; (ii) 1Q, 3Q / V, 9E, 15I, 22Y, 24N, 28A, 30T, 35A, 37T, 45I, 4 8I, 68S, 71A, 74G, 76K, 77D / H / N / S, 78I, 86H / N / R / S, 87S / T, 95A / Q, 96S, 98 K, 99L / S, 100N, 108H / K, 114N, 115F, 120A, 123I, 125N, 126Q, 127F / T, 128P / S, 129T, 130S, 143V, 146A / S, 147L, 151G, 155A / E / G, 165A / E / Q / S, 183G, 1 84Q, 187P, 193D, 194E, 202V, 203E / N, 210P, 211N, 217S, 234I / W, 238R / T, 239K / S / T, 242G, 247W, 250G, 255R, 258P, 259P, 260W and 274A; (iii) 1Q, 3V, 15I, 35A, 68S, 71A, 77N, 86H, 87S, 95A, 99S, 115F, 123I, 127T, 128P / S, 147L, 155G, 165Q / S, 184Q, 202V, 210P, 217S, 234W, 239S, 242G, 250G, 258P and 274A;or (iv) 3V, 68S, 77N, 86H, 99S, 115F, 123I, 127T, 128P, 165Q, 184Q, 202V, 210P, 217S, and 258P; provided that one or more of the two, three, or four or more substitutions is a non-naturally occurring amino acid; wherein the variant has 85% or more amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3, and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13, and motif 14; and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2.;

[0066] Still further embodiments relate to one or more subtilisin variants comprising an amino acid sequence comprising two, three, or four or more amino acid substitutions at two, three, or four or more positions corresponding to SEQ ID NO: 2, wherein the substitutions are selected from: (i) 1Q, 3Q / V, 9E, 10M, 15I, 22Y, 24N / Q, 26Q, 27R, 28A, 29S, 30T, 31I, 35A, 37T, 40E, 43A, 45I / Q / R, 48 I / T / V, 52R, 68S, 71A, 76K, 77D / E / H / N / Q / S, 78I, 86H / N / R, 87S / T, 95A / F / Q, 96S, 98H / R, 99A / E / Q / S, 100N, 101A, 102D / T, 103I, 105N, 108H / K, 111Q, 114N, 115F, 117S, 11 9Q, 123I, 125N, 126Q, 127E / F / Q / S / T / V, 128P / R / S, 129H / R, 130S, 136N / R, 143Q / V, 1 46A / S, 147L, 151G, 155E / G / N, 158D / N / Q, 160C / D / K, 161D / Q, 165A / E / Q / S, 184E / Q, 1 87E / P, 193D / Q, 202V, 203E / N / Q, 204I, 210E / H / I / P, 211S, 216Q, 217S, 234I / W, 238Q / R, 239K / S / T, 242G / N, 243I, 250G, 255R, 258D / E / P, 259E / P, 260W, 264H / Q / S and 274A;(ii) 1Q, 3Q / V, 9E, 10M, 15I, 22Y, 24N / Q, 26Q, 28A, 29S, 30T, 31I, 35A, 37T, 40E, 45I / Q / R, 48I / T / V, 52R, 68S, 71A, 76K, 77D / E / H / N / S, 78I, 86H / N / R, 87S / T, 95A / F / Q, 96S, 98K / R, 99A / E / Q / S, 100N, 101A, 102D / T, 105N, 108H / K, 111Q, 114N, 115F, 117S, 119Q, 123I, 125N, 126Q, 127E / F / Q / S / T / V, 128P / R / S, 129H / R, 130S, 136N, 143Q / V, 146A / S, 147L, 151G, 155E / G / N, 158D / N / Q, 160D / K, 161D / Q, 165A / E / Q / S, 184E / Q, 187E / P, 193D / Q, 194E, 202V, 203E / N / Q, 204I, 210I / P, 211S, 216Q, 217S, 234I / W, 238Q / R, 239K / S / T, 242G / N, 243I, 250G, 255R, 258D / P, 259E / P, 260W, 264H / Q / S and 274A; (iii) 1Q, 3V, 9E, 10M, 24Q, 26Q, 29S, 30T, 31I, 35A, 37T, 40E, 45I / Q / R, 48T / V, 52R, 68S, 71A, 77E / N, 78I, 86H / R, 87S, 95A / F, 96S, 98R, 99A / E / Q / S, 100N, 101A, 102D / T, 105N, 108H, 111Q, 115F, 117S, 119Q, 123I, 127E / Q / S / T / V, 128P / R, 129H / R, 130S, 136N, 143Q / V, 146A, 147L, 151G, 155G / N, 158D / N / Q, 160D / K, 161D / Q, 165A / E / Q, 184E / Q, 187E / P, 193Q, 202V, 203E / N / Q, 204I, 210I / P, 211S, 216Q, 217S, 234W, 238Q, 239S, 242G / N, 243I, 250G, 258D / P, 259E / P, 260W, 264H / Q / S and 274A;(iv) 3V, 9E, 10M, 35A, 68S, 77N, 78I, 86H, 87S, 99Q / S, 101A, 115F, 123I, 127S / T, 128P / R, 165E / Q, 184Q, 187E, 202V, 203E, 210P, 217S, 242G, 250G, 258D / P, and 264H; or (v) 3V, 68S, 77N, 78I, 123I, 127S, 128P, 165Q, 184Q, 202V, 210P, 217S, and 258D / P; provided that one or more of the two, three or four or more substitutions are artificial substitutions; wherein the variant has at least 85% but less than 100% amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14; and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2. ;

[0067] Still further embodiments provide one or more subtilisin variants comprising an amino acid sequence comprising two, three, or four or more amino acid substitutions selected from the group consisting of: (i) A / G1Q, T3Q / V, P9E / T, L / Q10M, K15I / V, Q17H, Q19E, K22Y, A24N, N25D, V26R, K27R, G / V28A, I / V30T, I35A, A / S37T, S38G, N / K43A, V45I / R, A48I, A68S, V71A, L74G, N76K, S / T77D / H / N / Q / S, T78I, N / S86H / N / R / S, V87S / T, A / I91G, L95A / Q, N96S, S98H / K / R, G99L / S, S100N / R, S / T102R, Y103I, S108H / K, E111Q, T114N, A / Q / T115F, V120A, M123I, L125N, G126Q, G127F / T, A / P / T128P / S, S129T, G / V130S, Q136A / R, A / S143V , A / I / V146A / S, V147L, A / S151G, S155A / E / G, N / S / Y160C / R, G165A / E / Q / S, G / K / N / S183A / G, N184Q, S 187P, A / P / S193D, K194E, A / S202V, G / S203E / N, S / T210E / H / P, S211N, N217S, L234I / W, P238R / T, A / N / T239K / S / T, A242G, D / N / T247W, S250G, R / S251E, N / Y255R, D / S258E / H / P, S259P, F260W and Q274A / F;(ii)A / G1Q, T3Q / V, P9E, K15I, K22Y, A24N, G / V28A, I / V30T, I35A, A / S37T, V45I, A48I, A68S, V71A, L74G, N76K, S / T77D / H / N / S, T78I, N / S86 H / N / R / S, V87S / T, L95A / Q, N96S, S98K, G99L / S, S100N, S108H / K, T114N, A / Q / T115F, V120A, M123I, L125N, G126Q, G127F / T, A / P / T128P / S, S 129T, G / V130S, A / S143V, A / I / V146A / S, V147L, A / S151G, S155A / E / G, G165A / E / Q / S, K / S183G, N184Q, S187P, A / P / S193D, K194E, A / S202V, G / S203E / N, S / T210P, S211N, N217S, L234I / W, P238R / T, A / N / T239K / S / T, A242G, D / N / T247W, S250G, N / Y255R, D / S258P, S259P, F260W and Q274A; (iii)A / G1Q, T3V, K15I, I35A, A68S, V71A, S / T77N, N / S86H, V87S, L95A, G99S, A / Q / T115F, M123I, G127T, A / P / T128P / S, V147L, S155G, G165 Q / S, N184Q, A / S202V, S / T210P, N217S, L234W, A / N / T239S, A242G, S250G, D / S258P and Q274A; or (iv) T3V, A68S, S / T77N, N / S86H, G99S, A / Q / T11 5F, M123I, G127T, A / T128P, G165Q, N184Q, A / S202V, S / T210P, N217S and D / S258P; provided that one or more of the two, three or four or more substitutions are non-naturally occurring amino acids; wherein the variant has 85% or more amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14;and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2. Still further embodiments provide one or more subtilisin protease variants comprising an amino acid sequence comprising two, three, or four or more amino acid substitutions selected from the group consisting of: (i) A / G1Q, T3Q / V, P9E / T, L10M, K15I / V, Q17H, Q19E, K22Y, A24N, N25D, V26R, K27R, G / V28A, I / V30T, I35A, A / S37T, S38G, N / K43A, V45I / R, A48I, A68S, V71A, L74G, N76K, T77D / H / N / Q / S, T78I, N / S86H / N / R / S, V87S / T, A91G, L95A / Q, N96S, S98H / K / R, G99L / S, S100N / R, S / T102R, Y103I, S108H / K, E111Q, T114N, Q / T115F, V120A, M123I, L125N, G126Q, G127F / T, A / P128P / S, S129T, G / V130S, Q136A / R, A143V, A / I / V146A / S, V147L, A / S151G, S155A / E / G, N / S160C / R, G165A / E / Q / S, K / S183A / G, N184Q , S187P, A / S193D, K194E, A202V, G / S203E / N, S / T210E / H / P, S211N, N217S, L234I / W, P238R / T, N / T239K / S / T, A242G, N / T247W, S250G, R / S251E, N / Y255R, D / S258E / H / P, S259P, F260W and Q274A / F;(ii)A / G1Q, T3Q / V, P9E, K15I, K22Y, A24N, G / V28A, I / V30T, I35A, A / S37T, V45I, A48I, A68S, V71A, L74G, N76K, T77D / H / N / S, T78I, N / S86 H / N / R / S, V87S / T, L95A / Q, N96S, S98K, G99L / S, S100N, S108H / K, T114N, Q / T115F, V120A, M123I, L125N, G126Q, G127F / T, A / P128P / S, S12 9T, G / V130S, A143V, A / I / V146A / S, V147L, A / S151G, S155A / E / G, G165A / E / Q / S, K / S183G, N184Q, S187P, A / S193D, K194E, A202V, G / S203E (iii) A / G 1Q, T3V, K15I, I35A, A68S, V71A, T77N, N / S86H, V87S, L95A, G99S, Q / T115F, M123I, G127T, A / P128P / S, V147L, S155G, G165Q / S, N184Q, A2 02V, S / T210P, N217S, L234W, N / T239S, A242G, S250G, D / S258P and Q274A; or (iv) T3V, A68S, T77N, N / S86H, G99S, Q / T115F, M123I, G127T, A128 P, G165Q, N184Q, A202V, S / T210P, N217S and D / S258P; provided that one or more of the two, three or four or more substitutions is a non-naturally occurring amino acid; wherein the variant has 85% or more amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14; and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2. ;

[0068] Even further embodiments relate to one or more subtilisin variants comprising an amino acid sequence comprising two, three or four or more amino acid substitutions selected from the group consisting of: (i) A / G1Q, T3Q / V, P / S9E, L / Q10M, K15I, K22Y, A24N / Q, V26Q, K27R, G / V28A, A / G29S, I / V30T, I / L31I, I35A, A / S37T, P / S / T40E, N / K43A, S / V45I / Q / R, A48I / T / V, A / S52R, A68S, V71A, N76K, S / T77D / E / H / N / Q / S, T78I, N / S86H / N / R, V87S / T, L95A / F / Q, N96S, S9 8H / R, G99A / E / Q / S, S100N, G101A, S / T102D / T, Y103I, A / G105N, S108H / K, E111Q, T114N, A / Q / S / T115F, G / N117S, D119Q, M123I, L125N, G126Q, G127E / F / Q / S / T / V, A / P / S128P / R / S, S129H / R, G / V130S, Q136N / R, A / S143Q / V, A / I / V146A / S, V147L, A / S151G, S155E / G / N, S158D / N / Q, N / S / Y160C / D / K, A / S / Q / T161D / Q, G165A / E / Q / S, N / Q184E / Q, S187E / P, A / P / S193D / Q, A / S202V, A / G / S203E / N / Q, V204I, S / T210E / H / I / P, N211S, L216Q, N217S, L234I / W, P238 Q / R, A / N / T239K / S / T, A242G / N, S243I, S250G, N / Y255R, D / S258D / E / P, S259E / P, F260W, K / N / R264H / Q / S and Q274A;(ii) A1Q, T3Q / V, P9E, L10M, K15I, K22Y, A24N / Q, V26Q, K27R, V28A, A29S, V30T, L31I, I35A, A37T, P40E, N43A, V45I / Q / R, A48I / T / V, A52R, A68S, V71A, N76K, S77D / E / H / N / Q / S, T78I, S86H / N / R, V87S / T, L95A / F / Q, N96S, S98H / R, G99A / E / Q / S, S100N, G101A, S102D / T, Y103I, G105N, S108H / K, E111Q, T114N, T115F, G117S, D119Q, M123I, L125N, G126Q, G127E / F / Q / S / T / V, A128P / R / S, S129H / R, G130S, Q136N / R, A143Q / V, V146A / S, V147L, A151G, S155E / G / N, S158D / N / Q, N160C / D / K, T161D / Q, G165A / E / Q / S, N184E / Q, S187E / P, A193D / Q, A202V, G203E / N / Q, V204I, T210E / H / I / P, N211S, L216Q, N217S, L234I / W, P238Q / R, N239K / S / T, A242G / N, S243I, S250G, Y255R, S258D / E / P, S259E / P, F260W, K264H / Q / S, and Q274A;(iii) A / G1Q, T3Q / V, P / S9E, L / Q10M, K15I, K22Y, A24N / Q, V26Q, G / V28A, A / G29S, I / V30T, I / L31I, I35A, A / S37T, P / S / T40E, S / V45I / Q / R, A48I / T / V, A / S52R, A68S, V71A, N76K, S / T77D / E / H / N / S, T78I, N / S86H / N / R, V87S / T, L95A / F / Q, N96S, S98R, G99A / E / Q / S, S100N, G101A, S / T102D / T, A / G105N, S108H / K, E111Q, T114N, A / Q / S / T115F, G / N117S, D119Q, M123I, L125N, G126Q, G127E / F / Q / S / T / V, A / P / S128P / R / S, S129H / R, G / V130S, Q136N, A / S143Q / V, A / I / V146A / S, V147L, A / S151G, S155E / G / N, S158D / N / Q, N / S / Y160D / K, A / S / Q / T161D / Q, G165A / E / Q / S, N / Q184E / Q, S187E / P, A / P / S193D / Q, A / S202V, A / G / S203E / N / Q, V204I, S / T210I / P, N211S, L216Q, N217S, L234I / W, P238Q / R, A / N / T239K / S / T, A242G / N, S243I, S250G, N / Y255R, D / S258D / P, S259E / P, F260W, K / N / R264H / Q / S, and Q274A;(iv) A1Q, T3Q / V, P9E, L10M, K15I, K22Y, A24N / Q, V26Q, V28A, A29S, V30T, L31I, I35A, A37T, P40E, V45I / Q / R, A48I / T / V, A52R, A68S, V71A, N76K, T77D / E / H / N / S, T78I, S86H / N / R, V87S / T, L95A / F / Q, N96S, S98R, G99A / E / Q / S, S100N, G101A, S102D / T, G105N, S108H / K, E111Q, T114N, T115F, G117S, D119Q, M123I, L125N, G126Q, G127E / F / Q / S / T / V, A128P / R / S, S129H / R, G130S, Q136N, A143Q / V, V146A / S, V147L, A151G, S155E / G / N, S158D / N / Q, N160D / K, T161D / Q, G165A / E / Q / S, N184E / Q, S187E / P, A193D / Q, A202V, G203E / N / Q, V204I, T210I / P, N211S, L216Q, N217S, L234I / W, P238Q / R, N239K / S / T, A242G / N, S243I, S250G, Y255R, S258D / P, S259E / P, F260W, K264H / Q / S and Q274A;(v) A / G1Q, T3V, P / S9E, L / Q10M, A24Q, V26Q, A / G29S, I / V30T, I / L31I, I35A, A / S37T, P / S / T40E, S / V45I / Q / R, A48T / V, A / S52R, A68S, V71A, S / T77E / N, T78I, N / S86H / R, V87S, L95A / F, N96S, S98R, G99A / E / Q / S, S100N, G101A, S / T102D / T, A / G105N, S108H, E111Q, A / Q / S / T115F, G / N117S, D119Q, M123I, G127E / Q / S / T / V, A / P / S128P / R, S129H / R, G / V130S, Q136N, A / S143Q / V, A / I / V146A, V147L, A / S151G, S155G / N, S158D / N / Q, N / S / Y160D / K, A / S / Q / T161D / Q, G165A / E / Q, N184E / Q, S187E / P, A / P / S193Q, A / S202V, A / G / S203E / N / Q, V204I, S / T210I / P, N211S, L216Q, N217S, L234W, P238Q, A / N / T239S, A242G / N, S243I, S250G, D / S258D / P, S259E / P, F260W, K / N / R264H / Q / S, and Q274A;(vi) A1Q, T3V, P9E, L10M, A24Q, V26Q, A29S, V30T, L31I, I35A, A37T, P40E, V45I / Q / R, A48T / V, A52R, A68S, V71A, T77E / N, T78I, S86H / R, V87S, L95A / F, N96S, S98R, G99A / E / Q / S, S100N, G101A, S102D / T, G105N, S108H, E111Q, T115F, G117S, D119Q, M123I, G127E / Q / S / T / V, A128P / R, S129H / R, G130S, Q136N, A143Q / V, V146A, V147L, A151G, S155G / N, S158D / N / Q, N160D / K, T161D / Q, G165A / E / Q, N184E / Q, S187E / P, A193Q, A202V, G203E / N / Q, V204I, T210I / P, N211S, L216Q, N217S, L234W, P238Q, N239S, A242G / N, S243I, S250G, S258D / P, S259E / P, F260W, K264H / Q / S and Q274A; (vii) T3V, P / S9E, L / Q10M, I35A, A68S, S / T77N, T78I, N / S86H, V87S, G99Q / S, G101A, A / Q / S / T115F, M123I, G127S / T, A / P / S128P / R, G165E / Q, N184Q, S187E, A / S202V, A / G / S203E, S / T210P, N217S, A242G, S250G, D / S258D / P and K / N / R264H; (viii) T3V, P9E, L10M, I35A, A68S, T77N, T78I, S86H, V87S, G99Q / S, G101A, T115F, M123I, G127S / T, A128P / R, G165E / Q, N184Q, S187E, A202V, G203E, T210P, N217S, A242G, S250G, S258D / P and K264H; (ix) T3V, A68S, S / T77N, T78I, M123I, G127S, A / S128P, G165Q, N184Q, A / S202V, S / T210P, N217S and D / S258D / P; or (x) T3V, A68S, T77N, T78I, M123I, G127S, A128P, G165Q, N184Q, A202V, T210P, N217S and S258D / P;Provided that one or more of the two, three or four or more substitutions are artificial substitutions; wherein the variant has at least 85% but less than 100% amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14; and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2. ;

[0069] Another embodiment relates to one or more subtilisin variants comprising an amino acid sequence comprising one or more amino acid substitution groups selected from the group consisting of: (i) T77N-G165Q, T77N-N217S, G165Q-N217S, A68S-T77N, N184Q-N217S, A68S-G165Q, G165Q-N184Q, T77N-A128P, T77N-N184Q, A68S-N217S, A128P-N217S, A128P-G165Q, A68S-A128P, A68S-N184Q, A128P-N184Q, T77N-A128 -N217S, T3V-T77N, T3V-N184Q, N184Q-A202V, G165Q-A202V, M123I-G165Q, T3V-G165Q, T3V-A128P, T3V-A68S, T77N-A202V, M123I-N217S, A68S-A202V , M123I-N184Q, A128P-A202V, T77N-M123I, T3V-A202V, N217S-S258P, A68S -M123I, N184Q-S258P, G165Q-S258P, M123I-A128P, T77N-S258P, T3V-M123 I. A128P-S258P, A202V-S258P, A68S-S258P, T3V-S258P, M123I-A202V, M12 3I-S258P, A68S-G127S, G127S-A128P, T77N-G127S, G127S-A202V, G127S-N 184Q, G127S-N217S, G127S-G165Q, T3V-G127S, G127S-S258P, M123I-G127S , T77N-T210P, G165Q-T210P, T210P-N217S, A128P-T210P, A68S-T210P, N18 4Q-T210P, T210P-S258P, A202V-T210P, M123I-T210P, T3V-T210P, G127S-T 210P, G127T-N184Q, G127T-N217S, V87S-G165Q, M123I-Q136R, A68S-G127T , G127T-A128P, T77N-V87S, G127T-G165Q, T77N-G127T, T78I-A202V, A68S- S258D, G165Q-S258D, T77N-S258D, T77N-T78I, T78I-G165Q, N184Q-S258D,N217S-S258D、P9E-M123I、A128P-A242G、A202V-S258D、A68S-T78I、G165Q-A242G、T77N-A242G、T78I-A128P、T78I-G127S、P9E-G165Q、Q136R-G165Q、T78I-N184Q、V87S-A128P、V87S-N217S、A68S-V87S、G127T-A202V、I35A-T77N、T3V-T78I、T78I-N217S、A68S-A242G、A68S-G99S、G99S-A128P、G99S-G165Q、I35A-G165Q、L10M-A202V、T3V-G127T、T77N-G99S、T78I-S258P、V45R-M123I、V87S-M123I、M123I-N239S、P9E-N184Q、S155G-G165Q、T78I-M123I、V45R-Q136R、V87S-A202V、A128P-S258D、I35A-A202V、M123I-G127T、M123I-S258D、N184Q-N239S、P9E-N217S、Q136R-S258E、S86H-S258P、T3V-A242G、T3V-S258D、G99S-N184Q、G99S-N217S、I35A-V87S、N217S-A242G、P9E-Q136R、G127T-S258P、I35A-A128P、I35A-A68S、I35A-N217S、L10M-M123I、P9E-S258P、P9E-T210P、P9E-T77N、S86H-A202V、S86H-G165Q、S86H-N184Q、T77N-S155G、T77N-V147L、T78I-T210P、V87S-S155G、A202V-A242G、A242G-S258P、A68S-G165E、G127S-A242G、G165E-N184Q、G165E-N217S、M123I-S258E、N184Q-A242G、S86H-T115F、T3V-G99S、V147L-G165Q、A128R-G165Q、A128R-S258D、A68S-A128R、A68S-V147L、G127S-S258D、G165E-A202V、G165Q-S258E、G165S-T210P、G203E-T210P、G99S-G127S、G99S-M123I、G99S-T210P、L10M-A128P、L10M-A68S、L10M-N184Q、L10M-N217S、L10M-S258P、L10M-T77N、M123I-A242G、M123I-G165E、M123I-S187P、N239S-S258P、P9E-A128P、P9E-A202V、P9E-A68S、P9E-V45R、Q136R-N184Q、Q136R-N239S、S86H-N217S、T115F-S258P、T210P-A242G、T3V-G165E、T3V-L10M、T3V-P9E、T77N-A128R、T77N-G165E、T78I-A242G、V45R-G165Q、V87T-M123I、V87T-N184Q、V87T-N239S、V87T-Q136R、A68S-G101A、A68S-V71A、G101A-A128P、G101A-G165Q、G101A-S258P、G165A-N184Q、G165E-S258P、G99S-S258P、I35A-M123I、K15I-G165S、K15I-T210P、L10M-G127S、L10M-G165Q、Q136R-S187P、S187E-A202V、S187P-S258P、S250G-S258P、T115F-A202V、T115F-G165Q、T3V-G101A、T3V-I35A、T3V-V87S、T77N-G101A、T77N-G203E、T78I-S258D、V71A-G165Q、V71A-T77N、V87S-S258P、V87S-V147L、A128P-G165E、A128P-S155G、A128P-V147L、A128R-A202V、A128R-N184Q、A128R-N217S、A1Q-S86H、A68S-G99Q、A68S-S155G、G101A-A202V、G101A-G127S、G101A-N184Q、G101A-N217S、G165Q-G203E、G165Q-N239S、G165S-N239S、G99Q-A128P、G99Q-G165Q、G99Q-N184Q、G99Q-N217S、G99S-A242G、I35A-N184Q、I35A-T78I、I35A-V147L、L10M-T210P、L10M-T78I、M123I-G165S、N184Q-G203E、N184Q-S187P、N43A-M123I、P9E-G127S、P9E-S258E、Q136R-S258P、T115F-N217S、T3V-S86H、T77H-M123I、T77H-N184Q、T77H-N239S、T77H-Q136R、T77H-V87T、T77N-G99Q、T77N-S250G、T78I-G99S、T78I-V87S、V146S-G165S、V146S-N239S、V146S-T210P、V45R-S258E、V87S-A242G、V87S-N184Q、A128S-T210P、A143V-A202V、A143V-G165Q、A143V-S258P、A1Q-N184Q、A1Q-S258P、A68S-G203E、A68S-S187E、A68S-T114N、G101A-M123I、G101A-T210P、G165A-N217S、G165Q-S187P、G99Q-A202V、G99S-A202V、I35A-A242G、I35A-S258P、L10M-I35A、L10M-V87S、M123I-A128R、M123I-V146S、N184Q-S187E、N217S-L234W、N239S-S250G、N43A-Q136R、N43A-V45R、P9E-N239S、P9E-V87T、S155G-N217S、S187E-N217S、S86H-A143V、S86H-Q274A、S86H-S250G、T114N-A128P、T114N-G165Q、T114N-N184Q、T114N-N217S、T115F-A143V、T115F-N184Q、T210P-N239S、T210P-S250G、T3V-A128R、T3V-G99Q、T77H-G165Q、T77N-S187E、T77N-T114N、V147L-N217S、V45R-N184Q、V71A-N184Q、V71A-N217S、V71A-T210P、T77N-G165Q-N217S、A68S-T77N-G165Q、T77N-N184Q-N217S、A68S-T77N-N217S、T77N-A128P-G165Q、T77N-A128P-N217S、A68S-T77N-A128P、G165Q-N184Q-N217S、T77N-G165Q-N184Q、A68S-A128P-G165Q、A68S-G165Q-N217S、A128P-N184Q-N217S、A128P-G165Q-N217S、A68S-A128P-N217S、A68S-N184Q-N217S、A68S-T77N-N184Q、T77N-A128P-N184Q、A68S-G165Q-N184Q、A128P-G165Q-N184Q、A68S-A128P-N184Q、T3V-N184Q-N217S、T3V-T77N-N217S、N184Q-A202V-N217S、T3V-T77N-N184Q、T3V-A128P-N217S、T3V-T77N-A128P、G165Q-A202V-N217S、T3V-A128P-N184Q、T77N-A202V-N217S、T3V-G165Q-N217S、T3V-T77N-G165Q、T3V-A68S-N217S、T3V-A68S-T77N、A68S-A202V-N217S、T3V-G165Q-N184Q、A128P-A202V-N217S、G165Q-N184Q-A202V、T77N-G165Q-A202V、T77N-N184Q-A202V、T3V-A128P-G165Q、T3V-A68S-A128P、T3V-A68S-N184Q、A128P-N184Q-A202V、A68S-N184Q-A202V、A68S-T77N-A202V、T3V-A68S-G165Q、T77N-A128P-A202V、A128P-G165Q-A202V、A68S-A128P-A202V、A68S-G165Q-A202V、M123I-G165Q-N217S、M123I-N184Q-N217S、T77N-M123I-G165Q、T77N-M123I-N217S、T3V-A202V-N217S、M123I-G165Q-N184Q、T77N-M123I-N184Q、T3V-N184Q-A202V、A68S-M123I-N217S、T3V-T77N-A202V、T3V-A128P-A202V、T3V-G165Q-A202V、M123I-A128P-N217S、T3V-A68S-A202V、A68S-M123I-N184Q、A68S-T77N-M123I、M123I-A128P-N184Q、T3V-M123I-N217S、T77N-M123I-A128P、A68S-M123I-G165Q、M123I-A128P-G165Q、A68S-M123I-A128P、N184Q-N217S-S258P、T3V-M123I-N184Q、T3V-M123I-G165Q、T3V-T77N-M123I、T77N-N217S-S258P、G165Q-N217S-S258P、T3V-A68S-M123I、T3V-M123I-A128P、T77N-N184Q-S258P、A128P-N184Q-S258P、A128P-N217S-S258P、T77N-A128P-S258P、T77N-G165Q-S258P、A202V-N217S-S258P、G165Q-N184Q-S258P、N184Q-A202V-S258P、A68S-A128P-S258P、A68S-N184Q-S258P、A68S-N217S-S258P、A68S-T77N-S258P、M123I-A202V-N217S、T3V-N217S-S258P、A128P-G165Q-S258P、G165Q-A202V-S258P、T3V-A128P-S258P、T3V-N184Q-S258P、A68S-G165Q-S258P、T3V-M123I-A202V、T3V-T77N-S258P、A128P-A202V-S258P、M123I-N184Q-A202V、T77N-A202V-S258P、A68S-A202V-S258P、M123I-G165Q-A202V、T77N-M123I-A202V、T3V-A68S-S258P、A68S-M123I-A202V、M123I-A128P-A202V、T3V-A202V-S258P、T3V-G165Q-S258P、A68S-G127S-A128P、A68S-T77N-G127S、T77N-G127S-A128P、A68S-G127S-A202V、A68S-G127S-N184Q、A68S-G127S-N217S、G127S-A128P-A202V、G127S-A128P-N184Q、G127S-A128P-N217S、A68S-G127S-G165Q、G127S-A128P-G165Q、G127S-A202V-N217S、G127S-N184Q-A202V、G127S-N184Q-N217S、M123I-N217S-S258P、T77N-G127S-A202V、T77N-G127S-N184Q、T77N-G127S-N217S、M123I-N184Q-S258P、T77N-G127S-G165Q、T3V-A68S-G127S、T3V-G127S-A128P、G127S-G165Q-A202V、G127S-G165Q-N184Q、G127S-G165Q-N217S、M123I-A202V-S258P、M123I-G165Q-S258P、T3V-M123I-S258P、T3V-T77N-G127S、A68S-G127S-S258P、G127S-A128P-S258P、A68S-M123I-S258P、M123I-A128P-S258P、T3V-G127S-A202V、T3V-G127S-N184Q、T3V-G127S-N217S、T77N-M123I-S258P、T3V-G127S-G165Q、T77N-G127S-S258P、G127S-A202V-S258P、G127S-N184Q-S258P、G127S-N217S-S258P、T3V-G127S-S258P、G127S-G165Q-S258P、A68S-M123I-G127S、M123I-G127S-A128P、M123I-G127S-A202V、M123I-G127S-N184Q、M123I-G127S-N217S、T77N-M123I-G127S、T3V-M123I-G127S、M123I-G127S-G165Q、M123I-G127S-S258P、T77N-G165Q-T210P、A68S-T77N-T210P、A68S-G165Q-T210P、A68S-A128P-T210P、T77N-A128P-T210P、T77N-T210P-N217S、A68S-N184Q-T210P、N184Q-T210P-N217S、T77N-N184Q-T210P、A128P-G165Q-T210P、A68S-T210P-N217S、G165Q-N184Q-T210P、G165Q-T210P-N217S、A128P-N184Q-T210P、A128P-T210P-N217S、T77N-T210P-S258P、G165Q-T210P-S258P、T3V-A128P-T210P、T3V-A68S-T210P、T3V-T77N-T210P、A128P-T210P-S258P、A202V-T210P-N217S、A68S-T210P-S258P、N184Q-A202V-T210P、T210P-N217S-S258P, T3V-G165Q-T210P, T77N-A202V-T210P, A202V-T210P -S258P, G165Q-A202V-T210P, T3V-N184Q-T210P, A128P-A202V-T210P, A68 S-A202V-T210P, N184Q-T210P-S258P, T3V-A202V-T210P, T3V-T210P-N217 S, T3V-T210P-S258P, M123I-T210P-N217S, T77N-M123I-T210P, A68S-M123I -T210P, M123I-A128P-T210P, M123I-N184Q-T210P, A68S-G127S-T210P, G1 27S-A128P-T210P, T77N-G127S-T210P, G127S-T210P-S258P, M123I-G165Q- T210P, T3V-G127S-T210P, G127S-G165Q-T210P, T3V-M123I-T210P, G127S- A202V-T210P, G127S-N184Q-T210P, G127S-T210P-N217S, G127T-N184Q-N21 7S, M123I-A202V-T210P, M123I-T210P-S258P, A68S-G127T-N184Q, A68S-G 127T-N217S, G127T-A128P-N184Q, G127T-A128P-N217S, T77N-V87S-G165Q, A68S-G127T-A128P, G127T-G165Q-N184Q, G127T-G165Q-N217S, T77N-G127 T-N184Q, T77N-G127T-N217S, A68S-G165Q-S258D, A68S-T77N-G127T, A68S- T77N-S258D, G127T-A128P-G165Q, T77N-G127T-A128P and T77N-G165Q-S258D; or (ii) N184Q-N217S, T77N-N217S, T77N-N184Q, T77N-G165Q, G165Q-N184Q, G165Q-N217S, A128P-N217S, A68S-T77N, T77N-A128P, A68S-N217S, A128P- N184Q, A68S-N184Q, A68S-G165Q, A128P-G165Q, A68S-A128P, A202V-N217S,N184Q-A202V、G165Q-A202V、T3V-N217S、T3V-N184Q、T3V-T77N、T77N-A202V、A68S-A202V、T3V-A128P、T3V-G165Q、A128P-A202V、T3V-A68S、T3V-A202V、N184Q-S258P、N217S-S258P、M123I-N217S、M123I-N184Q、T3V-M123I、T77N-S258P、A128P-S258P、G165Q-S258P、T77N-M123I、A202V-S258P、M123I-G165Q、A68S-M123I、A68S-S258P、M123I-A128P、M123I-A202V、T3V-S258P、A68S-G127S、G127S-A128P、T77N-G127S、G127S-A202V、G127S-N184Q、G127S-N217S、G127S-G165Q、M123I-S258P、T3V-G127S、G127S-S258P、M123I-G127S、T77N-T210P、A128P-T210P、A202V-T210P、A68S-T210P、G165Q-T210P、T210P-S258P、T3V-T210P、N184Q-T210P、T210P-N217S、G127S-T210P、M123I-T210P、T78I-A202V、A68S-S258D、G165Q-S258D、T77N-S258D、T77N-T78I、T78I-G165Q、G127T-N184Q、G127T-N217S、N184Q-S258D、N217S-S258D、A202V-S258D、A68S-T78I、T78I-A128P、T78I-G127S、A68S-G127T、T78I-N184Q、G127T-A128P、G127T-A202V、T3V-T78I、T78I-N217S、G127T-G165Q、L10M-A202V、T77N-G127T、T78I-S258P、T3V-G127T、T78I-M123I、V87S-A202V、A128P-A242G、A128P-S258D、G165Q-A242G、I35A-A202V、M123I-S258D、T3V-A242G、T3V-S258D、T77N-A242G、S86H-S258P、G127T-S258P、P9E-T77N、S86H-A202V、S86H-G165Q、S86H-N184Q、T78I-T210P、A202V-A242G、A242G-S258P、A68S-A242G、A68S-G165E、G127S-A242G、G165E-N184Q、G165E-N217S、I35A-V87S、L10M-M123I、M123I-G127T、N184Q-A242G、N217S-A242G、A128R-G165Q、A128R-S258D、A68S-A128R、A68S-G99S、G127S-S258D、G165E-A202V、G99S-A128P、G99S-G127S、G99S-G165Q、I35A-G165Q、I35A-T77N、L10M-A128P、L10M-A68S、L10M-N184Q、L10M-N217S、L10M-S258P、L10M-T77N、M123I-A242G、P9E-A128P、P9E-A202V、P9E-A68S、P9E-G165Q、P9E-N184Q、P9E-N217S、P9E-S258P、S86H-T115F、T210P-A242G、T3V-G165E、T3V-G99S、T3V-L10M、T3V-P9E、T77N-A128R、T77N-G165E、T77N-G99S、T77N-V87S、T78I-A242G、V87S-A128P、V87S-G165Q、V87S-M123I、V87S-N217S、A68S-G101A、G101A-A128P、G101A-G165Q、G101A-S258P、G165E-S258P、G99S-S258P、I35A-M123I、I35A-N217S、L10M-G127S、L10M-G165Q、M123I-G165E、P9E-T210P、S187E-A202V、S86H-N217S、T115F-A202V、T115F-G165Q、T115F-S258P、T3V-G101A、T3V-V87S、T77N-G101A、T77N-G203E、T78I-S258D、A128P-G165E、A128R-A202V、A128R-N184Q、A128R-N217S、A68S-G99Q、G101A-A202V、G101A-G127S、G101A-N184Q、G101A-N217S、G165Q-G203E、G99Q-A128P、G99Q-G165Q、G99Q-N184Q、G99Q-N217S、G99S-A242G、G99S-T210P、I35A-A128P、I35A-T78I、L10M-T210P、L10M-T78I、P9E-G127S、P9E-M123I、T3V-I35A、T77N-G99Q、T78I-G99S、T78I-V87S、V87S-N184Q、A143V-A202V、A143V-G165Q、A143V-S258P、A1Q-N184Q、A1Q-S86H、A68S-G203E、A68S-S187E、A68S-V87S、G101A-M123I、G101A-T210P、G99Q-A202V、G99S-A202V、G99S-M123I、G99S-N184Q、G99S-N217S、I35A-N184Q、L10M-I35A、L10M-V87S、M123I-A128R、N184Q-G203E、N184Q-S187E、S187E-N217S、S250G-S258P、S86H-A143V、T115F-A143V、T115F-N184Q、T115F-N217S、T3V-A128R、T3V-G99Q、T3V-S86H、T77N-S187E、T77N-N184Q-N217S、A128P-N184Q-N217S、T77N-A128P-N217S、A68S-N184Q-N217S、A68S-T77N-N217S、G165Q-N184Q-N217S、T77N-A128P-N184Q、T77N-G165Q-N217S、T77N-G165Q-N184Q、A68S-T77N-G165Q、A68S-T77N-N184Q、T77N-A128P-G165Q、A128P-G165Q-N217S、A68S-A128P-N217S、A68S-T77N-A128P、A68S-G165Q-N217S、A128P-G165Q-N184Q、A68S-A128P-N184Q、A68S-A128P-G165Q、A68S-G165Q-N184Q、N184Q-A202V-N217S、G165Q-A202V-N217S、T77N-A202V-N217S、T3V-N184Q-N217S、A68S-A202V-N217S、T3V-T77N-N217S、A128P-A202V-N217S、G165Q-N184Q-A202V、T77N-N184Q-A202V、T3V-A128P-N217S、T3V-T77N-A128P、T3V-T77N-N184Q、T77N-G165Q-A202V、A128P-N184Q-A202V、A68S-N184Q-A202V、T3V-A128P-N184Q、A68S-T77N-A202V、T77N-A128P-A202V、T3V-G165Q-N217S、T3V-T77N-G165Q、T3V-A68S-N217S、T3V-A68S-T77N、A128P-G165Q-A202V、A68S-A128P-A202V、T3V-G165Q-N184Q、A68S-G165Q-A202V、T3V-A128P-G165Q、T3V-A68S-A128P、T3V-A68S-N184Q、T3V-A68S-G165Q、T3V-A202V-N217S、T3V-N184Q-A202V、T3V-T77N-A202V、T3V-A128P-A202V、T3V-G165Q-A202V、T3V-A68S-A202V、N184Q-N217S-S258P、T3V-M123I-N217S、M123I-N184Q-N217S、T77N-M123I-N217S、A128P-N184Q-S258P、A128P-N217S-S258P、A68S-M123I-N217S、M123I-G165Q-N217S、T77N-A128P-S258P、T77N-M123I-N184Q、T77N-N184Q-S258P、M123I-A128P-N217S、T3V-M123I-N184Q、T77N-N217S-S258P、A202V-N217S-S258P、A68S-M123I-N184Q、M123I-A128P-N184Q、N184Q-A202V-S258P、T3V-M123I-G165Q,T3V-T77N-M123I、T77N-M123I-G165Q、A68S-A128P-S258P、A68S-T77N-M123I、M123I-G165Q-N184Q、T3V-A68S-M123I、A68S-N184Q-S258P、G165Q-N184Q-S258P、G165Q-N217S-S258P、M123I-A202V-N217S、T3V-M123I-A128P、T77N-M123I-A128P、A128P-G165Q-S258P、A68S-N217S-S258P、M123I-A128P-G165Q、T3V-N217S-S258P、T77N-G165Q-S258P、A68S-M123I-A128P、A68S-M123I-G165Q、A68S-T77N-S258P、T3V-A128P-S258P、T3V-N184Q-S258P、G165Q-A202V-S258P、T3V-M123I-A202V、A128P-A202V-S258P、M123I-N184Q-A202V、T3V-T77N-S258P、A68S-A202V-S258P、A68S-G165Q-S258P、M123I-G165Q-A202V、T77N-A202V-S258P、T77N-M123I-A202V、A68S-M123I-A202V、M123I-A128P-A202V、T3V-A202V-S258P、T3V-A68S-S258P、T3V-G165Q-S258P、A68S-G127S-A128P、A68S-T77N-G127S、T77N-G127S-A128P、A68S-G127S-A202V、A68S-G127S-N184Q、A68S-G127S-N217S、G127S-A128P-A202V、G127S-A128P-N184Q、G127S-A128P-N217S、A68S-G127S-G165Q、G127S-A128P-G165Q、G127S-A202V-N217S、G127S-N184Q-A202V、G127S-N184Q-N217S、T77N-G127S-A202V、T77N-G127S-N184Q、T77N-G127S-N217S、T77N-G127S-G165Q、T3V-A68S-G127S、T3V-G127S-A128P、G127S-G165Q-A202V、G127S-G165Q-N184Q、G127S-G165Q-N217S、M123I-A202V-S258P、M123I-N217S-S258P、M123I-N184Q-S258P、T3V-M123I-S258P、T3V-T77N-G127S、A68S-G127S-S258P、G127S-A128P-S258P、M123I-A128P-S258P、T3V-G127S-A202V、T3V-G127S-N184Q、T3V-G127S-N217S、A68S-M123I-S258P、T3V-G127S-G165Q、T77N-G127S-S258P、M123I-G165Q-S258P、T77N-M123I-S258P、G127S-A202V-S258P、G127S-N184Q-S258P、G127S-N217S-S258P、T3V-G127S-S258P、G127S-G165Q-S258P、A68S-M123I-G127S、M123I-G127S-A128P、M123I-G127S-A202V、M123I-G127S-N184Q、M123I-G127S-N217S、T77N-M123I-G127S、T3V-M123I-G127S、M123I-G127S-G165Q、M123I-G127S-S258P、A68S-A128P-T210P、A68S-T77N-T210P、T77N-A128P-T210P、N184Q-A202V-T210P、T3V-A128P-T210P、T3V-A68S-T210P、T3V-T77N-T210P、T77N-G165Q-T210P、T77N-T210P-S258P、A128P-G165Q-T210P、A128P-T210P-S258P、A202V-T210P-N217S、A68S-G165Q-T210P、A68S-T210P-S258P、N184Q-T210P-N217S、T3V-G165Q-T210P、T77N-A202V-T210P、A128P-A202V-T210P、A128P-N184Q-T210P、A128P-T210P-N217S、A202V-T210P-S258P、A68S-A202V-T210P、A68S-N184Q-T210P、A68S-T210P-N217S、G165Q-A202V-T210P、G165Q-T210P-S258P、T3V-A202V-T210P、T3V-N184Q-T210P、T3V-T210P-S258P、T77N-N184Q-T210P、T77N-T210P-N217S、G165Q-N184Q-T210P、N184Q-T210P-S258P、T210P-N217S-S258P、T3V-T210P-N217S、G165Q-T210P-N217S、A68S-G127S-T210P、G127S-A128P-T210P、T77N-G127S-T210P、G127S-T210P-S258P、T3V-G127S-T210P、G127S-G165Q-T210P、A68S-M123I-T210P、G127S-A202V-T210P、G127S-N184Q-T210P、G127S-T210P-N217S、M123I-A128P-T210P、M123I-A202V-T210P、M123I-N184Q-T210P、M123I-T210P-N217S、T3V-M123I-T210P、T77N-M123I-T210P、M123I-G165Q-T210P、M123I-T210P-S258P、A68S-G165Q-S258D、A68S-T77N-S258D、T77N-G165Q-S258D、T77N-T78I-G165Q、A68S-N184Q-S258D、A68S-N217S-S258D、G127T-N184Q-N217S、G165Q-N184Q-S258D、G165Q-N217S-S258D、M123I-G127S-T210P、T77N-N184Q-S258D、T77N-N217S-S258D、A68S-T77N-T78I、A68S-T78I-A128P、A68S-T78I-G127S、A68S-T78I-G165Q、T77N-T78I-A128P、T77N-T78I-G127S、T78I-A128P-G165Q、T78I-G127S-A128P、T78I-G127S-G165Q、A68S-G127T-N184Q、A68S-G127T-N217S、N184Q-N217S-S258D、T77N-T78I-N184Q、T78I-G165Q-N184Q、A202V-N217S-S258D、A68S-A202V-S258D、G127T-A128P-N184Q、G127T-A128P-N217S、G127T-A202V-N217S、G127T-N184Q-A202V、G165Q-A202V-S258D、T3V-T77N-T78I、T3V-T78I-G165Q、T77N-A202V-S258D、T77N-T78I-A202V、T77N-T78I-N217S、T78I-G165Q-A202V、T78I-G165Q-N217S、T78I-N184Q-A202V、T78I-N184Q-N217S、A68S-G127T-A128P、A68S-T78I-A202V、A68S-T78I-N184Q、A68S-T78I-N217S、G127T-G165Q-N184Q、G127T-G165Q-N217S、T3V-A68S-T78I、T3V-T78I-A128P、T3V-T78I-G127S、T77N-G127T-N184Q、T77N-G127T-N217S、T77N-T78I-S258P、T78I-A128P-A202V、T78I-A128P-N184Q、T78I-A128P-N217S、T78I-A202V-N217S、T78I-G127S-A202V、T78I-G127S-N184Q、T78I-G127S-N217S、T78I-G165Q-S258P、A68S-G127T-A202V、A68S-T77N-G127T、G127T-A128P-G165Q、N184Q-A202V-S258D、T3V-G127T-N184Q、T3V-G127T-N217S、T77N-G127T-A128P、T78I-M123I-A202V、A128P-G165Q-A242G、A128P-G165Q-S258D、A128P-N184Q-S258D、A128P-N217S-S258D、A68S-A128P-S258D、A68S-G127T-G165Q、A68S-T78I-S258P、G127T-A128P-A202V、T3V-A128P-A242G、T3V-A68S-G127T、T3V-A68S-S258D、T3V-G127T-A202V、T3V-G165Q-A242G、T3V-G165Q-S258D、T3V-T77N-A242G、T3V-T77N-S258D、T3V-T78I-N184Q、T3V-T78I-N217S、T3V-T78I-S258P、T77N-A128P-A242G、T77N-A128P-S258D、T77N-G127T-G165Q、T77N-G165Q-A242G、T78I-A128P-S258P、T78I-G127S-S258P、A68S-M123I-S258D、G127T-G165Q-A202V、M123I-G165Q-S258D、T3V-G127T-A128P、T3V-N217S-S258D、T3V-T78I-A202V、T77N-G127T-A202V、T77N-M123I-S258D、T77N-T78I-M123I、T78I-M123I-G165Q、T78I-M123I-N184Q、T78I-N184Q-S258P, A68S-T78I-M123I, A68S-T78I-T210P, G127T-N184Q-S258P , G127T-N217S-S258P, M123I-A202V-S258D, M123I-N217S-S258D, T3V-A202V-S2 58D, T3V-M123I-S258D, T3V-N184Q-S258D, T3V-T77N-G127T, T3V-T78I-T210P, T 77N-T78I-T210P, T78I-A128P-T210P, T78I-A202V-S258P, T78I-G127S-T210P, T 78I-G165Q-T210P, T78I-M123I-A128P, T78I-M123I-G127S, T78I-M123I-N217S and T78I-N217S-S258P; provided that one or more of the two, three or four or more substitutions are artificial substitutions; wherein the variant has at least 85% but less than 100% amino acid sequence identity with AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14; and wherein the amino acid positions of the variant are numbered by corresponding to the amino acid sequence of SEQ ID NO: 2. 、

[0070] Another embodiment relates to one or more subtilisin variants comprising an amino acid sequence comprising one or more amino acid substitutions selected from the group consisting of:

[0071] M123I-Q136R; P9E-M123I; V45R-M123I; G127T-G165S-T210E;

[0072] M123I-G165S-N239S; M123I-Q136R-G165Q; M123I-Q136R-S258E;

[0073] M123I-V146S-T210E; P9E-V45R-M123I; Q136R-G165Q-S258E;

[0074] V45R-M123I-Q136R; G127T-V146S-G165S-N239S;

[0075] L10M-K27R-S98H-M123I;M123I-Q136R-G165Q-S258E;

[0076] M123I-V146S-G165S-T210P;M123I-V146S-T210P-S250G;

[0077] P9E-M123I-Q136R-G165Q;P9E-Q136R-G165Q-S258E;

[0078] P9E-V45R-M123I-Q136R;V45R-M123I-Q136R-G165Q;

[0079] V45R-M123I-Q136R-S258E;V45R-Q136R-G165Q-S258E;

[0080] K27R-N43A-S98H-M123I-T210H;P9E-M123I-Q136R-G165Q-S258E;

[0081] P9E-V45R-M123I-Q136R-G165Q;P9E-V45R-M123I-Q136R-S258E;

[0082] V45R-M123I-Q136R-G165Q-S258E;Y103I-G127T-V146S-G165S-N239S;

[0083] T77H-V87T-M123I-Q136R-N184Q-N239S;

[0084] V26R-V45R-S98R-E111Q-M123I-Q136R;

[0085] T77H-V87T-M123I-Q136R-G165Q-N184Q-N239S;

[0086] T77S-V87S-M123I-Q136R-G165Q-S187P-S258P;

[0087] T77S-V87S-M123I-Q136R-N160C-S187P-S258P;

[0088] T77H-V87T-M123I-Q136R-N184Q-S187P-N239S-S258P;

[0089] <h2 style=";text-align:left;direction:ltr">T3Q-K22Y-A24N-V45I-A48I-T77N-N217S-L234I-Y255R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0090] <h2 style=";text-align:left;direction:ltr"> P9E-T77Q-V87T-M123I-Q136R-G165Q-N184Q-N217S-N239S-S258E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0091] <h2 style=";text-align:left;direction:ltr"> N43A-V45R-T77H-V87T-M123I-Q136R-G165A-N184Q-S187P-N239S-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0092] <h2 style=";text-align:left;direction:ltr"> P9E-N43A-V45R-T77S-V87S-M123I-Q136R-G165Q-S187P-N217S-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0093] <h2 style=";text-align:left;direction:ltr"> P9E-N43A-V45R-T77H-V87T-M123I-Q136R-G165Q-N184Q-S187P-N239S-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0094] <h2 style=";text-align:left;direction:ltr"> P9E-N43A-V45R-T77Q-V87T-M123I-Q136R-G165Q-N184Q-N217S-N239S-S258E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0095] <h2 style=";text-align:left;direction:ltr"> P9E-N43A-V45R-T77H-V87T-M123I-Q136R-G165A-N184Q-S187P-N217S-N239S-S258E;T77N-N217S-L234W;T77N-N217S,T77N-G126Q-G165Q-T210P-N217S-L234W-S258P,T77N-G127F-G165Q-A202V-T210P-N217S-L234W-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0096] <h2 style=";text-align:left;direction:ltr"> A1Q-T3V-V28A-I35A-A68S-T77N-S86H-T115F-N217S-N239S-S250G-S258P-Q274A;T77N-M123I-T210P-N239S-S250G-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0097] <h2 style=";text-align:left;direction:ltr"> V45I-A68S-T77N-M123I-L125N-G165Q-N217S-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0098] <h2 style=";text-align:left;direction:ltr"> A68S-V71A-T77N-L95A-G165Q-N184Q-T210P-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0099] M123I-T210P-N217S-S258P;G127F-G165Q-N217S-S258P;

[0100] T77N-G165Q-N217S-S258P;T77S-G165A-N184Q-T210P-N217S;

[0101] A68S-T77N-A128P-G165Q-N184Q-N217S;V71A-T77S-N184Q-S258P;

[0102] T77N-M123I-G165Q-N184Q-N217S;V71A-L95A-A128S-T210P;

[0103] V71A-M123I-A128S-T210P;V71A-M123I-A128S-G165E;

[0104] T77S-A128P-N184Q-N217S-F260W;T77H-A128S-G165Q-T210P;

[0105] T77D-S86R-S155E-G165A-N184Q;

[0106] A68S-V71A-T77N-A128S-G165Q-T210P-N217S;

[0107] V28A-I35A-A68S-T77N-A128S-G165Q-N184Q-G203N-T210P;

[0108] A128P-T210P;T210P-S258P;A128P-T210P-S259P;A128P-S187P-T210P;

[0109] A128P-S187P-T210P-S258P;P9E-T77S-V87T-T210P-N239K;

[0110] K15I-L95Q-M123I-G165S-T210P-N217S;

[0111] K15I-M123I-V146S-G165S-T210P-N239S;

[0112] K15I-N76K-M123I-V146S-G165S-T210P-N239S;

[0113] K15I-V146S-G165S-T210P-N239S;

[0114] P009E-V146S-N184Q-G203E-T210P-P238R-N239S;

[0115] M123I-G165S-N217S;K15I-L95Q-S108K-G165S-G203E-T210P-N217S;

[0116] K15I-L95Q-S108K-G165S-G203E-T210P;

[0117] P9E-K15I-G165S-A193D-G203E-T210P;T77N-M123I-G165Q-N217S;

[0118] T77H-G165Q;T77N-G165Q-N217S;A68S-V71A-T77N-G165Q-N217S;

[0119] T77N-V87S-M123I-S155G-G165Q-N217S;T77H-V87S-S155G-G165Q;

[0120] T77N-V87S-S155G-G165Q-N217S;T77N-M123I-G165Q;T77N-G165Q;

[0121] A68S-V71A-T77N-G165Q;T77N-V87S-M123I-S155G-G165Q;

[0122] I35A-A68S-T77N-V87S-V147L-G165Q;

[0123] A68S-T77N-V87S-V147L-S155E-G165Q;

[0124] T77N-V87S-M123I-V147L-S155G-G165Q;

[0125] A68S-T77N-M123I-G165Q-N184Q-N217S;

[0126] T77N-M123I-A128P-G165Q-N184Q-N217S;

[0127] A68S-V71A-T77N-S100N-A128P-G165Q-N184Q-T210P-N217S-N239T;

[0128] T77N-A128P-G165Q-N184Q-N217S;

[0129] A68S-A128P-G165Q-N184Q-N217S;A68S-T77N-G165Q-N184Q-N217S;

[0130] A68S-T77N-A128P-N184Q-N217S;A68S-T77N-A128P-G165Q-N217S;

[0131] A68S-T77N-A128P-G165Q-N184Q;M123I-G165Q-N184Q-N217S;

[0132] T77N-G165Q-N184Q-N217S;T77N-M123I-N184Q-N217S;

[0133] T77N-M123I-G165Q-N184Q;

[0134] T3V-A68S-T77N-A128P-G165Q-N184Q-N217S;

[0135] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-N217S;

[0136] T3V-T77N-M123I-G165Q-N184Q-N217S;

[0137] A68S-T77N-S108H-A128P-G165Q-N184Q-N217S;

[0138] A68S-T77N-G99S-A128P-G165Q-N184Q-N217S;

[0139] A68S-T77N-T114N-A128P-G165Q-N184Q-N217S;

[0140] A68S-T77N-M123I-A128P-G165Q-N184Q-N217S;

[0141] A68S-T77N-G127T-A128P-G165Q-N184Q-N217S;

[0142] A68S-T77N-A128P-G165Q-N184Q-T210P-N217S;

[0143] A68S-T77N-G99S-M123I-A128P-G165Q-N184Q-N217S;

[0144] A68S-T77N-G99S-G127T-A128P-G165Q-N184Q-N217S;

[0145] A68S-T77N-G99S-A128P-G165Q-N184Q-T210P-N217S;

[0146] A68S-T77N-T114N-M123I-A128P-G165Q-N184Q-N217S;

[0147] A68S-T77N-T114N-G127T-A128P-G165Q-N184Q-N217S;

[0148] A68S-T77N-T114N-A128P-G165Q-N184Q-T210P-N217S;

[0149] A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-N217S;

[0150] A68S-T77N-M123I-A128P-G165Q-N184Q-T210P-N217S;

[0151] A68S-T77N-G127T-A128P-G165Q-N184Q-T210P-N217S;

[0152] A68S-T77N-G99S-M123I-A128P-G165Q-N184Q-T210P-N217S;

[0153] A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-T210P-N217S;

[0154] T3V-A68S-T77N-G99S-M123I-A128P-G165Q-N184Q-N217S;

[0155] T3V-A68S-T77N-T114N-M123I-A128P-G165Q-N184Q-N217S;

[0156] T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-N217S;

[0157] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-T210P-N217S;

[0158] S86H-T115F-A143V-G165Q-A202V-S258P;

[0159] S86H-L95A-N184Q-A202V-T210P-N217S-L234W-S250G-S258P;

[0160] V30T-I35A-T77N-G165Q-A202V-T210P-S250G-S258P;

[0161] A1Q-T3V-I35A-T77N-T78I-S86H-T115F-G165Q-N184Q-N217S-L234W-S258P;

[0162] A1Q-A37T-T77N-T78I-S86H-M123I-G165Q-N184Q-A202V-N239S-S250G-S258P-Q274A;P9E-T77N-N96S-G165Q-G203E-S250G;

[0163] P9E-T77N-N96S-G165E-G203E-T210P-S250G;

[0164] A1Q-T3V-S86H-G130S-S155G-G165Q-N184Q-A202V-G203N-T210P-Q274A;A1Q-T3V-S86H-G165Q-N184Q-N239S-S250G-S258P;

[0165] A1Q-T77N-S86H-V147L-A151G-N184Q-N217S-S258P;

[0166] S86H-T115F-S155G-G165Q-N184Q-A202V-Q274A;

[0167] A68S-S86H-T115F-N184Q-A202V-G203N-N239S-S250G-S258P-Q274A;

[0168] A68S-T77N-V87S-A128P-S155G-G165Q-N217S;

[0169] A68S-T77N-A128P-G165Q;A68S-T77N-V87S-A128P-S155G-G165Q;

[0170] I35A-A68S-T77N-V87S-A128P-V147L-G165Q-N217S;

[0171] A68S-T77N-V87S-A128P-S155G-G165Q-N217S-A242G;

[0172] A68S-T77N-V87S-A128P-V147L-S155E-G165Q;

[0173] A68S-T77N-V87S-A128P-S155E-G165Q-N217S;

[0174] A68S-T77N-S86N-V87S-A128P-S155G-G165Q-N217S-A242G;

[0175] I35A-A68S-T77N-A128P-V147L-G165Q-N217S;

[0176] I35A-A68S-T77N-A128P-S155G-G165Q-A242G;

[0177] I35A-A68S-T77N-A128P-V147L-G165Q-A242G;

[0178] A68S-T77N-V87S-A128P-S155G-G165Q-A242G;

[0179] A068S-T077N-N086S-G165Q-K194E-S211N-N217S;

[0180] A068S-T077N-N086S-V087S-S155G-G165Q-K194E-S211N-N217S;

[0181] A068S-T077N-N086S-G165Q-K194E-S211N;

[0182] A068S-T077N-N086S-V087S-S155G-G165Q-K194E-S211N;

[0183] I035A-A068S-T077N-N086S-V087S-V147L-G165Q-K194E-S211N-N217S;

[0184] A068S-T077N-N086S-V087S-S155G-G165Q-K194E-S211N-N217S-A242G;A068S-T077N-N086S-V087S-V147L-S155E-G165Q-K194E-S211N;

[0185] A068S-T077N-N086S-V087S-S155E-G165Q-K194E-S211N-N217S;

[0186] A068S-T077N-V087S-S155G-G165Q-K194E-S211N-N217S-A242G;

[0187] I035A-A068S-T077N-N086S-V147L-G165Q-K194E-S211N-N217S;

[0188] I035A-A068S-T077N-N086S-S155G-G165Q-K194E-S211N-A242G;

[0189] 0I35A-A068S-T077N-N086S-V147L-G165Q-K194E-S211N-A242G;

[0190] A068S-T077N-N086S-V087S-S155G-G165Q-K194E-S211N-A242G;

[0191] A68S-T77N-G127T-A128P-G165Q-N184Q-A202V-N217S;

[0192] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-N217S;

[0193] A68S-T77N-G127T-A128P-G165Q-N184Q-N217S-S258P;

[0194] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-N217S-S258P;

[0195] A68S-T77N-G127T-A128P-G165Q-N184Q-G203Q-N217S;

[0196] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-G203Q-N217S;

[0197] A68S-T77N-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0198] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0199] A68S-T77N-A128P-G165Q-N184Q-G203Q-N217S-S258P;

[0200] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-G203Q-N217S-S258P;

[0201] S86H-T115F-G127T-A143V-G165Q-N184Q-A202V-N217S-S258P;

[0202] T3V-S86H-T115F-M123I-A143V-G165Q-A202V-N217S-S258P;

[0203] A68S-T77N-A128P-S129H-G165Q-N184Q-A202V-N217S;

[0204] T3V-A68S-T77N-M123I-A128P-S129H-G165Q-N184Q-A202V-N217S;

[0205] S86H-T115F-S129H-A143V-G165Q-N184Q-A202V-N217S-S258P;

[0206] T3V-S86H-T115F-M123I-S129H-A143V-G165Q-A202V-N217S-S258P;

[0207] A68S-T77N-A128P-S155N-G165Q-N184Q-A202V-N217S;

[0208] T3V-A68S-T77N-M123I-A128P-S155N-G165Q-N184Q-A202V-N217S;

[0209] I35A-A68S-T77N-V87S-A128P-V147L-G165Q-A202V-N217S;

[0210] T3V-I35A-A68S-T77N-V87S-M123I-A128P-V147L-G165Q-A202V-N217S;A68S-T77N-A128P-Q136N-S155N-G165Q-N184Q-N217S-A242N;

[0211] T3V-A68S-T77N-M123I-A128P-Q136N-S155N-G165Q-N184Q-N217S-A242N;A68S-V71A-T77N-A128P-G165Q-N184Q-A202V-N217S;

[0212] T3V-A68S-V71A-T77N-M123I-A128P-G165Q-N184Q-A202V-N217S;

[0213] A68S-T77N-A128P-G165Q-N184Q-S187P-A202V-N217S;

[0214] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-S187P-A202V-N217S;

[0215] A68S-T77N-A128R-G165Q-N184Q-A202V-N217S-S258D;

[0216] T3V-A68S-T77N-M123I-A128R-G165Q-N184Q-A202V-N217S-S258D;A68S-T77N-A128P-S129R-G165Q-N184Q-N217S-S258D;

[0217] T3V-A68S-T77N-M123I-A128P-S129R-G165Q-N184Q-N217S-S258D;

[0218] A68S-T77N-A128R-G165Q-N184Q-A202V-N217S-S258D-K264H;

[0219] T3V-A68S-T77N-M123I-A128R-G165Q-N184Q-A202V-N217S-S258D-K264H;A68S-T77N-A128R-G165Q-N184Q-G203E-S258D;

[0220] T3V-A68S-T77N-M123I-A128R-G165Q-N184Q-G203E-S258D;

[0221] T3V-A68S-T77N-M123I-A128R-G165Q-N184E-A202V-N217S-S258D;T3V-A52R-A68S-T77N-M123I-A128R-G165Q-N184E-A202V-N217S-S258D;

[0222] T3V-T77N-S100N-G105N-D119Q-A128P-S158N-N160D-T161Q-G165A-N184Q-N217S-S258P-K264S;

[0223] T3V-T77N-S100N-G105N-A128P-S158D-T161Q-G165A-N184Q-N217S-S258P;

[0224] T3V-T77N-S100N-G105N-A128P-S158N-N160D-T161Q-G165A-N184Q-N217S-S258P;

[0225] A24Q-V45I-A68S-T77N-S98R-E111Q-M123I-S158Q-G165E-N184Q-S187E-V204I-T210I-N217S-S259E;

[0226] P9E-A52R-S86R-E111Q-S129R-S158Q-G165E-V204I-T210I-S258D-S259E-K264Q;

[0227] T3V-A68S-T77N-G127T-A128P-G165Q-N184Q-A202V-N217S;

[0228] T3V-I35A-T77N-V87S-M123I-A128P-G165Q-N184Q-A202V-N217S-A242G;T3V-A68S-T77N-G127E-A128P-G165Q-N184Q-A202V-N217S;

[0229] T3V-A68S-T77N-M123I-G127V-A128P-G165Q-N184Q-N217S;

[0230] T3V-A68S-T77N-M123I-A128P-A151G-G165Q-N184Q-N217S;

[0231] T3V-A68S-T77N-G127T-A128P-A151G-G165Q-N184Q-N217S;

[0232] T3V-T77N-M123I-A128P-A151G-G165Q-N184Q-A202V-N217S;

[0233] T3V-A68S-T77N-L95F-A128P-G165Q-N184Q-A202V-N217S;

[0234] T3V-A68S-T77N-L95A-M123I-A128P-G165Q-N184Q-N217S;

[0235] T3V-A68S-T77N-L95F-M123I-A128P-G165Q-N184Q-N217S;

[0236] T3V-A68S-T77N-G99A-A128P-G165Q-N184Q-A202V-N217S;

[0237] T3V-A68S-T77N-G99E-A128P-G165Q-N184Q-A202V-N217S;

[0238] T3V-A68S-T77N-G99Q-A128P-G165Q-N184Q-A202V-N217S;

[0239] T3V-A68S-T77N-G99A-M123I-A128P-G165Q-N184Q-N217S;

[0240] T3V-A68S-T77N-S108H-A128P-G165Q-N184Q-N217S;

[0241] T3V-I35A-T77N-V87S-M123I-A128P-V146A-G165Q-N184Q-A202V-N217S-A242G;

[0242] T3V-A68S-T77N-G127T-A128P-G165E-N184Q-A202V-N217S;

[0243] T3V-A68S-T77N-M123I-A128P-V146A-G165Q-N184Q-N217S;

[0244] T3V-A68S-T77N-M123I-A128P-T161D-G165Q-N184Q-A202V-N217S;T3V-V45R-A68S-T77N-S102D-A128P-T161D-G165Q-N184Q-A202V-N217S;

[0245] T3V-A68S-T77N-G127T-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0246] T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0247] T3V-A68S-T77N-M123I-G127V-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0248] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0249] T3V-I35A-T77N-V87S-G127T-A128P-G165Q-N184Q-A202V-N217S-A242G-S258P;

[0250] T3V-T77N-M123I-G127Q-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0251] T3V-T77N-M123I-G127V-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0252] T3V-T77N-M123I-G127E-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0253] T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-A202V-N217S;

[0254] T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-A202V-T210P-N217S;

[0255] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0256] T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-A202V-N217S-S258D;L10M-I35A-T78I-V87S-M123I-A202V-S258D-F260W;

[0257] L10M-I35A-T78I-V87S-M123I-A202V-F260W-K264H;

[0258] L10M-I35A-T78I-V87S-A202V-S258D;

[0259] L10M-I35A-T78I-V87S-S187E-A202V;L10M-T78I-V87S-S187E-A202V;L10M-I35A-T78I-S187E-A202V;

[0260] T3V-L10M-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0261] T3V-I35A-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0262] T3V-A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0263] T3V-A68S-T77N-V87S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0264] T3V-A68S-T77N-M123I-G127S-A128P-G165E-N184Q-A202V-N217S-S258P;

[0265] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0266] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-L216Q-N217S-S258P;

[0267] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-A242G-S258P;

[0268] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0269] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P-F260W;

[0270] A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0271] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0272] T3V-L31I-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0273] A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0274] A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0275] A68S-T77N-G127T-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0276] T3V-A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0277] A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0278] T3V-A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0279] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0280] A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0281] T3V-A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0282] A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0283] T3V-A68S-T77N-S108H-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0284] T3V-A68S-T77N-G99Q-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0285] T3V-A68S-T77N-G99Q-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0286] T3V-A68S-T77N-G127S-A128P-G165E-N184Q-A202V-N217S-S258P;

[0287] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0288] T3V-P9E-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0289] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-S187E-A202V-T210P-N217S-S258P;

[0290] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-G203E-T210P-N217S-S258P;

[0291] T3V-L10M-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0292] T3V-P9E-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0293] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-S187E-A202V-N217S-S258P;

[0294] T3V-P9E-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0295] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-S187E-A202V-T210P-N217S-S258P;

[0296] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-G203E-T210P-N217S-S258P;

[0297] T3V-A68S-T77N-G101A-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0298] T3V-L10M-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0299] T3V-P9E-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0300] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-S187E-A202V-T210P-N217S-S258P;

[0301] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-G203E-T210P-N217S-S258P;

[0302] T3V-A68S-T77N-G101A-M123I-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0303] T3V-L10M-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0304] T3V-L10M-A68S-T77N-G101A-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0305] T3V-P9E-L10M-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0306] T3V-P9E-A68S-T77N-G99Q-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0307] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S243I-S258P;

[0308] T3V-A68S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0309] T3V-A68S-T77N-M123I-G127S-A128P-N184Q-A202V-N217S-S258P;

[0310] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-A202V-N217S-S258P;

[0311] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-S258P;

[0312] T3V-A68S-T77E-M123I-G127T-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0313] T3V-A68S-T77E-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0314] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P-K264H;

[0315] T3V-A68S-T77N-S102T-M123I-G127S-A128P-G165Q-N184Q-A202V-N211S-N217S-S258P;

[0316] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N211S-N217S-S258P;

[0317] T3V-A68S-T77N-S102T-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0318] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P-S259P;

[0319] T3V-A68S-T77N-M123I-G127S-A128P-T161Q-G165Q-N184Q-A202V-N217S-S258P;

[0320] T3V-A68S-T77N-M123I-G127S-A128P-S158Q-G165Q-N184Q-A202V-N217S-S258P;

[0321] T3V-V45Q-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0322] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A193Q-A202V-N217S-S258P;

[0323] T3V-A68S-T77N-M123I-G127S-A128P-A143Q-G165Q-N184Q-A202V-N217S-S258P;

[0324] T3V-A68S-T77N-G117S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0325] T3V-A48V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0326] T3V-A48T-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0327] T3V-A29S-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0328] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S;

[0329] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-N217S-S258P;

[0330] T3V-A68S-T77N-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0331] A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S;

[0332] A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0333] T3V-A68S-T77N-G99Q-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0334] T3V-A68S-T77N-T78I-V87S-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0335] T3V-A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-A242G-S258P;

[0336] T3V-V26Q-A68S-T77N-M123I-G127S-A128P-G165E-N184Q-A202V-T210P-N217S-S258P;

[0337] T3V-P9E-L10M-A68S-T77N-M123I-G127S-A128P-G165E-N184Q-A202V-T210P-N217S-S258P;

[0338] T3V-P9E-L10M-A68S-T77N-G101A-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0339] T3V-A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0340] T3V-A68S-T77N-T78I-M123I-G127S-A128P-N160K-G165Q-N184Q-A202V-T210P-N217S-A242G-S258P;

[0341] T3V-A68S-T77N-V87S-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0342] T3V-P40E-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0343] T3V-A68S-T77E-M123I-G127T-A128P-G165E-N184Q-A202V-N217S-P238Q-S258P;

[0344] T3V-A68S-T77N-G99S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0345] T3V-A68S-T77E-M123I-G127T-G165E-N184Q-A202V-N217S-S258P;

[0346] A68S-T77N-A128P-G165Q-N184Q-N217S-S258P;

[0347] T3V-A68S-T77N-G101A-S102T-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0348] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S;

[0349] T3V-A68S-T77N-T78I-G99S-G127S-A128P-G165Q-T210P-A242G-S258P-K264H;

[0350] T3V-A68S-T77N-T78I-G99S-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-A242G-S258P;

[0351] T3V-A68S-T77N-M123I-G127T-G165E-N184Q-A202V-N217S-P238Q-S258P;

[0352] T3V-A68S-T77N-T78I-G99S-G127S-A128P-G165Q-T210P-A242G-S258P;

[0353] T3V-A68S-T77N-T78I-G99S-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-A242G-S258D;I35A-M123I-G127T-T210P-S243I;

[0354] T3V-A68S-T77N-G99S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P-F260W;

[0355] T3V-A68S-T77N-T78I-G99S-G101A-G127S-A128P-G165Q-T210P-A242G-S258P;A68S-T77N-G99Q-A128P-G165Q-N184Q-N217S;

[0356] A68S-T77N-A128P-G165Q-N184Q-A202V-N217S;

[0357] A68S-T77N-A128P-G165Q-N184Q-N217S-S258D;

[0358] T3V-A68S-T77N-T78I-G99S-G101A-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-A242G-S258P; and V45R-S98R; provided that one or more of the two, three or four or more substitutions are artificial substitutions; wherein the variant has at least 85% but less than 100% amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14; and wherein the amino acid positions of the variant are identified by comparison with SEQ ID The amino acid sequence of NO:2 is numbered accordingly.

[0359] Another embodiment provides one or more subtilisin variants comprising an amino acid sequence comprising one or more amino acid substitution groups selected from the group consisting of: M123I-Q136R; P9E-M123I; V45R-M123I; G127T-G165S-T210E;

[0360] M123I-G165S-N239S; M123I-Q136R-G165Q; M123I-Q136R-S258E;

[0361] M123I-V146S-T210E; P9E-V45R-M123I; Q136R-G165Q-S258E;

[0362] V45R-M123I-Q136R; G127T-V146S-G165S-N239S;

[0363] L10M-K27R-S98H-M123I; M123I-Q136R-G165Q-S258E;

[0364] M123I-V146S-G165S-T210P; M123I-V146S-T210P-S250G;

[0365] P9E-M123I-Q136R-G165Q;P9E-Q136R-G165Q-S258E;

[0366] P9E-V45R-M123I-Q136R;V45R-M123I-Q136R-G165Q;

[0367] V45R-M123I-Q136R-S258E;V45R-Q136R-G165Q-S258E;

[0368] K27R-N43A-S98H-M123I-T210H;P9E-M123I-Q136R-G165Q-S258E;

[0369] P9E-V45R-M123I-Q136R-G165Q;P9E-V45R-M123I-Q136R-S258E;

[0370] V45R-M123I-Q136R-G165Q-S258E;Y103I-G127T-V146S-G165S-N239S;

[0371] T77H-V87T-M123I-Q136R-N184Q-N239S;

[0372] V26R-V45R-S98R-E111Q-M123I-Q136R;

[0373] T77H-V87T-M123I-Q136R-G165Q-N184Q-N239S;

[0374] T77S-V87S-M123I-Q136R-G165Q-S187P-S258P;

[0375] T77S-V87S-M123I-Q136R-N160C-S187P-S258P;

[0376] T77H-V87T-M123I-Q136R-N184Q-S187P-N239S-S258P;

[0377] T3Q-K22Y-A24N-V45I-A48I-T77N-N217S-L234I-Y255R;

[0378] P9E-T77Q-V87T-M123I-Q136R-G165Q-N184Q-N217S-N239S-S258E;

[0379] <h2 style=";text-align:left;direction:ltr">N43A-V45R-T77H-V87T-M123I-Q136R-G165A-N184Q-S187P-N239S-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0380] <h2 style=";text-align:left;direction:ltr"> P9E-N43A-V45R-T77S-V87S-M123I-Q136R-G165Q-S187P-N217S-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0381] <h2 style=";text-align:left;direction:ltr"> P9E-N43A-V45R-T77H-V87T-M123I-Q136R-G165Q-N184Q-S187P-N239S-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0382] <h2 style=";text-align:left;direction:ltr"> P9E-N43A-V45R-T77Q-V87T-M123I-Q136R-G165Q-N184Q-N217S-N239S-S258E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0383] <h2 style=";text-align:left;direction:ltr"> P9E-N43A-V45R-T77H-V87T-M123I-Q136R-G165A-N184Q-S187P-N217S-N239S-S258E;T77N-N217S-L234W;T77N-N217S,T77N-G126Q-G165Q-T210P-N217S-L234W-S258P,T77N-G127F-G165Q-A202V-T210P-N217S-L234W-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0384] <h2 style=";text-align:left;direction:ltr"> A1Q-T3V-V28A-I35A-A68S-T77N-S86H-T115F-N217S-N239S-S250G-S258P-Q274A;T77N-M123I-T210P-N239S-S250G-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0385] <h2 style=";text-align:left;direction:ltr"> V45I-A68S-T77N-M123I-L125N-G165Q-N217S-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0386] <h2 style=";text-align:left;direction:ltr"> A68S-V71A-T77N-L95A-G165Q-N184Q-T210P-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0387] <h2 style=";text-align:left;direction:ltr"> M123I-T210P-N217S-S258P;G127F-G165Q-N217S-S258P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0388] <h2 style=";text-align:left;direction:ltr"> T77N-G165Q-N217S-S258P;T77S-G165A-N184Q-T210P-N217S;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0389] A68S-T77N-A128P-G165Q-N184Q-N217S;V71A-T77S-N184Q-S258P;

[0390] T77N-M123I-G165Q-N184Q-N217S;V71A-L95A-A128S-T210P;

[0391] V71A-M123I-A128S-T210P;V71A-M123I-A128S-G165E;

[0392] T77S-A128P-N184Q-N217S-F260W;T77H-A128S-G165Q-T210P;

[0393] T77D-S86R-S155E-G165A-N184Q;

[0394] A68S-V71A-T77N-A128S-G165Q-T210P-N217S;

[0395] V28A-I35A-A68S-T77N-A128S-G165Q-N184Q-G203N-T210P;

[0396] A128P-T210P;T210P-S258P;A128P-T210P-S259P;A128P-S187P-T210P;

[0397] A128P-S187P-T210P-S258P;P9E-T77S-V87T-T210P-N239K;

[0398] K15I-L95Q-M123I-G165S-T210P-N217S;

[0399] K15I-M123I-V146S-G165S-T210P-N239S;

[0400] K15I-N76K-M123I-V146S-G165S-T210P-N239S;

[0401] K15I-V146S-G165S-T210P-N239S;

[0402] P009E-V146S-N184Q-G203E-T210P-P238R-N239S;

[0403] M123I-G165S-N217S;K15I-L95Q-S108K-G165S-G203E-T210P-N217S;

[0404] K15I-L95Q-S108K-G165S-G203E-T210P;

[0405] P9E-K15I-G165S-A193D-G203E-T210P;T77N-M123I-G165Q-N217S;

[0406] T77H-G165Q;T77N-G165Q-N217S;A68S-V71A-T77N-G165Q-N217S;

[0407] T77N-V87S-M123I-S155G-G165Q-N217S;T77H-V87S-S155G-G165Q;

[0408] T77N-V87S-S155G-G165Q-N217S;T77N-M123I-G165Q;T77N-G165Q;

[0409] A68S-V71A-T77N-G165Q;T77N-V87S-M123I-S155G-G165Q;

[0410] I35A-A68S-T77N-V87S-V147L-G165Q;

[0411] A68S-T77N-V87S-V147L-S155E-G165Q;

[0412] T77N-V87S-M123I-V147L-S155G-G165Q;

[0413] A68S-T77N-M123I-G165Q-N184Q-N217S;

[0414] T77N-M123I-A128P-G165Q-N184Q-N217S;

[0415] A68S-V71A-T77N-S100N-A128P-G165Q-N184Q-T210P-N217S-N239T;

[0416] T77N-A128P-G165Q-N184Q-N217S;

[0417] A68S-A128P-G165Q-N184Q-N217S;A68S-T77N-G165Q-N184Q-N217S;

[0418] A68S-T77N-A128P-N184Q-N217S;A68S-T77N-A128P-G165Q-N217S;

[0419] A68S-T77N-A128P-G165Q-N184Q;M123I-G165Q-N184Q-N217S;

[0420] T77N-G165Q-N184Q-N217S;T77N-M123I-N184Q-N217S;

[0421] T77N-M123I-G165Q-N184Q;

[0422] T3V-A68S-T77N-A128P-G165Q-N184Q-N217S;

[0423] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-N217S;

[0424] T3V-T77N-M123I-G165Q-N184Q-N217S;

[0425] A68S-T77N-S108H-A128P-G165Q-N184Q-N217S;

[0426] A68S-T77N-G99S-A128P-G165Q-N184Q-N217S;

[0427] A68S-T77N-T114N-A128P-G165Q-N184Q-N217S;

[0428] A68S-T77N-M123I-A128P-G165Q-N184Q-N217S;

[0429] A68S-T77N-G127T-A128P-G165Q-N184Q-N217S;

[0430] A68S-T77N-A128P-G165Q-N184Q-T210P-N217S;

[0431] A68S-T77N-G99S-M123I-A128P-G165Q-N184Q-N217S;

[0432] A68S-T77N-G99S-G127T-A128P-G165Q-N184Q-N217S;

[0433] A68S-T77N-G99S-A128P-G165Q-N184Q-T210P-N217S;

[0434] A68S-T77N-T114N-M123I-A128P-G165Q-N184Q-N217S;

[0435] A68S-T77N-T114N-G127T-A128P-G165Q-N184Q-N217S;

[0436] A68S-T77N-T114N-A128P-G165Q-N184Q-T210P-N217S;

[0437] A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-N217S;

[0438] A68S-T77N-M123I-A128P-G165Q-N184Q-T210P-N217S;

[0439] A68S-T77N-G127T-A128P-G165Q-N184Q-T210P-N217S;

[0440] A68S-T77N-G99S-M123I-A128P-G165Q-N184Q-T210P-N217S;

[0441] A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-T210P-N217S;

[0442] T3V-A68S-T77N-G99S-M123I-A128P-G165Q-N184Q-N217S;

[0443] T3V-A68S-T77N-T114N-M123I-A128P-G165Q-N184Q-N217S;

[0444] T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-N217S;

[0445] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-T210P-N217S;

[0446] S86H-T115F-A143V-G165Q-A202V-S258P;

[0447] S86H-L95A-N184Q-A202V-T210P-N217S-L234W-S250G-S258P;

[0448] V30T-I35A-T77N-G165Q-A202V-T210P-S250G-S258P;

[0449] A1Q-T3V-I35A-T77N-T78I-S86H-T115F-G165Q-N184Q-N217S-L234W-S258P;

[0450] A1Q-A37T-T77N-T78I-S86H-M123I-G165Q-N184Q-A202V-N239S-S250G-S258P-Q274A;P9E-T77N-N96S-G165Q-G203E-S250G;

[0451] P9E-T77N-N96S-G165E-G203E-T210P-S250G;

[0452] A1Q-T3V-S86H-G130S-S155G-G165Q-N184Q-A202V-G203N-T210P-Q274A;A1Q-T3V-S86H-G165Q-N184Q-N239S-S250G-S258P;

[0453] A1Q-T77N-S86H-V147L-A151G-N184Q-N217S-S258P;

[0454] S86H-T115F-S155G-G165Q-N184Q-A202V-Q274A;

[0455] A68S-S86H-T115F-N184Q-A202V-G203N-N239S-S250G-S258P-Q274A;

[0456] A68S-T77N-V87S-A128P-S155G-G165Q-N217S;

[0457] A68S-T77N-A128P-G165Q;A68S-T77N-V87S-A128P-S155G-G165Q;

[0458] I35A-A68S-T77N-V87S-A128P-V147L-G165Q-N217S;

[0459] A68S-T77N-V87S-A128P-S155G-G165Q-N217S-A242G;

[0460] A68S-T77N-V87S-A128P-V147L-S155E-G165Q;

[0461] A68S-T77N-V87S-A128P-S155E-G165Q-N217S;

[0462] A68S-T77N-S86N-V87S-A128P-S155G-G165Q-N217S-A242G;

[0463] I35A-A68S-T77N-A128P-V147L-G165Q-N217S;

[0464] I35A-A68S-T77N-A128P-S155G-G165Q-A242G;

[0465] I35A-A68S-T77N-A128P-V147L-G165Q-A242G;

[0466] A68S-T77N-V87S-A128P-S155G-G165Q-A242G;

[0467] A068S-T077N-N086S-G165Q-K194E-S211N-N217S;

[0468] A068S-T077N-N086S-V087S-S155G-G165Q-K194E-S211N-N217S;

[0469] A068S-T077N-N086S-G165Q-K194E-S211N;

[0470] A068S-T077N-N086S-V087S-S155G-G165Q-K194E-S211N;

[0471] I035A-A068S-T077N-N086S-V087S-V147L-G165Q-K194E-S211N-N217S;

[0472] A068S-T077N-N086S-V087S-S155G-G165Q-K194E-S211N-N217S-A242G; A068S-T077N-N086S-V087S-V147L-S155E-G165Q-K194E-S211N;

[0473] A068S-T077N-N086S-V087S-S155E-G165Q-K194E-S211N-N217S;

[0474] A068S-T077N-V087S-S155G-G165Q-K194E-S211N-N217S-A242G;

[0475] I035A-A068S-T077N-N086S-V147L-G165Q-K194E-S211N-N217S;

[0476] I035A-A068S-T077N-N086S-S155G-G165Q-K194E-S211N-A242G;

[0477] 0I35A-A068S-T077N-N086S-V147L-G165Q-K194E-S211N-A242G; and A068S-T077N-N086S-V087S-S155G-G165Q-K194E-S211N-A242G; provided that one or more of the two, three or four or more variations are non-naturally occurring amino acids; wherein the variant has 85% or more amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14; and wherein the amino acid positions of the variant are identified by comparison with SEQ ID The amino acid sequence of NO:2 is numbered accordingly.

[0478] Still even further embodiments relate to one or more subtilisin variants comprising an amino acid sequence comprising one or more amino acid substitution groups selected from the group consisting of: A68S-T77N-G127T-A128P-G165Q-N184Q-A202V-N217S;

[0479] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-N217S;

[0480] A68S-T77N-G127T-A128P-G165Q-N184Q-N217S-S258P;

[0481] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-N217S-S258P;

[0482] A68S-T77N-G127T-A128P-G165Q-N184Q-G203Q-N217S;

[0483] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-G203Q-N217S;

[0484] A68S-T77N-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0485] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0486] A68S-T77N-A128P-G165Q-N184Q-G203Q-N217S-S258P;

[0487] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-G203Q-N217S-S258P;

[0488] S86H-T115F-G127T-A143V-G165Q-N184Q-A202V-N217S-S258P;

[0489] T3V-S86H-T115F-M123I-A143V-G165Q-A202V-N217S-S258P;

[0490] A68S-T77N-A128P-S129H-G165Q-N184Q-A202V-N217S;

[0491] T3V-A68S-T77N-M123I-A128P-S129H-G165Q-N184Q-A202V-N217S;

[0492] S86H-T115F-S129H-A143V-G165Q-N184Q-A202V-N217S-S258P;

[0493] T3V-S86H-T115F-M123I-S129H-A143V-G165Q-A202V-N217S-S258P;

[0494] A68S-T77N-A128P-S155N-G165Q-N184Q-A202V-N217S;

[0495] T3V-A68S-T77N-M123I-A128P-S155N-G165Q-N184Q-A202V-N217S;

[0496] I35A-A68S-T77N-V87S-A128P-V147L-G165Q-A202V-N217S;

[0497] T3V-I35A-A68S-T77N-V87S-M123I-A128P-V147L-G165Q-A202V-N217S;A68S-T77N-A128P-Q136N-S155N-G165Q-N184Q-N217S-A242N;

[0498] T3V-A68S-T77N-M123I-A128P-Q136N-S155N-G165Q-N184Q-N217S-A242N;A68S-V71A-T77N-A128P-G165Q-N184Q-A202V-N217S;

[0499] T3V-A68S-V71A-T77N-M123I-A128P-G165Q-N184Q-A202V-N217S;

[0500] A68S-T77N-A128P-G165Q-N184Q-S187P-A202V-N217S;

[0501] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-S187P-A202V-N217S;

[0502] A68S-T77N-A128R-G165Q-N184Q-A202V-N217S-S258D;

[0503] T3V-A68S-T77N-M123I-A128R-G165Q-N184Q-A202V-N217S-S258D;

[0504] A68S-T77N-A128P-S129R-G165Q-N184Q-N217S-S258D;

[0505] T3V-A68S-T77N-M123I-A128P-S129R-G165Q-N184Q-N217S-S258D;

[0506] A68S-T77N-A128R-G165Q-N184Q-A202V-N217S-S258D-K264H;

[0507] T3V-A68S-T77N-M123I-A128R-G165Q-N184Q-A202V-N217S-S258D-K264H;A68S-T77N-A128R-G165Q-N184Q-G203E-S258D;

[0508] T3V-A68S-T77N-M123I-A128R-G165Q-N184Q-G203E-S258D;

[0509] T3V-A68S-T77N-M123I-A128R-G165Q-N184E-A202V-N217S-S258D;

[0510] T3V-A52R-A68S-T77N-M123I-A128R-G165Q-N184E-A202V-N217S-S258D;

[0511] T3V-T77N-S100N-G105N-D119Q-A128P-S158N-N160D-T161Q-G165A-N184Q-N217S-S258P-K264S;

[0512] T3V-T77N-S100N-G105N-A128P-S158D-T161Q-G165A-N184Q-N217S-S258P;

[0513] T3V-T77N-S100N-G105N-A128P-S158N-N160D-T161Q-G165A-N184Q-N217S-S258P;

[0514] A24Q-V45I-A68S-T77N-S98R-E111Q-M123I-S158Q-G165E-N184Q-S187E-V204I-T210I-N217S-S259E;

[0515] P9E-A52R-S86R-E111Q-S129R-S158Q-G165E-V204I-T210I-S258D-S259E-K264Q;

[0516] T3V-A68S-T77N-G127T-A128P-G165Q-N184Q-A202V-N217S;

[0517] T3V-I35A-T77N-V87S-M123I-A128P-G165Q-N184Q-A202V-N217S-A242G;T3V-A68S-T77N-G127E-A128P-G165Q-N184Q-A202V-N217S;

[0518] T3V-A68S-T77N-M123I-G127V-A128P-G165Q-N184Q-N217S;

[0519] T3V-A68S-T77N-M123I-A128P-A151G-G165Q-N184Q-N217S;

[0520] T3V-A68S-T77N-G127T-A128P-A151G-G165Q-N184Q-N217S;

[0521] T3V-T77N-M123I-A128P-A151G-G165Q-N184Q-A202V-N217S;

[0522] T3V-A68S-T77N-L95F-A128P-G165Q-N184Q-A202V-N217S;

[0523] T3V-A68S-T77N-L95A-M123I-A128P-G165Q-N184Q-N217S;

[0524] T3V-A68S-T77N-L95F-M123I-A128P-G165Q-N184Q-N217S;

[0525] T3V-A68S-T77N-G99A-A128P-G165Q-N184Q-A202V-N217S;

[0526] T3V-A68S-T77N-G99E-A128P-G165Q-N184Q-A202V-N217S;

[0527] T3V-A68S-T77N-G99Q-A128P-G165Q-N184Q-A202V-N217S;

[0528] T3V-A68S-T77N-G99A-M123I-A128P-G165Q-N184Q-N217S;

[0529] T3V-A68S-T77N-S108H-A128P-G165Q-N184Q-N217S;

[0530] T3V-I35A-T77N-V87S-M123I-A128P-V146A-G165Q-N184Q-A202V-N217S-A242G;

[0531] T3V-A68S-T77N-G127T-A128P-G165E-N184Q-A202V-N217S;

[0532] T3V-A68S-T77N-M123I-A128P-V146A-G165Q-N184Q-N217S;

[0533] T3V-A68S-T77N-M123I-A128P-T161D-G165Q-N184Q-A202V-N217S;

[0534] T3V-V45R-A68S-T77N-S102D-A128P-T161D-G165Q-N184Q-A202V-N217S;

[0535] T3V-A68S-T77N-G127T-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0536] T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0537] T3V-A68S-T77N-M123I-G127V-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0538] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0539] T3V-I35A-T77N-V87S-G127T-A128P-G165Q-N184Q-A202V-N217S-A242G-S258P;

[0540] T3V-T77N-M123I-G127Q-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0541] T3V-T77N-M123I-G127V-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0542] T3V-T77N-M123I-G127E-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0543] T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-A202V-N217S;

[0544] T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-A202V-T210P-N217S;

[0545] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0546] T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-A202V-N217S-S258D;L10M-I35A-T78I-V87S-M123I-A202V-S258D-F260W;

[0547] L10M-I35A-T78I-V87S-M123I-A202V-F260W-K264H;

[0548] L10M-I35A-T78I-V87S-A202V-S258D;

[0549] L10M-I35A-T78I-V87S-S187E-A202V;L10M-T78I-V87S-S187E-A202V;

[0550] L10M-I35A-T78I-S187E-A202V;

[0551] T3V-L10M-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0552] T3V-I35A-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0553] T3V-A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0554] T3V-A68S-T77N-V87S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0555] T3V-A68S-T77N-M123I-G127S-A128P-G165E-N184Q-A202V-N217S-S258P;

[0556] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0557] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-L216Q-N217S-S258P;

[0558] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-A242G-S258P;

[0559] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0560] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P-F260W;

[0561] A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0562] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0563] T3V-L31I-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0564] A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0565] A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0566] A68S-T77N-G127T-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0567] T3V-A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0568] A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0569] T3V-A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0570] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0571] A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0572] T3V-A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0573] A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;

[0574] T3V-A68S-T77N-S108H-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0575] T3V-A68S-T77N-G99Q-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0576] T3V-A68S-T77N-G99Q-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0577] T3V-A68S-T77N-G127S-A128P-G165E-N184Q-A202V-N217S-S258P;

[0578] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0579] T3V-P9E-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0580] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-S187E-A202V-T210P-N217S-S258P;

[0581] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-G203E-T210P-N217S-S258P;

[0582] T3V-L10M-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0583] T3V-P9E-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0584] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-S187E-A202V-N217S-S258P;

[0585] T3V-P9E-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0586] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-S187E-A202V-T210P-N217S-S258P;

[0587] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-G203E-T210P-N217S-S258P;

[0588] T3V-A68S-T77N-G101A-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0589] T3V-L10M-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0590] T3V-P9E-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0591] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-S187E-A202V-T210P-N217S-S258P;

[0592] T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-G203E-T210P-N217S-S258P;

[0593] T3V-A68S-T77N-G101A-M123I-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0594] T3V-L10M-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0595] T3V-L10M-A68S-T77N-G101A-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0596] T3V-P9E-L10M-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0597] T3V-P9E-A68S-T77N-G99Q-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0598] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S243I-S258P;

[0599] T3V-A68S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0600] T3V-A68S-T77N-M123I-G127S-A128P-N184Q-A202V-N217S-S258P;

[0601] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-A202V-N217S-S258P;

[0602] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-S258P;T3V-A68S-T77E-M123I-G127T-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0603] T3V-A68S-T77E-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0604] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P-K264H;

[0605] T3V-A68S-T77N-S102T-M123I-G127S-A128P-G165Q-N184Q-A202V-N211S-N217S-S258P;

[0606] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N211S-N217S-S258P;

[0607] T3V-A68S-T77N-S102T-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0608] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P-S259P;

[0609] T3V-A68S-T77N-M123I-G127S-A128P-T161Q-G165Q-N184Q-A202V-N217S-S258P;

[0610] T3V-A68S-T77N-M123I-G127S-A128P-S158Q-G165Q-N184Q-A202V-N217S-S258P;

[0611] T3V-V45Q-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0612] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A193Q-A202V-N217S-S258P;

[0613] T3V-A68S-T77N-M123I-G127S-A128P-A143Q-G165Q-N184Q-A202V-N217S-S258P;

[0614] T3V-A68S-T77N-G117S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0615] T3V-A48V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0616] T3V-A48T-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0617] T3V-A29S-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0618] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S;

[0619] T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-N217S-S258P;

[0620] T3V-A68S-T77N-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0621] A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S;

[0622] A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0623] T3V-A68S-T77N-G99Q-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0624] T3V-A68S-T77N-T78I-V87S-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0625] T3V-A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-A242G-S258P;

[0626] T3V-V26Q-A68S-T77N-M123I-G127S-A128P-G165E-N184Q-A202V-T210P-N217S-S258P;

[0627] T3V-P9E-L10M-A68S-T77N-M123I-G127S-A128P-G165E-N184Q-A202V-T210P-N217S-S258P;

[0628] T3V-P9E-L10M-A68S-T77N-G101A-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0629] T3V-A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0630] T3V-A68S-T77N-T78I-M123I-G127S-A128P-N160K-G165Q-N184Q-A202V-T210P-N217S-A242G-S258P;

[0631] T3V-A68S-T77N-V87S-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;

[0632] T3V-P40E-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0633] T3V-A68S-T77E-M123I-G127T-A128P-G165E-N184Q-A202V-N217S-P238Q-S258P;

[0634] T3V-A68S-T77N-G99S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0635] T3V-A68S-T77E-M123I-G127T-G165E-N184Q-A202V-N217S-S258P;

[0636] A68S-T77N-A128P-G165Q-N184Q-N217S-S258P;

[0637] T3V-A68S-T77N-G101A-S102T-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;

[0638] T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S;T3V-A68S-T77N-T78I-G99S-G127S-A128P-G165Q-T210P-A242G-S258P-K264H;

[0639] T3V-A68S-T77N-T78I-G99S-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-A242G-S258P;

[0640] T3V-A68S-T77N-M123I-G127T-G165E-N184Q-A202V-N217S-P238Q-S258P;

[0641] T3V-A68S-T77N-T78I-G99S-G127S-A128P-G165Q-T210P-A242G-S258P;

[0642] T3V-A68S-T77N-T78I-G99S-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-A242G-S258D;I35A-M123I-G127T-T210P-S243I;

[0643] T3V-A68S-T77N-G99S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P-F260W;

[0644] T3V-A68S-T77N-T78I-G99S-G101A-G127S-A128P-G165Q-T210P-A242G-S258P; A68S-T77N-G99Q-A128P-G165Q-N184Q-N217S;

[0645] A68S-T77N-A128P-G165Q-N184Q-A202V-N217S;

[0646] A68S-T77N-A128P-G165Q-N184Q-N217S-S258D;

[0647] T3V-A68S-T77N-T78I-G99S-G101A-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-A242G-S258P; and V45R-S98R; provided that one or more of the two, three or four or more substitutions are artificial substitutions; wherein the variant has at least 85% but less than 100% amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14; and wherein the amino acid positions of the variant are identified by comparison with SEQ ID The amino acid sequence of NO:2 is numbered accordingly.

[0648] In additional embodiments, one or more subtilisin protease variants described herein further comprise one or more substitutions selected from the group consisting of 3T, 4I, 9R, 15T, 67A, 75D, 86S, 97S, 98A / D / G, 100G / M / R, 102A, 103N / Y, 105A, 117R / V, 119D / N, 122S, 127L, 128Q, 129A, 159D, 166A, 169S, 193P, 194E, 198M, 216D, 217D, 221S, 231V, 234L, 235H, 244R, 251K, and 273A, and / or one or more insertions selected from the group consisting of a D at position 36, an E at position 96, and a D between positions 98 and 99, wherein the amino acid positions of the variant are identified by comparison with those of SEQ ID NO: The amino acid sequence of NO:2 is numbered accordingly.

[0649] In one embodiment, one or more subtilisin variants described herein are from a parent having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to an AprL-clade subtilisin comprising motif A, motif B or a combination thereof. In another embodiment, one or more subtilisin variants described herein are from a parent having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to an AprL-clade subtilisin comprising motif A, motif B or a combination thereof. In yet another embodiment, one or more subtilisin variants described herein are derived from a parent having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with an AprL-clade subtilisin comprising motif A and motif B. In even further embodiments, one or more subtilisin variants described herein are derived from a parent having 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with an AprL-clade subtilisin comprising motif A and motif B. The AprL-clade of subtilisins is more fully described in International Patent Application No. PCT / US16 / 32514 filed on May 13, 2016. In some embodiments, the AprL-clade subtilisin is selected from one of the enzymes listed in Figure 1. In another embodiment, the AprL-clade subtilisin comprises the amino acid sequence of SEQ ID NO: 2, 9, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, or 205. In still further embodiments, the AprL-clade subtilisin comprises the amino acid sequence of SEQ ID NO: 2, 9, 189, 198, 202, 203, or 205. In still further embodiments, the AprL-clade subtilisin comprises the amino acid sequence of SEQ ID NO: 2 or 9.

[0650] In one embodiment, the AprL-clade enzyme comprises motif A, motif B, or a combination thereof. In other embodiments, the AprL-clade enzyme comprises motif A and motif B. In some embodiments, one or more subtilisin variants described herein comprise motif A, motif B, or a combination thereof. In still other embodiments, one or more subtilisin variants described herein comprise motif A and motif B. In another embodiment, motif A is selected from motif 1, motif 2, motif 3, and motif 4. In still other embodiments, motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13, and motif 14. In still other embodiments, the AprL-clade enzyme comprises motif 1 between Asp32 and His63, wherein the amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO: 2. In even still other embodiments, the AprL-clade enzyme comprises motif 6 or motif 7 between Lys236 and Arg246, wherein these amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:2. In still further embodiments, the AprL-clade enzyme comprises (i) motif 1 between Asp32 and His63, and (ii) motif 6 or motif 7 between Lys236 and Arg246, wherein these amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:2. In still even further embodiments, one or more subtilisin variants described herein comprise motif 1 between Asp32 and His63, wherein these amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:2. In still even further embodiments, one or more subtilisin variants described herein comprise motif 6 or motif 7 between Lys236 and Arg246, wherein these amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO:2. In yet another embodiment, one or more subtilisin protease variants described herein comprise (i) motif 1 between Asp32 and His63, and (ii) motif 6 or motif 7 between Lys236 and Arg246, wherein these amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:2.

[0651] In some embodiments, the AprL-clade enzyme comprises a YNT (SEQ ID NO: 166) motif ("motif 1"). In other embodiments, the AprL-clade enzyme comprises a Y56N57T58 (SEQ ID NO: 167) motif, wherein the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2 ("motif 2"). In further embodiments, the AprL-clade enzyme comprises a FVX1GEX2X3YNT (SEQ ID NO: 168) motif, wherein X1 is A or S, X2 is absent or is any amino acid, and X3 is A or S ("motif 3"). In still further embodiments, the AprL-clade enzyme comprises a F50V51X152G53E54X355Y56N57T58 (SEQ ID NO: 169) motif, wherein X1 is A or S and X3 is A or S; and further wherein the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2 ("motif 4"). In some embodiments, the AprL-clade enzyme comprises a loop region between residues 52-60, wherein the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2.

[0652] In other embodiments, the AprL-clade enzyme comprises a LSX4S (SEQ ID NO: 170) motif, wherein X4 is A or G ("motif 5"). In still other embodiments, the AprL-clade enzyme comprises a LSAS (SEQ ID NO: 171) motif ("motif 6"). In another embodiment, the AprL-clade enzyme comprises a LSGS (SEQ ID NO: 172) motif ("motif 7"). In some embodiments, the AprL-clade enzyme comprises a L240S241A242S243 (SEQ ID NO: 173) motif, wherein the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2 ("motif 8"). In further embodiments, the AprL-clade enzyme comprises a L240S241G242S243 (SEQ ID NO: 174) motif, wherein the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2 ("motif 9"). In still further embodiments, the AprL-clade enzyme comprises a KXXXLSX4SQX5R (SEQ ID NO: 175) motif, wherein X is any amino acid, X4 is A or G, and X5 is I or V ("motif 10"). In still further embodiments, the AprL-clade enzyme comprises a KXXXLSASQX5R (SEQ ID NO: 176) motif, wherein X is any amino acid and X5 is I or V ("motif 11"). In even still further embodiments, the AprL-clade enzyme comprises a KXXXLSGSQX5R (SEQ ID NO: 177) motif, wherein X is any amino acid and X5 is I or V ("motif 12"). In still further embodiments, the AprL-clade enzyme comprises a K236XXXLSASQX5R246 (SEQ ID NO: 178) motif, wherein X is any amino acid and X5 is I or V; and further wherein the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2 ("motif 13"). In even still further embodiments, the AprL-clade enzyme comprises a K236XXXLSGSQX5R246 (SEQ ID NO: 179) motif, wherein X is any amino acid and X5 is I or V; and further wherein the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2 ("motif 14").

[0653] In some embodiments, one or more subtilisin variants described herein comprise a YNT (SEQ ID NO: 166) motif ("motif 1"). In other embodiments, one or more subtilisin variants described herein comprise a Y56N57T58 (SEQ ID NO: 167) motif, wherein these amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2 ("motif 2"). In further embodiments, one or more subtilisin variants described herein comprise a FVX1GEX2X3YNT (SEQ ID NO: 168) motif, wherein X1 is A or S, X2 is absent or is any amino acid, and X3 is A or S ("motif 3"). In still other embodiments, one or more subtilisin variants described herein comprise a F50V51X152G53E54X355Y56N57T58 (SEQ ID NO: 169) motif, wherein X1 is A or S and X3 is A or S; and further wherein the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2 ("motif 4"). In some embodiments, one or more subtilisin variants described herein comprise a loop region between residues 52-60, wherein the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2.

[0654] In other embodiments, one or more subtilisin variants described herein comprise a LSX4S (SEQ ID NO: 170) motif, wherein X4 is A or G ("motif 5"). In still other embodiments, one or more subtilisin variants described herein comprise a LSAS (SEQ ID NO: 171) motif ("motif 6"). In another embodiment, one or more subtilisin variants described herein comprise a LSGS (SEQ ID NO: 172) motif ("motif 7"). In some embodiments, one or more subtilisin variants described herein comprise a L240S241A242S243 (SEQ ID NO: 173) motif, wherein these amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO: 2 ("motif 8"). In further embodiments, one or more subtilisin variants described herein comprise a L240S241G242S243 (SEQ ID NO: 174) motif, wherein these amino acid positions are numbered by corresponding to the amino acid sequence of SEQ ID NO: 2 ("motif 9"). In still further embodiments, one or more subtilisin variants described herein comprise a KXXXLSX4SQX5R (SEQ ID NO: 175) motif, wherein X is any amino acid, X4 is A or G, and X5 is I or V ("motif 10"). In still further embodiments, one or more subtilisin variants described herein comprise a KXXXLSASQX5R (SEQ ID NO: 176) motif, wherein X is any amino acid and X5 is I or V ("motif 11"). In even still further embodiments, one or more subtilisin variants described herein comprise a KXXXLSGSQX5R (SEQ ID NO: 177) motif, wherein X is any amino acid and X5 is I or V ("motif 12"). In still further embodiments, one or more subtilisin variants described herein comprise a K236XXXLSASQX5R246 (SEQ ID NO: 178) motif, wherein X is any amino acid and X5 is I or V; and further wherein the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2 ("motif 13"). In even still further embodiments, one or more subtilisin variants described herein comprise a K236XXXLSGSQX5R246 (SEQ ID NO: 179) motif, wherein X is any amino acid and X5 is I or V; and further wherein the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2 ("motif 14").

[0655] In another embodiment, one or more subtilisin protease variants described herein comprise two, three or four or more variations described herein relative to SEQ ID NO: 2, provided that (i) the variations relative to SEQ ID NO: 2 are not insertions or deletions; (ii) when the amino acid sequence of the variant comprises a variation at one or more of positions 37, 187, 193 and 258 relative to SEQ ID NO: 2, the variation is not proline; (iii) when the amino acid sequence of the variant comprises a variation at two or more of positions 26, 38, 234 and 250 relative to SEQ ID NO: 2, the variation is not 26R, 38S, 234R or 250E / N; and / or (iv) when the amino acid sequence of the variant comprises a variation at position 128, the variation relative to SEQ ID NO: 2 is not at position 130 or 193; wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 2.

[0656] In yet another embodiment, one or more subtilisin protease variants described herein comprise two, three or four or more variations at positions described herein relative to SEQ ID NO: 2, provided that (i) the variations at positions relative to SEQ ID NO: 2 are not insertions or deletions; (ii) when the amino acid sequence of the variant comprises a variation at one or more corresponding SEQ ID NO: 2 positions selected from positions 37, 187, 193 and 258, the variation is not proline; (iii) when the amino acid sequence of the variant comprises a variation at two or more corresponding SEQ ID NO: 2 positions selected from positions 26, 38, 234 and 250, the variation is not 26R, 38S, 234R, or 250E / N; (iv) when the amino acid sequence of the variant comprises a variation at position 128, the variation at the position corresponding to SEQ ID NO: 2 is not at position 130 or 193; and / or (v) the variant is not selected from SEQ ID NO: 5 in WO 2016074925 or JP 2016074925. The amino acid sequence is shown in SEQ ID NO:110 in 201300714.

[0657] In still further embodiments, one or more subtilisin protease variants described herein comprise two, three or four or more substitutions described herein, provided that (i) when the amino acid sequence of the variant comprises a substitution at one or more of positions 37, 187, 193 and 258, the substitution is not proline; (ii) when the amino acid sequence of the variant comprises a substitution at two or more of positions 26, 38, 234 and 250, the substitution is not 26R, 38S, 234R or 250E / N; and / or (iii) when the amino acid sequence of the variant comprises a substitution at position 128, the substitution is not at position 130 or 193; wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:2.

[0658] In yet another embodiment, one or more subtilisin variants described herein comprise two, three or four or more substitutions described herein, provided that (i) when the amino acid sequence of the variant comprises a substitution at one or more of positions 37, 187, 193 and 258, the substitution is not proline; (ii) when the amino acid sequence of the variant comprises a substitution at two or more of positions 26, 38, 234 and 250, the substitution is not 26R, 38S, 234R or 250E / N; (iii) when the amino acid sequence of the variant comprises a substitution at position 128, the substitution is not at position 130 or 193; and / or (iv) the variant is not composed of the amino acid sequence shown in SEQ ID NO: 5 in WO 2016074925 or SEQ ID NO: 110 in JP 201300714.

[0659] In one embodiment, one or more of the subtilisin protease variants described herein comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% amino acid sequence identity to the amino acid sequence of the AprL-clade subtilisin protease identified in Figure 1. In some embodiments, one or more of the subtilisin protease variants described herein comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2 or 9. In other embodiments, one or more subtilisin variants described herein comprise an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% amino acid sequence identity to (i) the amino acid sequence of the AprL-clade subtilisin identified in Figure 1, or (ii) the amino acid sequence of SEQ ID NO: 2 or 9. In another embodiment, one or more subtilisin variants described herein comprise an amino acid sequence having at least 89% but less than 100% amino acid sequence identity to the amino acid sequence of the AprL-clade subtilisin identified in Figure 1. In other embodiments, one or more subtilisin variants described herein comprise an amino acid sequence having at least 89% but less than 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2 or 9. In further embodiments, one or more subtilisin variants described herein comprise an amino acid sequence having at least 90% but less than 100% amino acid sequence identity to the amino acid sequence of the AprL-clade subtilisin identified in Figure 1. In some embodiments, one or more subtilisin variants described herein comprise an amino acid sequence having at least 90% but less than 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2 or 9.In still other embodiments, one or more subtilisin variants described herein comprise an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% amino acid sequence identity to the amino acid sequence of the AprL-clade subtilisin identified in Figure 1. In still yet other embodiments, one or more subtilisin variants described herein comprise an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2 or 9. In even still yet other embodiments, one or more subtilisin variants described herein are isolated, recombinant, substantially pure or non-naturally occurring subtilisin enzymes having subtilisin activity or caseinolytic activity (e.g., dimethylcaseinolytic activity).

[0660] In one embodiment, one or more subtilisin protease variants as described herein have enzymatic activity (for example, protease activity) and therefore can be used in cleaning applications, and this cleaning application includes but is not limited to the method for cleaning tableware articles, desktop utensil articles, fabrics and articles with hard surfaces (for example, the hard surfaces of tables, desktops, walls, furniture articles, floors, ceilings, etc.). The exemplary cleaning compositions comprising one or more subtilisin protease variants as described herein are described below. The enzymatic activity (for example, protease activity) of one or more subtilisin protease variants as described herein can be easily determined using a program well known to those of ordinary skill in the art. The example provided below describes the method for evaluating enzymatic activity and cleaning performance. Can easily use the program well known in the art and / or by using the program listed in the example to determine the performance of the polypeptide enzyme of the present invention in removing stains (for example, protein stains such as blood / milk / ink, pigment / milk / ink or yolk), cleaning hard surfaces or cleaning clothing, tableware or one or more desktop utensil articles.

[0661] In some embodiments, when compared to a reference subtilisin, one or more subtilisin variants described herein have one or more improved properties, wherein the improved properties are selected from improved protease activity, improved detergent cleaning performance, improved detergent thermal stability, and improved detergent maturation clothing cleaning, wherein the detergent is optionally a boron-free composition. In other embodiments, when compared to a reference subtilisin, one or more subtilisin variants described herein have one or more improved properties, wherein the improved properties are selected from improved protease activity, improved detergent cleaning performance, improved detergent thermal stability, and improved detergent maturation clothing cleaning, wherein the detergent is optionally a boron-free composition, wherein the reference subtilisin comprises an amino acid sequence of SEQ ID NO: 2, 9, or 63. In still other embodiments, one or more subtilisin variants described herein have one or more improved properties compared to a reference subtilisin, wherein the improved properties are selected from improved protease activity, improved detergent cleaning performance, improved detergent thermal stability, and improved detergent maturation laundry cleaning, wherein the detergent is optionally a boron-free composition, wherein the reference subtilisin comprises an amino acid sequence of SEQ ID NO: 2 or 9. In one embodiment, compared to a reference subtilisin, one or more subtilisin variants described herein are more stable in a longer wash period. In another embodiment, compared to a reference subtilisin, one or more subtilisin variants described herein are more stable in a short cold water wash cycle or a long hot water wash cycle. In still further embodiments, the one or more improved properties are (i) improved protease activity, wherein the variant has a PI>1 for N-suc-AAPF-pNA or dimethylcasein substrates; (ii) improved detergent cleaning performance, wherein the variant has a BMI, POM, egg and / or egg yolk cleaning PI>1; (iii) improved detergent thermal stability, wherein the variant has a stability PI>1; and / or (iv) improved detergent mature clothing cleaning, wherein the variant has a mature clothing cleaning PI>1; wherein the detergent is optionally a boron-free composition.In yet other embodiments, the one or more improved properties are (i) improved protease activity, wherein the variant has a PI>1 for N-suc-AAPF-pNA or dimethylcasein substrates; (ii) improved detergent cleaning performance, wherein the variant has a BMI, POM, egg and / or egg yolk cleaning PI>1; (iii) improved detergent thermal stability, wherein the variant has a stability PI>1 or a residual activity %≥5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 8%, 99%, 100% or more; and / or (iv) improved detergent mature laundry cleaning, wherein the variant has (a) a mature laundry cleaning PI>1, (b) a residual cleaning activity after 24 hours at 55°C of>10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 ... %, 98%, 99%, 100% or more, and / or (c) residual cleaning activity>10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more after 3 days or 3 weeks at 37°C; wherein the detergent is optionally a boron-free composition. In even further embodiments, one or more subtilisin variants described herein have improved protease activity, wherein the variant has a PI>1 for N-suc-AAPF-pNA or dimethylcasein substrates. In even further embodiments, one or more subtilisin variants described herein have improved detergent cleaning performance, wherein the variant has a BMI, POM and / or egg yolk cleaning PI>1, wherein the detergent is optionally a boron-free composition. In another embodiment, one or more subtilisin variants described herein have improved detergent thermostability, wherein the variant has a stability PI>1, wherein the detergent is optionally a boron-free composition.In still further embodiments, one or more subtilisin variants described herein have improved detergent thermostability, wherein after 15-20 minutes at a stress temperature of 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or more, the variant has a stability PI>1 or a residual activity % ≥5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more, wherein the detergent is optionally a boron-free composition. In another embodiment, one or more subtilisin variants described herein have improved detergent mature laundry cleaning, wherein the variant has (a) a mature laundry cleaning PI>1, (b) a residual cleaning activity>10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more after 24 hours at 55°C, and / or (c) a residual cleaning activity>10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more after 3 days or 3 weeks at 37°C, wherein the detergent is optionally a boron-free composition. In another embodiment, one or more subtilisin variants described herein have improved detergent maturation laundry cleaning, wherein the variant has a maturation laundry cleaning PI>1, wherein the detergent is optionally a boron-free composition. In another embodiment, one or more subtilisin variants described herein have improved protease activity, wherein the variant has a PI>1 for N-suc-AAPF-pNA or dimethylcasein substrates, and the PI is measured according to the protease activity assay of Example 1. In a further embodiment, one or more subtilisin variants described herein have improved detergent cleaning performance, wherein the variant has a BMI, POM, egg and / or egg yolk cleaning PI>1 and the PI is measured according to the cleaning performance in the laundry (HDL and HDD) and ADW detergent assays of Example 1. In even further embodiments, one or more subtilisin variants described herein have improved detergent thermal stability, wherein the variant has a stability PI>1 and the PI is measured according to the universal sample setting of the stability assay of Example 1.In yet even further embodiments, one or more subtilisin variants described herein have improved detergent thermal stability, wherein the variant has a residual activity %≥5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more after 15-20 minutes at a stress temperature of 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or more and the residual activity is measured according to the universal sample setting of the stability assay of Example 1. In still further embodiments, one or more subtilisin variants described herein have improved detergent mature laundry cleaning, wherein the variant has a mature laundry cleaning PI>1 and the PI is measured according to the mature laundry cleaning assay in Example 1. In even still further embodiments, one or more subtilisin variants described herein have improved detergent mature laundry cleaning, wherein the variant has a residual cleaning activity of>10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more after 24 hours at 55°C and / or has a residual cleaning activity of>10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more after 3 days or 3 weeks at 37°C; and the residual cleaning activity is measured according to the mature laundry cleaning assay of Example 1.

[0662] In some embodiments, one or more subtilisin variants described herein show cleaning performance in cleaning compositions. Cleaning compositions generally include ingredients that are harmful to the stability and performance of enzymes, and these ingredients make cleaning compositions a harsh environment in which enzymes such as serine proteases retain functions. Therefore, it is important to place enzymes in cleaning compositions and expect to exert enzyme functions (such as serine protease activity, such as those shown by cleaning performance). In some embodiments, one or more subtilisin variants described herein show cleaning performance in automatic dishwashing (ADW) detergent compositions. In some embodiments, the cleaning performance in ADW detergent compositions includes cleaning egg yolk stains. In some embodiments, one or more subtilisin variants described herein show cleaning performance in laundry detergent compositions. In some embodiments, the cleaning performance in laundry detergent compositions includes cleaning blood / milk / ink, eggs, yolks and / or POM stains. In each cleaning composition, one or more subtilisin variants described herein show cleaning performance with or without bleaching components. In even still further embodiments, one or more ADW or laundry detergent compositions described herein comprise one or more subtilisin variants described herein, wherein the variant is stable in the presence of one or more adjunct materials and / or one or more additional enzymes and / or further wherein the variant is stable to autolysis.

[0663] One or more subtilisin variants described herein can be made to undergo multiple changes, such as one or more amino acid insertions, deletions and / or substitutions (conservative or non-conservative), including situations where such changes do not substantially change the enzymatic activity of the variant. Similarly, nucleic acids of the present invention can also undergo multiple changes, such as one or more substitutions of one or more nucleotides in one or more codons, so that specific codons encode identical or different amino acids, leading to silent variation (for example, when the encoded amino acid is not changed by nucleotide mutation) or non-silent variation, one or more deletions of one or more nucleic acids (or codons) in the sequence, one or more additions or insertions of one or more nucleic acids (or codons) in the sequence, and / or one or more truncated cuttings of one or more nucleic acids (or codons) in the sequence. Compared with the polypeptide enzyme encoded by the original nucleic acid sequence, many such changes in the nucleic acid sequence may substantially not change the enzymatic activity of the polypeptide enzyme encoded by the obtained. Nucleotide sequences described herein can also be modified to include one or more codons, which provide optimal expression in an expression system (for example, a bacterial expression system), while, if desired, the one or more codons still encode one or more identical amino acids.

[0664] Described herein are one or more separated, non-naturally occurring or recombinant polynucleotides, which comprise a nucleic acid sequence encoding one or more subtilisin variants described herein or its recombinant polypeptide or active fragment. One or more nucleic acid sequences described herein can be used for the recombinant production (e.g., expression) of one or more subtilisin variants described herein, typically by expressing a plasmid expression vector comprising a sequence encoding one or more subtilisin variants described herein or its fragment. One embodiment provides nucleic acids encoding one or more subtilisin variants described herein, wherein the variant is a mature form with proteolytic activity. In certain embodiments, one or more subtilisin variants described herein are recombinantly expressed with homologous propeptide sequences. In other embodiments, one or more subtilisin variants described herein are recombinantly expressed with heterologous propeptide sequences (e.g., GG36 propeptide sequences).

[0665] One or more nucleotide sequences described herein can be produced by using any suitable synthesis, operation and / or separation technology or its combination.For example, one or more polynucleotides described herein can be produced using standard nucleic acid synthesis techniques well known to those skilled in the art, such as solid phase synthesis techniques.In such technology, typically synthesize fragments up to 50 or more nucleotide bases, then connect (such as by enzyme or chemical connection method) to form any desired continuous nucleic acid sequence.It is also possible to promote the synthesis of one or more polynucleotides described herein by any suitable method known in the art, including but not limited to the chemical synthesis using the following method: classical phosphoramidite method (see, for example, Beaucage et al., Tetrahedron Letters [tetrahedron express] 22: 1859-69 (1981)), or the method described by Matthes et al., EMBO J. [European Molecular Biology Association Magazine] 3: 801-805 (1984), as typically practiced in automatic synthesis methods.One or more polynucleotides described herein can also be produced by using an automatic DNA synthesizer. Custom nucleic acids can be ordered from a variety of commercial sources (e.g., The Midland Certified Reagent Company, Great American Gene Company, Operon Technologies Inc., and DNA 2.0). Other techniques and related principles for synthesizing nucleic acids are described, for example, by Itakura et al., Ann. Rev. Biochem. [Annual Book of Biochemistry] 53:323 (1984) and Itakura et al., Science [Science] 198:1056 (1984).

[0666] Recombinant DNA techniques for modifying nucleic acids are well known in the art, such as restriction endonuclease digestion, ligation, reverse transcription and cDNA production, and polymerase chain reaction (e.g., PCR). One or more polynucleotides described herein can also be obtained by screening a cDNA library using one or more oligonucleotide probes, which can be hybridized with or PCR-amplified polynucleotides encoding one or more AprL clade variants described herein or their recombinant polypeptides or active fragments. Methods for screening and separating cDNA clones and PCR amplification procedures are well known to those skilled in the art and are described in standard references known to those skilled in the art. One or more polynucleotides described herein can be obtained by, for example, changing the naturally occurring polynucleotide backbone (e.g., the polynucleotide backbone encoding one or more subtilisin variants described herein or reference subtilisin) through known mutagenesis procedures (e.g., site-directed mutagenesis, site-saturation mutagenesis, and in vitro recombination). A variety of methods suitable for generating modified polynucleotides described herein encoding one or more subtilisin variants described herein are known in the art, including, but not limited to, site-saturation mutagenesis, scanning mutagenesis, insertion mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis, and directed evolution, as well as various other recombination methods.

[0667] Another embodiment relates to one or more vectors comprising one or more subtilisin variants described herein (e.g., polynucleotides encoding one or more subtilisin variants described herein); expression vectors or expression cassettes comprising one or more nucleic acid or polynucleotide sequences described herein; isolated, substantially pure, or recombinant DNA constructs comprising one or more nucleic acid or polynucleotide sequences described herein; isolated or recombinant cells comprising one or more polynucleotide sequences described herein; and compositions comprising one or more such vectors, nucleic acids, expression vectors, expression cassettes, DNA constructs, cells, cell cultures, or any combination or mixture thereof.

[0668] Some embodiments relate to one or more recombinant cells comprising one or more vectors (e.g., expression vectors or DNA constructs) as described herein, comprising one or more nucleic acids or polynucleotide sequences as described herein. Some such recombinant cells are transformed or transfected using at least one such vector, although other methods are available and known in the art. Such cells are typically referred to as host cells. Some such cells comprise bacterial cells, including but not limited to Bacillus cells, such as Bacillus subtilis cells. Other embodiments relate to recombinant cells (e.g., recombinant host cells) comprising one or more subtilisins as described herein.

[0669] In some embodiments, one or more vectors described herein are expression vectors or expression cassettes comprising one or more polynucleotide sequences described herein operatively linked to one or more additional nucleic acid segments required for efficient gene expression (e.g., operatively linked to a promoter of one or more polynucleotide sequences described herein). The vector may include a transcription terminator and / or a selection gene, such as an antibiotic resistance gene that enables continuous culture maintenance of plasmid-infected host cells by growth in a medium containing an antimicrobial agent.

[0670] Expression vectors may be derived from plasmids or viral DNA, or in alternative embodiments, contain elements of both. Exemplary vectors include, but are not limited to, pC194, pJH101, pE194, pHP13 (see Harwood and Cutting [eds.], Chapter 3, Molecular Biological Methods for Bacillus, John Wiley & Sons (1990); suitable replicating plasmids for Bacillus subtilis include those listed on page 92). (See also, Perego, Integrational Vectors for Genetic Manipulations in Bacillus subtilis, Sonenshein et al. [Eds.]; Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular Genetics, American Society for Microbiology, Washington (1993), pp. 615-624; and p2JM103BBI).

[0671] In order to express and produce the target protein (e.g., one or more subtilisin variants described herein) in a cell, one or more expression vectors containing one or more copies (and in some cases multiple copies) of a polynucleotide encoding one or more subtilisin variants described herein are transformed into a cell under conditions suitable for variant expression. In some embodiments, the polynucleotide sequence encoding one or more subtilisin variants described herein (and other sequences contained in the vector) is integrated into the genome of the host cell; however, in other embodiments, a plasmid vector containing a polynucleotide sequence encoding one or more subtilisin variants described herein is maintained as an autonomous extrachromosomal element in the cell. Some embodiments provide extrachromosomal nucleic acid elements and imported nucleotide sequences integrated into the host cell genome. The vectors described herein can be used to produce one or more subtilisin variants described herein. In some embodiments, a polynucleotide construct encoding one or more subtilisin variants described herein is present in an integration vector that can integrate the polynucleotide encoding the variant into the host chromosome and optionally amplify the polynucleotide in the host chromosome. Examples of integration sites are well known to those skilled in the art. In some embodiments, transcription of the polynucleotide encoding one or more subtilisin variants described herein is achieved by a promoter that is a wild-type promoter of the parent subtilisin. In some other embodiments, the promoter is heterologous to the one or more subtilisins described herein, but functions in the host cell. Examples of exemplary promoters for bacterial host cells include, but are not limited to, amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpaII promoters; promoters of Bacillus stearothermophilus maltogenic amylase gene, promoters of Bacillus amyloliquefaciens (BAN) amylase gene, promoters of Bacillus subtilis alkaline protease gene, promoters of Bacillus clausii alkaline protease gene, promoters of Bacillus pumilus xylosidase gene, promoters of Bacillus thuringiensis cryIIIA and promoters of Bacillus licheniformis alpha-amylase gene. Other promoters include, but are not limited to, A4 promoter, and bacteriophage λPR or PL promoters and Escherichia coli lac, trp or tac promoters.

[0672] One or more subtilisin variants described herein can be produced in the host cell of any suitable microorganism (including bacteria and fungi).In certain embodiments, one or more subtilisin variants described herein can be produced in gram-positive bacteria.In certain embodiments, host cell is bacillus species (Bacillus spp.), Streptomyces species (Streptomyces spp.), Escherichia species (Escherichia spp.), Aspergillus species (Aspergillus spp.), Trichoderma species (Trichoderma spp.), Pseudomonas species (Pseudomonas spp.), Corynebacterium species (Corynebacterium spp.), Saccharomyces species (Saccharomyces spp.) and Pichia species (Pichia spp.).In certain embodiments, one or more subtilisin variants described herein are produced by bacillus host cells. Examples of Bacillus host cells that can be used for the production of one or more subtilisin variants described herein include, but are not limited to: Bacillus licheniformis, Bacillus lentus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, alkaliphilic Bacillus, Bacillus coagulans, Bacillus circulans, Bacillus brevis, Bacillus thuringiensis, Bacillus clausii and Bacillus megaterium, and other organisms within the genus Bacillus. In certain embodiments, Bacillus subtilis host cells are used to produce variants described herein. USPN 5,264,366 and 4,760,025 (RE 34,606) describe various Bacillus host strains that can be used to produce one or more subtilisin variants described herein, but other suitable strains can be used.

[0673] Several bacterial strains that can be used to produce one or more subtilisin variants as described herein include non-recombinant (i.e. wild-type) Bacillus strains, and naturally occurring bacterial strains and / or recombinant strain variants. In certain embodiments, the host strain is a recombinant strain, wherein the polynucleotides encoding one or more subtilisin variants as described herein have been introduced into the host. In certain embodiments, the host strain is a subtilis host strain, particularly a recombinant subtilis host strain. Many strains of Bacillus subtilis are known, including, but not limited to, e.g., 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753 to PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP211 strains (see, e.g., Hoch et al., Genetics 73:215-228 (1973); see also U.S. Pat. No. 4,450,235; U.S. Pat. No. 4,302,544; and EP 0134048). The use of Bacillus subtilis as an expression host cell is well known in the art (see, e.g., Palva et al., Gene 19:81-87 (1982); Fahnestock and Fischer, J. Bacteriol., 165:796-804 (1986); and Wang et al., Gene 69:39-47 (1988)).

[0674] In some embodiments, the Bacillus host cell is a Bacillus species comprising a mutation or deletion in at least one of the following genes: degU, degS, degR, and degQ. In some embodiments, the mutation is in the degU gene, and in some embodiments, the mutation is degU(Hy)32 (see, e.g., Msadek et al., J. Bacteriol. 172:824-834 (1990); and Olmos et al., Mol. Gen. Genet. 253:562-567 (1997)). In some embodiments, the Bacillus host comprises a mutation or deletion in scoC4 (see, e.g., Caldwell et al., J. Bacteriol. 183:7329-7340 (2001)); spoIIE (see, e.g., Arigoni et al., Mol. Microbiol. 31:1407-1415 (1999)); and / or oppA or other genes of the opp operon (see, e.g., Perego et al., Mol. Microbiol. 5:173-185 (1991)). Indeed, it is contemplated that any mutation in the opp operon that causes the same phenotype as a mutation in the oppA gene will be useful in some embodiments of the altered Bacillus strains described herein. In some embodiments, these mutations occur alone, while in other embodiments, there are combinations of mutations. In some embodiments, an altered Bacillus host cell strain that can be used to produce one or more subtilisin variants described herein is a Bacillus host strain that already contains a mutation in one or more of the above genes. In addition, Bacillus host cells comprising one or more mutations and / or deletions of endogenous protease genes can be used. In some embodiments, the Bacillus host cell comprises a deletion of the aprE and nprE genes. In other embodiments, the Bacillus host cell comprises a deletion of 5 protease genes, and in other embodiments, the Bacillus host cell comprises a deletion of 9 protease genes (see, e.g., US2005 / 0202535).

[0675] Use any suitable method known in the art to transform host cells with one or more nucleic acid sequences encoding one or more subtilisin variants as herein described. Utilize plasmid DNA construct or carrier to introduce nucleic acid (for example DNA) into bacillus cell or Escherichia coli cell and the method that such plasmid DNA construct or carrier are transformed into such cell is well known. In certain embodiments, plasmid is separated from Escherichia coli cell subsequently and is transformed into bacillus cell. However, there is no need to use intervention microorganism such as Escherichia coli, and in certain embodiments, DNA construct or carrier are directly introduced into bacillus host.

[0676] Exemplary methods for introducing one or more nucleic acid sequences described herein into Bacillus cells are described, for example, in Ferrari et al., "Genetics," in Hardwood et al. (eds.), Bacillus, Plenum Publishing Corp. (1989), pp. 57-72; Saunders et al., J. Bacteriol., 157:718-726 (1984); Hoch et al., J. Bacteriol., 93:1925-1937 (1967); Mann et al., Current Microbiol., 13:131-135 (1986); Holubova, Folia [Microbiol., 30:97 (1985); Chang et al., Mol. Gen. Genet., 168:11-115 (1979); Vorobjeva et al., FEMS Microbiol. Lett., 7:261-263 (1980); Smith et al., Appl. Env. Microbiol., 51:634 (1986); Fisher et al., Arch. Microbiol., 139:213-217 (1981); and McDonald, J. Gen. Microbiol., 130:203 (1984). In practice, transformation methods including protoplast transformation and transfection, transduction and protoplast fusion are well known and suitable for use herein. Methods known in the art for transforming Bacillus cells include methods such as plasmid marker rescue transformation, which involves the uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (see, Contente et al., Plasmid [Plasmid] 2: 555-571 (1979); Haima et al., Mol. Gen. Genet. [Molecular and General Genetics], 223: 185-191 (1990); Weinrauch et al., J. Bacteriol. [Journal of Bacteriology], 154: 1077-1087 (1983); and Weinrauch et al., J. Bacteriol. [Journal of Bacteriology], 169: 1205-1211 (1987)). In this method, the incoming donor plasmid recombines with homologous regions of a resident "helper" plasmid in a process that mimics chromosomal transformation.

[0677] In addition to commonly used methods, in some embodiments, a host cell is directly transformed with a DNA construct or vector comprising one or more nucleic acids encoding one or more subtilisin variants described herein (i.e., before introduction into the host cell, the intermediate cell is not used to amplify or otherwise process the DNA construct or vector). The introduction of a DNA construct or vector described herein into a host cell includes those physical and chemical methods known in the art for introducing a nucleic acid sequence (e.g., a DNA sequence) into a host cell without inserting it into the host genome. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, and liposomes. In another embodiment, a DNA construct or vector is co-transformed with a plasmid without inserting a plasmid. In another embodiment, a selection marker is deleted from a modified Bacillus strain by methods known in the art (see Stahl et al., J. Bacteriol. [Journal of Bacteriology] 158: 411-418 (1984); and Palmeros et al., Gene [Gene] 247: 255-264 (2000)).

[0678] In some embodiments, the transformed cells are cultured in a conventional nutrient medium. Suitable specific culture conditions, such as temperature, pH, etc. are known to those skilled in the art and are described in detail in the scientific literature. Some embodiments provide a culture (e.g., a cell culture) comprising one or more subtilisin variants or nucleic acid sequences as described herein.

[0679] In some embodiments, host cells transformed with one or more polynucleotide sequences encoding one or more subtilisin variants described herein are cultured in a suitable nutrient medium under conditions that allow expression of the variant, and the resulting variant is thereafter recovered from the culture. In some embodiments, variants produced by the cells are recovered from the culture medium by conventional procedures, including, but not limited to, for example, separation of host cells from the culture medium by centrifugation or filtration, precipitation of protein components of the supernatant or filtrate with the aid of salts (e.g., ammonium sulfate), and chromatography purification (e.g., ion exchange, gel filtration, affinity, etc.).

[0680] In certain embodiments, one or more subtilisin variants produced by recombinant host cells are secreted into the culture medium. The nucleic acid sequence encoding the purification promotion domain can be used to promote the purification of the variant. The vector or DNA construct comprising the polynucleotide sequence encoding one or more subtilisin variants described herein can further include the nucleic acid sequence encoding the purification promotion domain that promotes the purification of the variant (see, for example, Kroll et al., DNA Cell Biol. [DNA cell biology] 12: 441-53 (1993)). Such purification promotion domains include, but are not limited to, for example, metal chelating peptides, such as histidine-tryptophan modules (see Porath, Protein Expr. Purif. [protein expression and purification] 3: 263-281

[1992] ) that allow purification on immobilized metals; Protein A domains that allow purification on immobilized immunoglobulins; and the domains used in the FLAGS extension / affinity purification system. The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (eg, sequences available from Invitrogen, San Diego, CA) between the purification domain and the heterologous protein can also be used to facilitate purification.

[0681] A variety of methods can be used to determine the production level of one or more mature subtilisin variants as described herein in a host cell. Such methods include, but are not limited to, for example, methods using polyclonal or monoclonal antibodies specific for proteases. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), and fluorescence activated cell sorting (FACS). These and other assays are well known in the art (see, for example, Maddox et al., J. Exp. Med. [Journal of Experimental Medicine] 158: 1211 (1983)).

[0682] Some other embodiments provide methods for preparing or producing one or more mature subtilisin protease variants as described herein.Ripe subtilisin protease variants do not include signal peptide or leader peptide sequence.Some methods are included in recombinant bacterial host cells (for example, as Bacillus cells (for example, subtilis cells)) to prepare or produce one or more subtilisin protease variants as described herein.Other embodiments provide methods for producing one or more subtilisin protease variants as described herein, wherein the method is included in cultivating a recombinant host cell comprising a recombinant expression vector under conditions conducive to producing the variant, and the recombinant expression vector comprises a nucleotide sequence encoding one or more subtilisin protease variants as described herein.Some such methods further include reclaiming the variant from the culture.

[0683] Other embodiments provide methods for producing one or more subtilisin variants as described herein, wherein the method comprises: (a) introducing a recombinant expression vector comprising a nucleic acid encoding the variant into a cell population (e.g., a bacterial cell, e.g., a subtilis cell); and (b) culturing cells in a culture medium under conditions conducive to producing a variant encoded by the expression vector. Some such methods further comprise: (c) isolating the variant from the cell or from the culture medium.

[0684] Unless otherwise noted, all components or composition levels provided herein are given with reference to the activity level of the component or composition, and do not include impurities that may be present in commercially available sources, such as residual solvents or by-products. Enzyme component weight is based on total active protein. Unless otherwise noted, all percentages and ratios are calculated by weight. Unless otherwise noted, all percentages and ratios are calculated based on total composition. Compositions described herein include cleaning compositions, such as detergent compositions. In the exemplified detergent compositions, enzyme levels are represented by pure enzymes by weight of the total composition and, unless otherwise noted, detergent ingredients are represented by weight of the total composition.

[0685] In one embodiment, one or more subtilisin variants described herein can be used in cleaning applications, such as, but not limited to, for cleaning tableware items, tableware items, fabrics, medical devices, and items with hard surfaces (e.g., hard surfaces of tables, tabletops, walls, furniture items, floors, ceilings, etc.). In other embodiments, one or more subtilisin variants described herein can be used in disinfection applications, such as, but not limited to, disinfecting automatic dishwashing machines or washing machines.

[0686] Another embodiment relates to a composition comprising one or more subtilisin variants as described herein. In certain embodiments, the composition is a cleaning composition. In other embodiments, the composition is a detergent composition. In other embodiments, the composition is selected from laundry detergent compositions, automatic dishwashing (ADW) compositions, hand-washing (manual) dishwashing detergent compositions, hard surface cleaning compositions, glasses cleaning compositions, medical device cleaning compositions, disinfectants (e.g., malodors or microorganisms) compositions, and personal care cleaning compositions. In still other embodiments, the composition is a laundry detergent composition, an ADW composition, or a hand-washing (manual) dishwashing detergent composition. Even still other embodiments relate to fabric cleaning compositions, while other embodiments relate to non-fabric cleaning compositions. In certain embodiments, the cleaning composition does not contain boron. In other embodiments, the cleaning composition does not contain phosphate. In still other embodiments, the composition comprises one or more subtilisin variants as described herein and one or more excipients, auxiliary materials, and / or other enzymes.

[0687] In still further embodiments, the compositions described herein contain phosphates, do not contain phosphates, contain boron, do not contain boron, or a combination thereof. In other embodiments, the composition is a boron-free composition. In some embodiments, a boron-free composition is a composition to which a borate stabilizer is not added. In another embodiment, a boron-free composition is a composition containing less than 5.5% boron. In still further embodiments, a boron-free composition is a composition containing less than 4.5% boron. In still another embodiment, a boron-free composition is a composition containing less than 3.5% boron. In still further embodiments, a boron-free composition is a composition containing less than 2.5% boron. In even further embodiments, a boron-free composition is a composition containing less than 1.5% boron. In another embodiment, a boron-free composition is a composition containing less than 1.0% boron. In still further embodiments, a boron-free composition is a composition containing less than 0.5% boron. In still further embodiments, a boron-free composition is a composition substantially free of boron.

[0688] In another embodiment, one or more compositions described herein are in a form selected from gel, tablet, powder, granule, solid, liquid, unit dose and combinations thereof. In yet another embodiment, one or more compositions described herein are in a form selected from low water dense formula, low water HDL or UD, or high water formula or HDL. In some embodiments, the cleaning composition described herein is in unit dose form. In other embodiments, the unit dose form is selected from pills, tablets, capsules, caplets, sachets, pouches, multi-compartment pouches and pre-measured powders or liquids. In some embodiments, the unit dose form is designed to provide controlled release of ingredients in a multi-compartment pouch (or other unit dose form). Suitable unit doses and controlled release forms are described in, for example, EP 2100949; WO 02 / 102955; US 4,765,916; US 4,972,017; and WO 04 / 111178. In some embodiments, the unit dose form is a tablet or powder contained in a water-soluble film or pouch.

[0689] Exemplary laundry detergent compositions include, but are not limited to, for example, liquid and powder laundry detergent compositions. Exemplary hard surface cleaning compositions include, but are not limited to, for example, compositions for cleaning hard surfaces of non-tableware items, non-desktop utensil items, tables, desktops, furniture items, walls, floors, and ceilings. Exemplary hard surface cleaning compositions are described in, for example, USPN 6,610,642, 6,376,450, and 6,376,450. Exemplary personal care compositions include, but are not limited to, compositions for cleaning dentures, teeth, hair, contact lenses, and skin. Exemplary components of such oral care compositions include, for example, those described in US 6,376,450.

[0690] In certain embodiments, one or more subtilisin variants described herein clean at low temperatures. In other embodiments, one or more compositions described herein clean at low temperatures. In other embodiments, one or more compositions described herein comprise an effective amount of one or more subtilisin variants described herein, which are useful or effective for cleaning surfaces that require protein stain removal.

[0691] In some embodiments, adjunct materials are incorporated, for example, to assist or enhance cleaning performance (for treating substrates to be cleaned), or to change the aesthetics of the cleaning composition (e.g., with perfumes, pigments, dyes, etc.). One embodiment relates to a composition comprising one or more adjunct materials described herein and one or more subtilisin variants. Another embodiment relates to a composition comprising one or more adjunct materials described herein and one or more subtilisin variants, wherein the adjunct materials are selected from: bleach catalysts, additional enzymes, enzyme stabilizers (including, for example, enzyme stabilization systems), chelants, brighteners, soil release polymers, dye transfer agents, dispersants, foam inhibitors, dyes, perfumes, colorants, fillers, photoactivators, fluorescent agents, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, Fungicides, color embellishments, silver care agents, anti-tarnish agents, anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, pH control agents, surfactants, builders, chelating agents, dye transfer inhibitors, deposition aids, dispersants, catalytic materials, bleach activators, bleach boosters, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, polymer dispersants, clay soil removal / anti-redeposition agents, structure elasticizers, fabric softeners, carriers, hydrotropes, processing aids, pigments, and combinations thereof. Suitable examples of exemplary auxiliary materials and use levels can be found in USPN 5,576,282; 6,306,812; 6,326,348; 6,610,642; 6,605,458; 5,705,464; 5,710,115; 5,698,504; 5,695,679; 5,686,014 and 5,646,101. In embodiments where one or more cleaning auxiliary materials are incompatible with one or more subtilisin variants described herein, a method of keeping the auxiliary material and one or more variants separate (i.e., not in contact with each other) is used until the combination of the two components is suitable. Such separation methods include any suitable method known in the art (e.g., capsules, encapsulation, tablets, physical separation, etc.).

[0692] Some embodiments relate to a cleaning additive product comprising one or more subtilisin variants as described herein. In certain embodiments, the additive is encapsulated in a formulation for adding to a cleaning process. In certain embodiments, the additive is encapsulated in a formulation for adding to a cleaning process, in which a peroxide source is used and an increased bleaching effect is desired.

[0693] Exemplary fillers or carriers for granular compositions include, but are not limited to, various salts such as sulfates, carbonates, and silicates; talc; and clay. Exemplary fillers or carriers for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols, including polyols and diols (e.g., methanol, ethanol, propanol, and isopropanol). In some embodiments, the composition comprises from about 5% to about 90% of such fillers or carriers. Acidic fillers may be included in such compositions to reduce the pH of the resulting solution in the cleaning method or application.

[0694] In one embodiment, one or more cleaning compositions described herein comprise an effective amount of one or more subtilisin variants described herein, alone or in combination with one or more additional enzymes. Typically, the cleaning compositions comprise at least about 0.0001 to about 20 wt.%, from about 0.0001 to about 10 wt.%, from about 0.0001 to about 1 wt.%, from about 0.001 to about 1 wt.%, from about 0.001 to about 1 wt.%, or from about 0.01 to about 0.1 wt.% of one or more proteases. In another embodiment, one or more cleaning compositions described herein comprise from about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.01 to about 2 mg, about 0.01 to about 1 mg, about 0.05 to about 1 mg, about 0.5 to about 10 mg, about 0.5 to about 5 mg, about 0.5 to about 4 mg, about 0.5 to about 4 mg, about 0.5 to about 3 mg, about 0.5 to about 2 mg, about 0.5 to about 1 mg, about 0.1 to about 10 mg, about 0.1 to about 5 mg, about 0.1 to about 4 mg, about 0.1 to about 3 mg, about 0.1 to about 2 mg, about 0.1 to about 2 mg, about 0.1 to about 1 mg, or about 0.1 to about 0.5 mg of one or more proteases per gram of composition.

[0695] Typically the cleaning compositions described herein are formulated so that during use in aqueous cleaning operations, the wash water will have a pH from about 4.0 to about 11.5, or even from about 5.0 to about 11.5, or even from about 5.0 to about 8.0, or even from about 7.5 to about 10.5. Liquid product formulations are typically formulated to have a pH from about 3.0 to about 9.0, or even from about 3 to about 5. Granular laundry products are typically formulated to have a pH from about 9 to about 11. In some embodiments, the cleaning compositions of the present invention may be formulated to have an alkaline pH under washing conditions, such as a pH from about 8.0 to about 12.0, or from about 8.5 to about 11.0, or from about 9.0 to about 11.0. In some embodiments, the cleaning compositions of the present invention may be formulated to have a neutral pH under washing conditions, such as a pH from about 5.0 to about 8.0, or from about 5.5 to about 8.0, or from about 6.0 to about 8.0, or from about 6.0 to about 7.5. In some embodiments, neutral pH conditions can be measured when the cleaning composition is dissolved in deionized water at 1:100 (wt:wt) at 20° C., using a conventional pH meter. Techniques for controlling pH at recommended usage levels include the use of buffers, bases, acids, etc., and are well known to those of ordinary skill in the art.

[0696] In some embodiments, one or more subtilisin variants described herein are encapsulated to protect them from other components in the composition during storage and / or to control the availability of variants during cleaning. In certain embodiments, encapsulation enhances the performance of variants and / or other enzymes. In certain embodiments, the encapsulating material typically encapsulates at least a portion of subtilisin variants described herein. Typically, the encapsulating material is water-soluble and / or water-dispersible. In certain embodiments, the encapsulating material has a glass transition temperature (Tg) of 0 ° C or higher. Exemplary encapsulating materials include, but are not limited to: carbohydrates, natural or synthetic gums, chitin, chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin and combinations thereof. When the encapsulating material is a carbohydrate, it is typically selected from monosaccharides, oligosaccharides, polysaccharides and combinations thereof. In certain embodiments, the encapsulating material is starch (see, for example, EP 0922499, US4,977,252, US 5,354,559 and US 5,935,826). In some embodiments, the encapsulating material is a microsphere made of a plastic such as a thermoplastic, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile, and mixtures thereof. Exemplary commercial microspheres include, but are not limited to (Akzo Nobel Chemicals International, BV); PM6545, PM6550, PM7220, PM7228 and (Sphere One Corporation); and and (Potters Industries LLC).

[0697] In some embodiments, the present invention provides a kind of washing condition, and the washing condition of the present invention is suitable for washing at a temperature of from about 10 ℃ to about 40 ℃, from about 20 ℃ to about 30 ℃, or from about 15 ℃ to about 25 ℃, and at about 15 ℃ to about 35 ℃, and at a temperature of from 10 ℃ to 40 ℃, and at a temperature of from about 10 ℃ to about 40 ℃, and at a temperature of from about 10 ℃ to about 35 ℃, and at a temperature of from about 10 ℃ to about ...35 ℃, and at a temperature of from about 10 ℃ to about 40 ℃, and at a temperature of from about 10 ℃ to about 35 ℃, and at a temperature of from about 10 ℃ to about 35 ℃, and at a temperature of from about 10 ℃ to about 40 ℃, and at a temperature of from about 10 ℃ to about 35 ℃, and at a temperature of from about 10 ℃ to about 35 ℃, and

[0698] Different geographical locations have different water hardness. Hardness is the amount of calcium (Ca 2+ ) and magnesium (Mg 2+ ) is a measure of the amount of Ca mixed in the mixture. Usually measured in particles per gallon (gpg) 2+ / Mg 2+ to describe water hardness. In the United States, most water is hard water, but there are different levels of hardness. Moderately hard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 ppm (to convert ppm to particles / US gallon is to divide ppm by 17.1) of hardness minerals.

[0699] water Particles / Gallon Parts per million soft Less than 1.0 Less than 17 Slightly hard 1.0 to 3.5 17 to 60 Medium hard 3.5 to 7.0 60 to 120 hard 7.0 to 10.5 120 to 180 Very hard Greater than 10.5 More than 180

[0700] Other embodiments relate to one or more cleaning compositions comprising from about 0.00001% to about 10% by weight of the composition of one or more subtilisin variants described herein, and from about 99.999% to about 90.0% by weight of the composition of one or more auxiliary materials. In another embodiment, the cleaning composition comprises from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, or from about 0.005% to about 0.5% by weight of the composition of one or more subtilisin variants, and from about 99.9999% to about 90.0%, from about 99.999% to about 98%, from about 99.995% to about 99.5% by weight of the composition of one or more auxiliary materials.

[0701] In other embodiments, the compositions described herein comprise one or more subtilisin variants described herein and one or more additional enzymes. The one or more additional enzymes are selected from acyltransferases, α-amylases, β-amylases, α-galactosidases, arabinosidases, arylesterases, β-galactosidases, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, DNA enzyme or nuclease, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hyaluronidase, keratinase, laccase, lactase, The invention relates to a combination of enzymes (i.e., a "mixture") comprising a conventional enzyme (e.g., an amylase, a lipase, a cutinase, and / or a cellulase) and / or a combination of enzymes (e.g., a "mixture") of at least one subtilisin enzyme variant described herein and / or at least one additional protease.

[0702] In another embodiment, one or more compositions described herein comprise one or more subtilisin variants described herein and one or more additional proteases. In one embodiment, additional proteases are serine proteases. In another embodiment, additional proteases are microbial alkaline proteases or trypsin-like proteases. Suitable additional proteases include those of animal, plant or microbial origin. In certain embodiments, additional proteases are microbial proteases. In other embodiments, additional proteases are chemically or genetically modified mutants. In another embodiment, additional proteases are microbial alkaline proteases or trypsin-like proteases. Exemplary alkaline proteases include subtilisins derived from, for example, Bacillus (e.g., subtilisin slow, subtilisin amylolytic, subtilisin Gibbs, subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168). Exemplary metalloproteases include neutral metalloproteases nprE expressed in recombinant form in Bacillus subtilis (see, for example, WO 07 / 044993) and purified neutral metalloproteases PMN from Bacillus amyloliquefaciens.Exemplary additional proteases include, but are not limited to, those described in WO 92 / 21760, WO 95 / 23221, WO 2008010925, WO 09149200, WO 09149144, WO 09149145, WO10056640, WO 10056653, WO 20100566356, WO 2011072099, WO 201113022, WO 11140364, WO2012151534, WO 2015038792, WO 2015 / 089441, WO 2015089447, WO 2015143360, WO2016001449, WO 2016001450, WO 2016061438、WO 2016069544、WO 2016069548、WO2016069552、WO 2016069557、WO 2016069563、WO 2016069569、WO 2016087617、WO2016087619、WO 2016145428, WO 2016174234, WO 2016183509, WO 2016202835, WO2016205755, US20080090747, US 5,801,039, US 5,340,735, US 5,500,364, US 5,855,625,RE 34,606, US 5,955,340, US 5,700,676 US 6,312,936, US 6,482,628, US 8,530,219, US Provisional Application Nos. 62 / 331282, 62 / 351649, 62 / 437171, 62 / 437174 and 62 / 437509 and PCT Application Nos. PCT / CN2017 / 076749 and PCT / US2017 / 029307, and metalloproteases described in the following published patents: WO1999014341, WO 1999033960, WO 1999014342, WO 1999034003, WO 2007044993, WO2009058303, WO 2009058661, WO 2014071410, WO 2014194032, WO 2014194034, WO2014194054 and WO 2014194117. Exemplary additional proteases also include, but are not limited to, trypsin (e.g., of porcine or bovine origin) and the Fusarium protease described in WO 89 / 06270. Exemplary commercial proteases include, but are not limited to, MAXATASE, MAXACAL, MAXAPEM,. OXP, PREFERENZ TM Proteases (e.g. P100, P110, P280), EFFECTENZ TM Proteases (e.g. P1000, P1050, P2000), EXCELLENZ TM Proteases (e.g. P1000), and PURAFAST (Danisco, USA); ULTRA, 16L, ULTRA, PRIMASE、DURAZYM、 PROGRESS and (Novozymes); BLAP TM and BLAP TM Variant (Henkel); LAVERGY TM PRO 104L (BASF), and KAP (B. alkalophilus subtilisin (Kao Corporation).

[0703] Another embodiment relates to a composition comprising one or more subtilisin variants as described herein and one or more lipases. In certain embodiments, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% lipase by weight of the composition. Exemplary lipases can be chemically or genetically modified mutants. Exemplary lipases include, but are not limited to, those of, for example, bacterial or fungal origin, such as, for example, H. lanuginosa lipase (see, for example, EP 258068 and EP 305216), T. lanuginosus lipase (see, for example, WO 2014 / 059360 and WO 2015 / 010009), Rhizomucor miehei lipase (see, for example, EP 238023), Candida lipases such as C. antarctica lipase (e.g., C. antarctica lipase A or B) (see, for example, EP 238023). 214761), Pseudomonas lipases such as Pseudomonas alcaligenes and Pseudomonas pseudoalcaligenes lipases (see, e.g., EP 218272), Pseudomonas cepacia lipase (see, e.g., EP 331376), Pseudomonas stutzeri lipase (see, e.g., GB 1,372,034), Pseudomonas fluorescens lipase, Bacillus lipases (e.g., Bacillus subtilis lipase (Dartois et al., Biochem. Biophys. Acta [Biochemistry and Biophysics] 1131:253-260 (1993)), Bacillus stearothermophilus lipase (see, e.g., JP 64 / 744992), and Bacillus pumilus lipase (see, e.g., WO 114761), Pseudomonas lipases such as Pseudomonas alcaligenes and Pseudomonas pseudoalcaligenes lipases (see, e.g., EP 218272), Pseudomonas cepacia lipase (see, e.g., EP 331376), Pseudomonas stutzeri lipase (see, e.g., GB 1,372,034), Pseudomonas fluorescens lipase, 91 / 16422)).Exemplary cloned lipases include, but are not limited to, Penicillium camembertii lipase (see Yamaguchi et al., Gene 103:61-67 (1991)); Geotricum candidum lipase (see Schimada et al., J. Biochem., 106:383-388 (1989)); and various Rhizopus lipases such as R. delemar lipase (see Hass et al., Gene 109:117-113 (1991)); R. niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 (1992)) and R. oryzae lipase. Other lipolytic enzymes such as cutinases can also be used in one or more compositions described herein, including, but not limited to, for example, cutinases derived from Pseudomonas mendocina (see WO 88 / 09367) and / or Fusarium solani pisi (see WO 90 / 09446). Exemplary commercial lipases include, but are not limited to, M1 LIPASE, LUMA FAST, and LIPOMAX (Genencor). and ULTRA (Novozymes); and LIPASE P (Amano Pharmaceutical Co. Ltd).

[0704] Still other embodiments relate to compositions comprising one or more subtilisin variants as described herein and one or more amylases. In one embodiment, the composition comprises by composition weight from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% amylase. Any amylase (e.g., alpha amylase and / or beta amylase) suitable for use in alkaline solutions may be used for inclusion in such compositions. Exemplary amylases may be chemically or genetically modified mutants. Exemplary proteases include, but are not limited to, those of bacterial or fungal origin, such as, for example, the amylases described in the following patent publications: GB 1,296,839, WO 91 / 00353, WO 94 / 02597, WO 94 / 183314, WO 95 / 10603, WO 95 / 26397, WO 95 / 35382, WO 96 / 05295, WO 96 / 23873, WO 96 / 23874, WO 96 / 30481, WO 97 / 10342, WO 97 / 41213, WO 97 / 43424, WO 98 / 13481, WO 98 / 26078, WO 99 / 02702, WO 99 / 09183, WO 99 / 19467, WO 99 / 23211、WO 99 / 29876、WO 99 / 42567、WO 99 / 43793、WO 99 / 43794、WO 99 / 46399、WO 00 / 29560、WO 00 / 60058、WO 00 / 60059、WO 00 / 60060、WO 01 / 14532、WO 01 / 34784、WO 01 / 64852、WO 01 / 66712、WO 01 / 88107、WO 01 / 96537、WO 02 / 092797、WO 02 / 10355、WO 02 / 31124、WO 2004055178、WO 2004113551、WO 2005001064, WO 2005003311, WO2005018336, WO 2005019443, WO 2005066338, WO 2006002643, WO 2006012899, WO2006012902, WO 2006031554, WO 2006063594、WO 2006066594、WO 2006066596、WO2006136161、WO 2008000825、WO 2008088493、WO 2008092919、WO 2008101894、WO2008112459、WO 2009061380、WO2011076897、WO2011080352、WO 2011080353、WO 2011080354、WO 2011082425、WO 2011082429、WO2011087836、WO 2011098531, WO 2013063460, WO 2013184577, WO 2014099523, WO2014164777 and WO 2015077126. Exemplary commercial amylases include, but are not limited to AMPLIFY BAN ULTRA, PLUS, ULTRA and (Novozymes); EFFECTENZ TM S1000, PREFERENZ TM S100、PREFERENZ TM S110、EXCELLENZ TM S2000, and P(Danisco Corporation of America).

[0705] Still other embodiments relate to compositions comprising one or more subtilisin variants as described herein and one or more cellulases. In one embodiment, the composition comprises cellulases from about 0.00001% to about 10%, 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Any suitable cellulase may be used in compositions as described herein. Exemplary cellulases may be chemically or genetically modified mutants. Exemplary cellulases include, but are not limited to, those cellulases of bacterial or fungal origin, such as described in the following published patents: WO 2005054475, WO 2005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP 0495257; and U.S. Provisional Application Nos. 62 / 296,678 and 62 / 435340. Exemplary commercial cellulases include, but are not limited to, PREMIUM, and (Novozymes); 100. 200 / 220 and 2000 (Danisco, Inc., USA); and KAC-500(B) (Kao Corporation). In some embodiments, the cellulase is incorporated as a portion or fragment of a mature wild-type or variant cellulase in which a portion of the N-terminus is deleted (see, e.g., US 5,874,276).

[0706] Still further embodiments relate to compositions comprising one or more subtilisin variants described herein and one or more mannanases. In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% mannanase by weight of the composition. Exemplary mannanases may be chemically or genetically modified mutants. Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as, for example, as described in WO2016007929, WO 2017 / 079756, and WO 2017 / 079751; and USPN 6566114, 6602842, and 6440991. Exemplary commercial mannanases include, but are not limited to (Novozymes) and EFFECTENZ TM M 1000, M 100, and PURABRITE (Danisco, USA).

[0707] Still even other embodiment relates to the composition comprising one or more subtilisin variants as herein described and one or more peroxidases and / or oxidases.In one embodiment, said composition comprises by composition weight from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2% or about 0.005% to about 0.5% peroxidase or oxidase.Peroxidase can be used in combination with hydrogen peroxide or its source (for example percarbonate, perborate or persulfate), and oxidase can be used in combination with oxygen.Peroxidase and oxidase combined with single or synergist are used for " solution bleaching " (i.e., preventing textile dyes from transferring to another fabric from a dyeing fabric when fabric is washed together in washing liquid) (see for example WO 94 / 12621 and WO 95 / 01426).Exemplary peroxidase and / or oxidase can be chemical or genetically modified mutants. Exemplary peroxidases / oxidases include, but are not limited to, those of plant, bacterial, or fungal origin.

[0708] Another embodiment relates to a composition comprising one or more subtilisin variants described herein and one or more perhydrolases, such as, for example, the perhydrolases described in WO 2005 / 056782, WO 2007 / 106293, WO 2008 / 063400, WO 2008 / 106214, and WO 2008 / 106215.

[0709] In yet another embodiment, one or more subtilisin variants described herein and one or more additional enzymes included in one or more compositions described herein may each independently vary to about 10%, with the balance of the cleaning composition being one or more adjunct materials.

[0710] In some embodiments, one or more compositions described herein can be used as detergent additives, wherein the additive is in solid or liquid form. Such additive products are intended to supplement and / or improve the performance of conventional detergent compositions and can be added at any stage of the cleaning process. In some embodiments, the density of the laundry detergent composition ranges from about 400 g / liter to about 1200 g / liter, while in other embodiments, it ranges from 500 g / liter to about 950 g / liter of composition measured at 20°C.

[0711] Some embodiments relate to laundry detergent compositions comprising one or more subtilisin variants described herein and one or more adjunct materials selected from the group consisting of surfactants, enzyme stabilizers, builder compounds, polymeric compounds, bleaching agents, additional enzymes, foam inhibitors, dispersants, lime soap dispersants, soil suspending agents, anti-redeposition agents, corrosion inhibitors, and combinations thereof. In some embodiments, the laundry composition further comprises a softener.

[0712] Additional embodiments relate to manual dishwashing compositions comprising one or more subtilisin variants described herein and one or more adjunct materials selected from the group consisting of surfactants, organic polymeric compounds, suds boosters, Group II metal ions, solvents, hydrotropes, and additional enzymes.

[0713] Other embodiments relate to one or more compositions described herein, wherein the composition is a compact granular fabric cleaning composition for colored fabric washing or providing softening through wash capacity, or a heavy duty liquid (HDL) fabric cleaning composition. Exemplary fabric cleaning compositions and / or methods of preparation are described in USPN 6,610,642 and 6,376,450. Other exemplary cleaning compositions are described in, for example, USPNs 6,605,458; 6,294,514; 5,929,022; 5,879,584; 5,691,297; 5,565,145; 5,574,005; 5,569,645; 5,565,422; 5,516,448; 5,489,392; and 5,486,303; 4,968,451; 4,597,898; 4,561,998; 4,550,862; 4,537,706; 4,515,707; and 4,515,705.

[0714] In some embodiments, the cleaning composition comprises acidified particles or aminocarboxylic acid builders. Examples of aminocarboxylic acid builders include aminocarboxylic acids, salts thereof, and derivatives thereof. In some embodiments, the aminocarboxylic acid builders are aminopolycarboxylic acid builders, such as glycine-N,N-diacetic acid or a builder having the general formula MOOC-CHR-N(CH2COOM)2 (wherein R is C 1-12alkyl and M is an alkali metal). In some embodiments, the aminocarboxylic acid builder can be methylglycine diacetic acid (MGDA), GLDA (glutamic acid-N,N-diacetic acid), iminodisuccinic acid (IDS), carboxymethyl inulin and its salts and derivatives, aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl) In some embodiments, the acidifying particles include 1,2-diamino-1,2-diamino-2,2-diamino-3,4-diamino-4,4-diamino-5,4-diamino-6,4-diamino-7,4-diamino-8,4-diamino-9,4-diamino-10,4-diamino-11,4-diamino-2,4-diamino-9,4-diamino-12,4-diamino-13,4-diamino-14,4-diamino-15,4-diamino-16,4-diamino-17,4-diamino-18,4-diamino-20,4-diamino-19,4-diamino-21,4-diamino-22,4-diamino-3,4-diamino-18,4-diamino-23,4-diamino-19,4-diamino-24,4-diamino-18,4-diamino-25,4-diamino-26,4-diamino-19,4-diamino-27,4-diamino-28,4-diamino-3,4-diamino-18,4-diamino-29,4-diamino-30,4-diamino-31,4-diamino-32,4-diamino-33,4-diamino-34,4-diamino-35,4-diamino-36,4-diamino-37,4-diamino-38,4-diamino-39,4-diamino-4,4-diamino-39,4-diamino-4,4-diamino-31,4-diamino-33,4-diamino-34,4-diamino-39,4-diamino-31,4-diamino-33,4-diamino-34,4-diamino-35,4-diamino-36,4-diamino-39,4-diamino-31,4-diamino-33,4-diamino-33,4-

[0715] In certain embodiments, the acidified particles can comprise any acid, including organic and inorganic acids. The organic acid can have one or two carboxyl groups, and can have up to 15 carbons in some cases, particularly up to 10 carbons, for example formic acid, acetic acid, propionic acid, capric acid, oxalic acid, succinic acid, adipic acid, maleic acid, fumaric acid, sebacic acid, malic acid, lactic acid, glycolic acid, tartaric acid and hydrated glyoxylic acid. In certain embodiments, the acid is citric acid. Inorganic acids include hydrochloric acid and sulfuric acid. In some cases, the acidified particles are high-activity particles comprising high-level aminocarboxylic acid builders. It has also been found that sulfuric acid further contributes to the stability of the final particles.

[0716] Additional embodiments relate to cleaning compositions comprising one or more subtilisin variants and one or more surfactants and / or surfactant systems, wherein the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof. In some embodiments, the surfactant is present at a level of from about 0.1% to about 60%, while in alternative embodiments, the level is from about 1% to about 50%, and in yet further embodiments, the level is from about 5% to about 40%, by weight of the cleaning composition.

[0717] In some embodiments, one or more compositions described herein comprise one or more detergent builders or builder systems. In one embodiment, the composition comprises from about 1%, from about 0.1% to about 80%, from about 3% to about 60%, from about 5% to about 40%, from about 10% to about 50% of a builder by weight of the composition. Exemplary builders include, but are not limited to, alkali metal, ammonium and alkanolammonium salts of polyphosphates; alkali metal silicates; alkaline earth and alkali metal carbonates; aluminosilicates; polycarboxylate compounds; ether hydroxypolycarboxylates; copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulfonic acid, and carboxymethyloxysuccinic acid; ammonium and substituted ammonium salts of polyacetic acids, such as ethylenediaminetetraacetic acid and nitrilotriacetic acid; polycarboxylates, such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid; and soluble salts thereof. In some such compositions, builders form water-soluble hardness ion complexes (e.g., chelating builders), such as citrates and polyphosphates, such as sodium tripolyphosphate, sodium tripolyphosphate hexahydrate, potassium tripolyphosphate, and mixed sodium tripolyphosphate and potassium tripolyphosphate. Exemplary builders are described in, for example, EP 2100949. In certain embodiments, builders include phosphate builders and non-phosphate builders. In certain embodiments, builders are phosphate builders. In certain embodiments, builders are non-phosphate builders. In certain embodiments, builders include mixtures of phosphate and non-phosphate builders. Exemplary phosphate builders include, but are not limited to, monophosphates, diphosphates, tripolyphosphates or oligomeric polyphosphates, including alkali metal salts of these compounds, including sodium salts. In certain embodiments, builders can be sodium tripolyphosphate (STPP). In addition, compositions can include carbonates and / or citrates. Other suitable non-phosphate builders include homopolymers and copolymers of polycarboxylic acids and partially or completely neutralized salts thereof, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts. In some embodiments, salts of the above compounds include ammonium salts and / or alkali metal salts, i.e., lithium salts, sodium salts, and potassium salts, including sodium salts. Suitable polycarboxylic acids include acyclic, alicyclic, heterocyclic, and aromatic carboxylic acids, wherein in some embodiments, they may contain at least two carboxyl groups, which are separated from each other in each case, and in some cases are separated by no more than two carbon atoms.

[0718] In some embodiments, one or more compositions described herein include one or more chelating agents. In one embodiment, the composition includes from about 0.1% to about 15% or about 3% to about 10% of a chelating agent by weight of the composition. Exemplary chelating agents include, but are not limited to, for example, copper, iron, manganese, and mixtures thereof.

[0719] In some embodiments, one or more compositions described herein comprise one or more deposition aids. Exemplary deposition aids include, but are not limited to, for example, polyethylene glycol; polypropylene glycol; polycarboxylates; soil release polymers, such as, for example, polyethylene terephthalate; clays, such as, for example, kaolinite, montmorillonite, attapulgite, illite, bentonite, and halloysite; and mixtures thereof.

[0720] In other embodiments, one or more compositions described herein include one or more anti-redeposition agents or nonionic surfactants (which can prevent the redeposition of soil) (see, e.g., EP 2100949). For example, in ADW compositions, nonionic surfactants can be used for surface modification purposes (particularly for thin sheets) to avoid filming and staining and to improve gloss. These nonionic surfactants can also be used to prevent the redeposition of soil. In some embodiments, the nonionic surfactant can be an ethoxylated nonionic surfactant, an epoxy-terminated poly(alkoxylated) alcohol, and an amine oxide surfactant.

[0721] In some embodiments, one or more compositions described herein include one or more dye transfer inhibitors. Exemplary polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones, polyvinylimidazoles, and mixtures thereof. In one embodiment, the composition includes from about 0.0001% to about 10%, about 0.01% to about 5%, or about 0.1% to about 3% of a dye transfer inhibitor by weight of the composition.

[0722] In some embodiments, one or more compositions described herein include one or more silicates. Exemplary include, but are not limited to, sodium silicates, such as sodium disilicate, sodium metasilicate, and crystalline phyllosilicates. In some embodiments, the silicate is present at a level of from about 1% to about 20% or from about 5% to about 15% by weight of the composition.

[0723] In some still further embodiments, one or more compositions described herein comprise one or more dispersants.Exemplary water-soluble organic materials include, but are not limited to, for example, homopolymeric or copolymeric acids or their salts, wherein the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.

[0724] In some other embodiments, one or more compositions described herein include one or more enzyme stabilizers. In certain embodiments, enzyme stabilizers are water-soluble sources of calcium and / or magnesium ions. In certain embodiments, enzyme stabilizers include oligosaccharides, polysaccharides and inorganic divalent metal salts, including alkaline earth metals, such as calcium salts. In certain embodiments, enzymes used herein are stabilized by being present in water-soluble sources of zinc (II), calcium (II) and / or magnesium (II) ions in the finished product compositions providing the following ions for these enzymes, and other metal ions (e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), tin (II), cobalt (II), copper (II), nickel (II) and vanadium (IV)). Chlorides and sulfates may also be used in certain embodiments. Exemplary oligosaccharides and polysaccharides (e.g., dextrins) are described in, for example, WO 07 / 145964. In some embodiments, reversible protease inhibitors may also be used, for example, in boron-containing compounds (e.g., borates, 4-formylphenylboronic acid, and phenylboronic acid derivatives (e.g., those described in WO 96 / 41859)) and / or peptide aldehydes (e.g., as further described in WO 2009 / 118375 and WO2013004636).

[0725] In some embodiments, one or more compositions described herein comprise one or more bleaching agents, bleach activators, and / or bleach catalysts. In some embodiments, one or more compositions described herein comprise one or more inorganic and / or organic bleaching compounds. Exemplary inorganic bleaching agents include, but are not limited to, perhydrate salts, such as perborates, percarbonates, perphosphates, persulfates, and persilicates. In some embodiments, the inorganic perhydrate salts are alkali metal salts. In some embodiments, inorganic perhydrate salts that are crystalline solids but without additional protection are included, but in some other embodiments, the salts are coated. Bleach activators are typically organic peracid precursors that enhance bleaching during cleaning at temperatures of 60° C. and below. Exemplary bleach activators include compounds that give aliphatic peroxycarboxylic acids having from about 1 to about 10 carbon atoms or from about 2 to about 4 carbon atoms under perhydrolysis conditions, and / or optionally substituted peroxybenzoic acid. Exemplary bleach activators are described, for example, in EP 2100949. Exemplary bleach catalysts include, but are not limited to, manganese triazacyclononane and related complexes, and cobalt, copper, manganese and iron complexes. Additional exemplary bleach catalysts are described in, for example, US 4,246,612; US 5,227,084; US 4,810,410; WO 99 / 06521; and EP 2100949.

[0726] In some embodiments, one or more compositions described herein include one or more catalytic metal complexes. In some embodiments, metal-containing bleaching catalysts can be used. In some embodiments, metal bleaching catalysts include a catalytic system, which includes: transition metal cations (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations) with limited bleaching catalytic activity, auxiliary metal cations (e.g., zinc or aluminum cations) with little or no bleaching catalytic activity, and chelates with limited stability constants for catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetrakis (methylenephosphonic acid) and their water-soluble salts (see, e.g., US 4,430,243). In some embodiments, one or more compositions described herein are catalyzed with manganese compounds. Such compounds and usage levels are described in, e.g., US 5,576,282. In other embodiments, cobalt bleaching catalysts can be used for and are included in one or more compositions described herein. Various cobalt bleaching catalysts are described in, e.g., USPN 5,597,936 and 5,595,967.

[0727] In some further embodiments, one or more compositions described herein comprise transition metal complexes of large polycyclic rigid ligands (MRLs). As a practical matter and not by way of limitation, in some embodiments, the compositions and cleaning methods described herein are adjusted to provide at least one part per hundred million, about 0.005 ppm to about 25 ppm, about 0.05 ppm to about 10 ppm, or about 0.1 ppm to about 5 ppm of active MRL in the washing liquid. Exemplary MRLs include, but are not limited to, special ultra-rigid ligands cross-linked and bridged, such as, for example, 5,12-diethyl-1,5,8,12-tetraazabicyclo (6.6.2) hexadecane. Exemplary metal MRLs are described, for example, in WO 2000 / 32601 and US 6,225,464.

[0728] In another embodiment, one or more compositions described herein include one or more metal care agents. In some embodiments, the composition includes from about 0.1% to about 5% of a metal care agent by weight of the composition. Exemplary metal care agents include, for example, aluminum, stainless steel, and non-ferrous metals (e.g., silver and copper). Other exemplary metal care agents are described, for example, in EP2100949, WO 94 / 26860, and WO 94 / 26859. In some compositions, the metal care agent is a zinc salt.

[0729] In some embodiments, the cleaning composition is a high density liquid (HDL) composition comprising one or more subtilisin variants described herein. The HDL liquid laundry detergent may comprise a cleaning surfactant (10%-40%) comprising: an anionic cleaning surfactant selected from the following groups: linear or branched or random chain, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and / or mixtures thereof; and optionally a nonionic surfactant selected from the following groups: linear or branched or random chain, substituted or unsubstituted alkyl alkoxylated alcohols, such as C8-C 18 Alkyl ethoxylated alcohols and / or C6-C 12 Alkylphenol alkoxylate, optionally wherein the weight ratio of anionic cleaning surfactant (hydrophilic index (HIc) from 6.0 to 9) to nonionic cleaning surfactant is greater than 1: 1. Suitable cleaning surfactants also include cationic cleaning surfactants (selected from alkyl pyridine compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl ternary sulfonium compounds and / or mixtures thereof); zwitterionic and / or amphoteric cleaning surfactants (selected from alkanolamine sulfobetaines); amphoteric surfactants; semi-polar nonionic surfactants; and mixtures thereof.

[0730] In another embodiment, the cleaning composition is a liquid or gel detergent (which is not unit dosed), which can be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70% water by weight, up to about 65% water by weight, up to about 55% water by weight, up to about 45% water by weight, or up to about 35% water by weight. Other types of liquids (including but not limited to alkanols, amines, glycols, ethers, and polyols) can be included in the aqueous liquid or gel. The aqueous liquid or gel detergent may contain from 0 to 30% of an organic solvent. The liquid or gel detergent may be non-aqueous.

[0731] The composition may optionally comprise a surfactant enhancing polymer consisting of an amphiphilic alkoxylated grease cleaning polymer selected from the group consisting of an alkoxylated polymer having branched hydrophilic and hydrophobic properties, such as an alkoxylated polyalkylene imine (in the range of 0.05 wt% to 10 wt%) and / or a random grafted polymer typically comprising a hydrophilic backbone containing monomers selected from the group consisting of unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyols (e.g. glycerol) and mixtures thereof; and one or more hydrophobic side chains selected from the group consisting of C4-C6 25Alkyl groups, polypropylene, polybutene, vinyl esters of saturated C2-C6 monocarboxylic acids, C1-C6 alkyl esters of acrylic or methacrylic acid, and mixtures thereof.

[0732] The composition may include additional polymers such as soil release polymers including, for example, anionically terminated polyesters such as SRP1; polymers in random or block configurations containing at least one monomer unit selected from the group consisting of saccharides, dicarboxylic acids, polyols, and combinations thereof; ethylene terephthalate-based polymers and copolymers thereof in random or block configurations such as Repel-o-tex SF, SF-2, and SRP6; Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300, and SRN325; Marloquest SL; anti-redeposition polymers (0.1 wt % to 10 wt %, including, for example, carboxylate polymers, such as polymers comprising at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid, and any mixture thereof; vinyl pyrrolidone homopolymer; and / or polyethylene glycol having a molecular weight in the range of 500 to 100,000 Da); cellulosic polymers (including, for example, alkyl cellulose; alkyl alkoxyalkyl cellulose; carboxyalkyl cellulose; alkyl carboxyalkyl cellulose, examples of which include carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose; and mixtures thereof); and polymeric carboxylates (e.g., such as maleate / acrylate random copolymers or polyacrylate homopolymers).

[0733] The composition may further comprise saturated or unsaturated fatty acids, preferably saturated or unsaturated C 12 -C 24 Fatty acids (0 wt% to -10 wt%); deposition aids in random or block configurations (including, for example, polysaccharides, cellulosic polymers, polydiallyldimethylammonium halides (DADMAC)), and copolymers of DADMAC and vinylpyrrolidone, acrylamide, imidazole, halogenated imidazolines, and mixtures thereof; cationic guar gum; cationic cellulose, such as cationic hydroxyethyl cellulose; cationic starch; cationic polyacrylamide; and mixtures thereof.

[0734] The composition may further comprise dye transfer inhibiting agents, examples of which include manganese phthalocyanine, peroxidase, polyvinyl pyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinyl pyrrolidone and N-vinylimidazole, polyvinyl oxazolidones and polyvinylimidazoles and / or mixtures thereof; chelating agents, examples of which include ethylenediaminetetraacetic acid (EDTA); diethylenetriaminepentamethylenephosphonic acid (DTPMP); hydroxyethanediphosphonic acid (HEDP); ethylenediamine N,N'-disuccinic acid (EDDS); methylglycine diacetic acid (MGDA); diethylenetriaminepentaacetic acid (DTPA); propylenediaminetetraacetic acid (PDT A); 2-hydroxypyridine-N-oxide (HPNO); or methylglycine diacetic acid (MGDA); glutamic acid N,N-diacetic acid (N,N-dicarboxymethylglutamate tetrasodium salt (GLDA); nitrilotriacetic acid (NTA); 4,5-dihydroxyisobenzene disulfonic acid; citric acid and any salt thereof; N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP) and its derivatives.

[0735] The composition may further comprise silicone-based or fatty acid-based foam inhibitors; enzyme stabilizers; hueing dyes, calcium and magnesium cations, visual signaling ingredients, anti-foaming agents (0.001 wt.% to about 4.0 wt.%) and / or structurants / thickeners (0.01 wt.% to -5 wt.%) (selected from the group consisting of diglycerides, triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose-based materials, microcellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof).

[0736] In some embodiments, the cleaning composition is a high density powder (HDD) composition comprising one or more subtilisin variants described herein. The HDD powder laundry detergent may include a cleaning surfactant, which includes an anionic cleaning surfactant (selected from linear or branched or random chain, substituted or unsubstituted alkyl sulfate, alkyl sulfonate, alkyl alkoxylated sulfate, alkyl phosphate, alkyl phosphonate, alkyl carboxylate and / or mixtures thereof); a nonionic cleaning surfactant (selected from linear or branched or random chain, substituted or unsubstituted C8-C 18 Alkyl ethoxylates and / or C6-C 12alkylphenol alkoxylate); cationic detersive surfactant (selected from alkylpyridinium compounds, alkylquaternary ammonium compounds, alkylquaternary phosphonium compounds, alkylternary sulfonium compounds and mixtures thereof); zwitterionic and / or amphoteric detersive surfactant (selected from alkanolamine sulfobetaines); amphoteric surfactant; semi-polar nonionic surfactant and mixtures thereof; builder (phosphate-free builder, such as zeolite builder, examples of which include zeolite A, zeolite X, zeolite P and zeolite MAP in the range of 0 to less than 10 wt.%); phosphate builder, such as sodium tripolyphosphate in the range of 0 to less than 10 wt.%; citric acid, citrate and suberic acid in the range of less than 15 wt.%. nitrilotriacetic acid or its salt; silicate (sodium silicate or potassium silicate or sodium metasilicate in the range of 0 to less than 10 wt.%) or layered silicate (SKS-6); carbonate (sodium carbonate and / or sodium bicarbonate in the range of 0 to less than 10 wt.%); and bleaching agent (photobleaching agent, such as sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, xanthene dye and mixtures thereof); hydrophobic or hydrophilic bleach activator (for example, dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or its salt, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine-TAED, and nonanoyloxybenzenesulfonate-NOBS, nitrile quaternary ammonium salt (nitrile quats), and mixtures thereof); hydrogen peroxide; a source of hydrogen peroxide (an inorganic perhydrate salt, such as the mono- or tetrahydrated sodium salt of perborate, percarbonate, persulfate, perphosphate or persilicate); a preformed hydrophilic and / or hydrophobic peracid (selected from percarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, and mixtures thereof); and / or a bleach catalyst (e.g., imine bleach boosters, such as iminium cations and polyions; iminium zwitterions, modified amines, modified amine oxides, N-sulfonylimines, N-phosphonylimines, N-acylimines, thiadiazole dioxides, perfluoroimines, cyclic sugar ketones, and mixtures thereof), a metal-containing bleach catalyst (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations and auxiliary metal cations (e.g., zinc or aluminum) and a chelating agent (e.g., ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and water-soluble salts thereof)).

[0737] The composition may further comprise additional detergent ingredients including perfume microcapsules, starch encapsulated perfume coordinators, enzyme stabilizers, hueing agents, additional polymers (including fabric integrity and cationic polymers), dye locking ingredients, fabric softeners, brighteners (e.g. CI fluorescent brighteners), flocculants, chelants, alkoxylated polyamines, fabric deposition aids and / or cyclodextrins.

[0738] In some embodiments, the cleaning composition is an ADW detergent composition comprising one or more subtilisin variants described herein. The ADW detergent composition may comprise two or more nonionic surfactants selected from the following: ethoxylated nonionic surfactants, alcohol alkoxylated surfactants, epoxy-terminated poly(oxyalkylated) alcohols and amine oxide surfactants, which are present in an amount of 0-10% by weight; a builder in the range of 5%-60% (by weight), the builder including: a phosphate builder (monophosphate, diphosphate, tripolyphosphate or oligophosphate), sodium tripolyphosphate-STPP or a phosphate-free builder (amino acid-based compounds, such as MGDA (methyl-glycine-diacetic acid) and its salts and derivatives, GLDA (glutamic acid) and its derivatives. acid-N,N-diacetic acid) and its salts and derivatives, IDS (iminodisuccinic acid) and its salts and derivatives, carboxymethyl inulin and its salts and derivatives and mixtures thereof, nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and B-alaninediacetic acid (B-ADA) and its salts), homopolymers and copolymers of polycarboxylic acids and their partially or fully neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts, which are in the range of 0.5%-50% (by weight); sulfonated / carboxylated polymers (providing dimensional stability to the product), which are in the range of about 0.1% to about 50% (by weight); about 0.1% to about 10% (by weight) Drying aids (selected from polyesters, in particular anionic polyesters, optionally together with further monomers having 3 to 6 functional groups (in particular acid, alcohol or ester functions) which assist the polycondensation, polycarbonate-, polyurethane- and / or polyurea-polyorganosiloxane compounds or their reactive cyclic carbonate and urea precursor compounds) in the range of about 1% to about 20% (by weight); silicates (sodium or potassium silicates, for example sodium disilicate, sodium metasilicate and crystalline phyllosilicates); inorganic bleaches (for example peroxyhydrate salts such as perborates, percarbonates, perphosphates, persulfates and persilicates) and organic bleaches (for example % to about 10% by weight; bleach catalyst (selected from manganese triazacyclononane and related complexes, Co, Cu, Mn and Fe bipyridylamine and related complexes, and pentaamine cobalt (III) acetate and related complexes); metal care agent (selected from benzotriazole, metal salts and complexes, and silicates) in the range of about 0.1%-5% (by weight); per gram of ADW detergent composition in the range of about 0.01 mg to about 5.0mg of active enzymes (acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, carrageenanase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exomannanase, galactanase, glucoamylase, hemicellulase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipase, lipoxygenase, mannanase, oxidase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, peroxidase, phenoloxidase, phosphatase, phospholipase, phytase, polygalacturonase, protease, pullulanase, reductase, rhamnogalacturonase, β-glucanase, tannase, transglutaminase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and mixtures thereof); and an enzyme stabilizer component (selected from oligosaccharides, polysaccharides and inorganic divalent metal salts).

[0739] More embodiments relate to compositions and methods for treating fabrics (e.g., desizing textiles) using one or more subtilisin variants described herein. Fabric treatment methods are well known in the art (see, e.g., US 6,077,316). For example, the feel and appearance of a fabric can be improved by methods including contacting the fabric with a variant described herein in a solution. The fabric can be treated with a solution under pressure.

[0740] One or more subtilisin variants described herein can be applied during or after the weaving of textiles, during the desizing stage or in one or more additional fabric processing steps. During the weaving of textiles, the line is exposed to considerable mechanical strain. Before weaving on a mechanical loom, warp yarns are often coated with sizing starch or starch derivatives to increase their tensile strength and prevent disconnection. One or more subtilisin variants described herein can be applied during or after weaving to remove sizing starch or starch derivatives. After weaving, the variant can be used to remove the sizing coating before further processing the fabric to ensure uniform and washable results. One or more subtilisin variants described herein can be used alone or together with other desizing chemicals and / or desizing enzymes to desize fabrics (including cotton-containing fabrics) as detergent additives (e.g., in aqueous compositions). Amylases can also be used in compositions and methods for producing a stone-ground appearance on indigo-dyed denim fabrics and clothing. For clothing production, fabrics can be cut and sewn into clothing or clothes, which are subsequently finished. In particular, for the production of denim, different enzymatic finishing methods have been developed. The finishing process of denim garments usually begins with an enzymatic desizing step, wherein the garments are subjected to the action of starch hydrolyzing enzymes to provide softness to the fabric and make the cotton more susceptible to subsequent enzymatic finishing steps. One or more subtilisin variants described herein can be used in the following methods: finishing denim garments (e.g., "biosanding process"), enzymatic desizing and providing softness and / or finishing methods for fabrics.

[0741] In further embodiments, one or more of the subtilisin variants described herein may be used in industrial cleaning and institutional cleaning applications, such as, for example, in industrial or institutional dishwashing systems, machines or processes; in industrial or institutional on premises laundry systems, machines or processes; and in industrial or institutional laundry systems, machines or processes.

[0742] One or more subtilisin variants described herein can be used for removing protein (e.g., feathers, skin, hair, and hides) from animals and for subsequent degradation or disposal thereof. In some cases, compared with traditional immersion in boiling water or dehairing processes, soaking animal carcasses in a solution comprising one or more subtilisin variants described herein can play a role in protecting the skin from damage. In one embodiment, feathers can be sprayed with one or more subtilisin variants described herein under conditions suitable for digestion or initiation of feather degradation. In certain embodiments, the variant can be used in combination with an oxidant.

[0743] In certain embodiments, the removal of oil or fat associated with unprocessed feathers can be assisted by one or more subtilisin variants as described herein. In certain embodiments, one or more subtilisin variants as described herein are used in a composition for cleaning feathers, and are used to disinfect and partially dehydrate fibers. In other embodiments, one or more subtilisin variants as described herein can be used to recycle protein from feathers. In some other embodiments, with or without about 0.5% (v / v) of a surfactant, one or more subtilisin variants as described herein are combined with 95% ethanol or other polar organic solvents and applied to a washing solution. In other embodiments, one or more subtilisin variants as described herein can be used alone or in combination with suitable feather processing and proteolysis methods (such as those disclosed in PCT / EP 2013 / 065362, PCT / EP 2013 / 065363 and PCT / EP2013 / 065364). In certain embodiments, the recovered protein can be used in animal or fish feeds subsequently.

[0744] In still other embodiments, one or more animal feed compositions, animal feed additives and / or pet foods contain one or more subtilisin variants as described herein. Other embodiments relate to methods for preparing such animal feed compositions, animal feed additive compositions and / or pet foods, the methods comprising mixing one or more subtilisin variants as described herein with one or more animal feed ingredients and / or animal feed additive ingredients and / or pet food ingredients.

[0745] The term "animal" includes all non-ruminants and ruminants. In a specific embodiment, the animal is a non-ruminant, such as a horse and a monogastric animal. Examples of monogastric animals include, but are not limited to: pigs (pig and swine), such as piglets, growing pigs, sows; poultry, such as turkeys, ducks, chickens, broilers, laying hens; fish, such as salmon, trout, tilapia, catfish and carp; and crustaceans, such as shrimp and prawns. In a further embodiment, the animal is a ruminant, including but not limited to cattle, calves, goats, sheep, giraffes, bison, moose, elk, yaks, buffalo, deer, camels, alpacas, llamas, antelopes, pronghorns and wildebeests.

[0746] In the context of the present invention, the term "pet food" is intended to be understood as meaning food for: household animals such as, but not limited to, dogs, cats, gerbils, hamsters, chinchillas, brown rats, guinea pigs; avian pets such as canaries, parakeets and parrots; reptile pets such as turtles, lizards and snakes; and aquatic pets such as tropical fish and frogs.

[0747] The terms "animal feed composition", "feed" and "feedstock" are used interchangeably and comprise one or more feed ingredients selected from the following group: a) cereals, such as small grains (e.g. wheat, barley, rye, oats and combinations thereof) and / or large grains, such as corn or sorghum; b) by-products from cereals, such as corn gluten meal, Distillers Dried Grain Solubles (DDGS), particularly corn-based Distillers Dried Grain Solubles (cDDGS), wheat bran, semolina, wheat flour, rice bran, rice hulls, oat hulls, palm kernels and citrus pulp; c) proteins obtained from sources such as soy, sunflower, peanut, lupin, pea, bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from plant and animal sources; and e) minerals and vitamins.

[0748] One or more subtilisin variants described herein can be further used for enzyme-assisted bleaching of pulp (e.g., chemical pulp, semi-chemical pulp, kraft pulp, mechanical pulp, or pulp prepared by a sulfite process). Generally, pulp is incubated with one or more subtilisin variants described herein under conditions suitable for bleaching pulp.

[0749] In certain embodiments, the pulp is chlorine-free pulp, which is bleached with oxygen, ozone, hydrogen peroxide or peroxyacid. In certain embodiments, one or more subtilisin variants described herein are used to exhibit enzyme-assisted bleaching of low-lignin pulp produced by a modified pulping process or a continuous pulping process. In some other embodiments, one or more subtilisin variants described herein are used alone or preferably in combination with xylanase and / or endoglucanase and / or alpha-galactosidase and / or cellobiohydrolase.

[0750] In other embodiments, one or more of the subtilisin variants described herein may be further used in enzyme-assisted debridement of tissue. This involves removing dead or damaged tissue, such as from a wound to aid healing.

[0751] In even other embodiments, one or more subtilisin variants as described herein can be further used for tissue culture. Specifically, one or more subtilisin variants as described herein can be used for (e.g., in a harvested cell process) to suspend or resuspend the cells attached to a cell culture wall. In another embodiment, one or more subtilisin variants as described herein can be used to cut the protein binding between culture cells and a culture dish, thereby allowing cells to be suspended in a solution.

[0752] In yet another embodiment, one or more of the subtilisin variants described herein may be further used as a food additive, a digestive aid, and / or a food processing aid.

[0753] In still yet another embodiment, one or more of the subtilisin variants described herein are further used in leather processing by removing hair from animal hides, soaking, degreasing, or bating, which are processes involving the degradation of non-structural proteins during leather manufacturing.

[0754] Example 1

[0755] Determination

[0756] The following assay was used in the examples that follow. Any deviations from the assay described below will be addressed in the examples where such deviations occurred.

[0757] Performance Index

[0758] The performance index (PI) of an enzyme compares the performance of a variant (measured value) with a parent enzyme (theoretical value or measured value) at the same protein concentration, the parent enzyme being selected from AprL wild type (WT) (SEQ ID NO: 2) (hereinafter referred to as "AprL parent"), BliD02339 WT (SEQ ID NO: 9) (hereinafter referred to as "BliD parent") or SQCBV35 variant (SEQ ID NO: 63) (hereinafter referred to as SQCBV35 parent). The theoretical concentration of the parent can be calculated using the parameters extracted from the Langmuir fit of the parent standard curve. A PI greater than 1 (PI>1) indicates that the variant has improved performance compared to the parent, while a PI of 1 (PI=1) identifies the variant as having the same performance as the parent, and a PI less than 1 (PI<1) identifies the variant as having worse performance than the parent.

[0759] Protein determination analysis

[0760] Protein determination of AprL or BliD variants in culture supernatants was performed using Agilent U-HPLC or HPLC systems. For the U-HPLC method, a calibration curve (0-500ppm) of purified parental protein was prepared and used to generate a standard curve. AprL or BliD variants in clarified culture supernatants were diluted 10 times in dilution buffer (Tris 25mM, pH 7.4, 5mM CaCl2) and then mixed with acetonitrile buffer (22.5mM Tris, pH 7.4, 4.5mM CaCl2, 9% acetonitrile) in a 1:1 ratio. Then, the sample was filtered using a 45 μm filter plate and loaded onto a reverse phase column (Zorbax 300SB-C3 column, 2.1x 100mm & 2.1x 50mm, all with 1.8 μm bead size) by an automatic sampler. The sample was eluted from the column with a gradient of buffer A (0.1% trifluoroacetic acid (TFA)) and buffer B (0.07% acetonitrile). The flow rate was 1 mL / min for a 4 minute run and column equilibration for 1 minute after the run. The absorbance was measured at 220 nm and the peaks were integrated using ChemStation software (Agilent Technologies). The protein concentration of the sample was calculated based on a standard curve of the purified parent. In some cases, the Bradford method for protein determination was used with HPLC. The Bradford reagent (BradfordQuickstart 1X dye reagent, BioRad) was used and the absorbance at 595 / 450 nm was measured on a Spectramax microplate reader.

[0761] Alternatively, protein determination of AprL variants from culture supernatants was performed by HPLC using an Agilent 1100 / 1200 system. Calibration curves (0-1500ppm) were prepared using purified parental proteins and used to generate standard curves. AprL variants from clarified culture supernatants were analyzed by one of two methods. In some cases, culture supernatants were diluted 2 times in dilution buffer (Tris 100mM, pH 8.6, 0.005% Tween 80) and loaded onto an Agilent C8 column (Agilent Poroshell 5 μm 300SB-C8 2.1x 75mm) pre-equilibrated at 69°C-70°C by an automatic sampler. Samples were eluted from the column with a gradient of buffer A (0.1% TFA) and buffer B (100% acetonitrile and 0.1% TFA). Flow rate was 2 mL / min for 2 min. In other cases, the clarified culture supernatant containing AprL variants was pretreated in an acid / SDS solution. 75 μL of sample was mixed with 6.4 μL of 4N HCl and mixed on ice for 10 minutes. 20.4 μL of 10% SDS solution was added to each well and mixed, the sample was filtered, and then loaded into an HPLC HIP-ALS Aquity125A USEC column set up at room temperature. The sample was eluted with a solution of 25 mM sodium acetate, pH 5.5, 100 mM NaCl, 0.5% SDS, with a flow rate of 0.35 mL / min and run for 8 min. The absorbance was measured at 220 nm or 230 nm, and the peaks were integrated using ChemStation (Agilent Technologies) software.

[0762] For all three protein assays, the protein concentration of each sample was calculated based on a standard curve of the purified parent enzyme.

[0763] Protease activity

[0764] The protease activity of the parent and its variants was tested by measuring the hydrolysis of N-suc-AAPF-pNA or dimethylcasein (DMC) substrates.

[0765] For the AAPF assay, the reagent solution used was: 100 mM Tris pH 8.6, 10 mM CalCl2, 0.005% -80 (Tris / Ca buffer) and 160mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388). To prepare the working solution, 1mL suc-AAPF-pNA stock solution was added to 100mL Tris / Ca buffer and mixed. The enzyme sample was added to a microtiter plate (MTP) containing 1mg / mL suc-AAPF-pNA working solution, and the activity was measured kinetically at room temperature over 3-5min at 405nm using a SpectraMax plate reader. The protease activity was expressed as mOD / min.

[0766] For the DMC assay, the reagent solutions used were: 2.5% DMC (Sigma) in 100 mM sodium carbonate (pH 9.5), 0.075% TNBSA (2,4,6-trinitrobenzenesulfonic acid, ThermoScientific) in reagent A. Reagent A: 45.4 g Na2B4O7.10H2O (Merck) in 15 mL 4N NaOH, brought to a final volume of 1000 mL in MQ water, diluent solution: 10 mM NaCl, 0.1 mM CaCl2, 0.005% Tween-80, 0.02% sodium azide. After adding 2.5 μL of 20 ppm protease supernatant, the MTP was filled with 47.5 μL of DMC substrate. Then 50 μL of TNBSA in reagent A was added while mixing slowly. Activity was measured kinetically at 405 nm over 5 min at room temperature using a SpectraMax plate reader. Activity was expressed in mOD / min as described above for the AAPF assay.

[0767] Evaluation of detergent compositions

[0768] The detergent formulations used in these studies are listed in Table 1 and include heavy-duty liquid laundry (HDL), heavy-duty powder laundry (HDD) and automatic dishwashing (ADW) formulations. Commercial detergents are heat-inactivated to remove enzyme activity and are administered as described in Table 1. Detergent treatments for enzyme inactivation are as follows. Enzyme-containing HDL laundry detergents (excluding test HDL 1, test HDL 2 and Persil Non-Bio HDL detergents) are inactivated by heating the pure liquid in a water bath at 95°C for 16 hours. The enzymes in the HDD detergents are inactivated by preparing a 10X concentrated solution relative to that used in the final cleaning assay and heating it at 95°C for 16 hours. The commercial ADW detergent formulation is heat-inactivated at 65°C for 30-45min as follows. The ADW detergent tablets are dissolved in 700mL 21GH water and the enzyme activity is inactivated by incubating the solution at 65°C for 30-45min. The enzyme-deactivated solution was diluted 10-fold in 21GH water before use in the assay. Protease activity was determined after inactivation using AAPF substrate to ensure complete inactivation of the protease. No protease activity was detected after heat treatment of HDD, ADW, and HDL detergents. HDL detergents: Persil Non-Bio, Test HDL 1, and Test HDL 2 were considered boron-free because they contained ≤5 mg / Kg of boron when tested for elemental boron content. The GSM-B powder formula was dissolved to 3 g / L for use and the pH was not adjusted.

[0769]

[0770]

[0771] *The pH of the wash solutions for detergents were adjusted, the values ​​are provided and the pH of the wash solutions for those labeled ND were not adjusted.

[0772] Detergent sources: ALL, Super Kirkland, and Kirkland Super Clean (Sun Products), Color Omni, Omni Klein & Clark Teague, Surf Excel, and SUN All-in-One Detergent Tablets (Unilever), Finish All-in-One and Finish Quantum Detergent Tablets (Reckitt Benckiser), and Blue Moon (Guangzhou Blue Moon). The detergents listed above were purchased from local supermarkets in 2012. Persil Small & Mighty Non-Bio Liquid Detergent "Persil Non-Bio" (Unilever) was purchased in 2014. Liby (Guangzhou Liby Enterprise) was purchased in 2016. Test HDL 1 and Test HDL 2 detergents were custom made in-house, and the respective compositions are listed in Tables 1.2 and 1.3, respectively. Table 1.4 shows the GSM-B phosphate-free ADW detergent (purchased without enzymes from WFK Testgewebe GmbH, Brüggen, Germany). www.testgewebe.de The GSM-B 9 formulation was prepared by adjusting the pH of a commercially available GSM-B phosphate-free detergent down to pH 9. Table 1.5 shows the composition of the custom liquid ADW detergent formulations adjusted to pH 7 and pH 9 used for testing.

[0773]

[0774]

[0775]

[0776]

[0777]

[0778] Cleaning performance of laundry (HDL and HDD) and ADW detergents

[0779] Variants were tested for cleaning performance relative to the parent for various technical soils: BMI (EMPA-116, blood / milk / ink on cotton), egg (CS-38, egg yolk and heat-ripened pigment) and POM (CFT-C10, pigment / oil / milk) for laundry-based applications; and for technical soils: egg yolk (PAS-38, egg yolk on polyacrylic fiber (ripened and colored with carbon black dye)), the cleaning performance of the variants was tested relative to the parent for tableware applications. EMPA-116, CS-38 and CFT-C10 samples were pre-rinsed with deionized water for 20 minutes and dried overnight at room temperature. PAS-38 fabrics were pre-rinsed with 10mM CAPS buffer (pH 11) at 60°C in an iEMS oscillator at 900rpm for 30min, rinsed with 2ml deionized water and dried overnight. For all stains, pre-punched swatches in MTP plates (Corning 3641) were prepared by the Center for Testmaterials BV, Vlaardingen, The Netherlands. Prior to the addition of the enzyme, these microswatch-containing plates were filled with detergent.

[0780] General sample setup for stability determination

[0781] The stability of the variants was tested under various stress conditions (buffer and detergent as shown in Table 2 below) by measuring the residual activity after incubation at an elevated temperature. The elevated temperature was set to obtain a residual activity of about 30% of the stressed sample (compared to the unstressed sample). The diluted enzyme samples were mixed in the stressor and the unstressed protease activity was measured. The diluted samples in the stressor were incubated at an elevated temperature for the time period listed in Table 2, and then the stress protease activity was measured by AAPF or DMC hydrolysis. Before being used for stability determination, the detergent enzyme was heat inactivated as described above.

[0782]

[0783] For non-stress conditions, the enzyme activity to AAPF or DMC was measured immediately (see above). For stress conditions, the PCR plate was sealed and incubated for 5 minutes at elevated temperatures using an Eppendorf 385 MasterCycler Pro thermal cycler or a PTC-200 Peltier thermal cycler, and then the activity was measured. As described above, the activity of stressed and unstressed was measured by hydrolysis of synthetic substrates or by the DMC method. % residual activity was calculated by taking the ratio of stress activation to non-stress activation and multiplying by 100. The stability PI was obtained by dividing the residual activity of the variant by the residual activity of the parent.

[0784] The enzyme aliquots are added to the detergent-filled MTP containing micro-samples to a final volume of 200 μL for laundry determination, with a final enzyme concentration of 0.5-5ppm. The laundry cleaning determination using HDL or HDD formula is carried out at 25 ° C for 15-30 minutes, while the ADW determination is carried out at 40 ° C for 30 minutes. After incubation, 100-150 μL of supernatant is transferred to a fresh MTP, and the absorbance of the EMPA-116 sample is read at 600nm using a SpectraMax microplate reader or the absorbance of CS-10, CS-38 and PAS-38 samples is read at 405nm. The absorbance results are obtained by subtracting the value of the blank control (no enzyme) from each sample value. The clean PI under each determination condition is obtained by dividing the absorbance value of a given variant by the absorbance value of the predicted parent of the same concentration. The parent value is determined by fitting the standard curve of the purified parent sample to a Langmuir fit.

[0785] Determination of cleaning of matured clothes

[0786] (A) MTP assay: Each well of a 96-well MTP was filled with a mini stir plate and then 270 μL of 100% Persil non-biological detergent was added. 18.1 μL of enzyme supernatant diluted to about 500 ppm was added to the detergent-filled MTP and mixed for 5 minutes using a magnetic roller mixing device (VP710 series from V&P Scientific). For the initial time point (T=0), 25 μL of enzyme-containing detergent was transferred to an MTP filled with 125 μL of buffer (5 mM HEPES, 250 ppm 3:1 Ca:Mg water hardness, pH 8). A 5 μL aliquot of the enzyme diluted in detergent was then added to 185 μL of buffer (5 mM HEPES, 250 ppm hardness, pH 8) that was pre-added to the MTP containing the EMPA-116 BMI microswatch. At time point 0 (T=0), a cleaning assay was performed in a similar manner as described above for fresh cleaning. For the remaining time points, the detergent enzyme plates were incubated at 37°C for 3 days or 3 weeks or at 55°C for: 1.5h, 5h, or 24h; aliquots were removed at the end of each time point; and a cleaning assay was performed in a similar manner as described above for fresh cleaning. Residual cleaning values ​​were obtained by comparing the cleaning at various remaining time points with the initial cleaning at T=0.

[0787] (B) Conical tube assay: Clarified enzyme culture supernatant and 100% test HDL 1 detergent were added to 15mL polystyrene centrifuge tubes with a final volume of 1.1mL to reach a final enzyme concentration of 88ppm in the detergent. 5μL samples of each variant were immediately taken out of the tube and frozen for later determination as the initial time point (T=0). The samples were incubated at 37°C for 3 days. After incubation, 5μL samples were taken out and frozen. In order to prepare samples for cleaning determination, all samples were thawed and 660μL buffer (5mM HEPES, 250ppm 3:1Ca:Mg water hardness, pH 8) was added to the samples. 200μL aliquots of diluted samples were added to MTP containing EMPA-116BMI microsamples. The laundry cleaning determination was performed at 25°C in an iEMS incubator oscillating at 1400rpm for 60min. After incubation, 120 μL of supernatant was transferred to a fresh MTP and the absorbance was read at 600 nm using a SpectraMax plate reader. The absorbance results were obtained by subtracting the value of the blank control (no enzyme) from each sample value. The residual cleaning of each variant was calculated as the ratio of the remaining cleaning after 3 days to the cleaning at T=0 and reported as the remaining cleaning percentage.

[0788] Example 2

[0789] Combinatorial design and expression of AprL clade protease variants

[0790] Bacillus sp. AprL and Bacillus licheniformis Bra7 BliD02339 subtilisin, both members of the AprL clade, were modified to generate AprL-clade subtilisin variants with improved cleaning performance and detergent stability. The AprL clade is more fully described in International Application No. PCT / US 16 / 32514, filed May 13, 2016, published as WO 2016183509.

[0791] The amino acid sequence of the mature AprL parent enzyme is shown in SEQ ID NO:2. The synthetic gene encoding the AprL parent protease (SEQ ID NO:1) was synthesized by GeneArt, Life Technologies and used to generate variant sequences using conventional molecular biology techniques (see, e.g., Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory Press). Genes encoding AprL variants were also synthesized. The various AprL genes were then cloned into the pHYT expression vector (derived from pHY300PLK (Takara-Clontech)). The expression vector contains the aprE promoter (SEQ ID NO:3), the aprE signal peptide (SEQ ID NO:4), the AprL propeptide (SEQ ID NO:5) and the BPN' terminator sequence (SEQ ID NO:6). DNA fragments encoding various mature protease sequences of interest (wild type and variants) were introduced using techniques known in the art. The expression plasmids containing the AprL parent and protease variant gene sequences were transformed into suitable Bacillus subtilis host strains. Cells were grown in a 96-well MTP in a medium (an enriched semi-defined medium based on MOP buffer, urea as the main nitrogen source, glucose as the main carbon source, supplemented with 1% soytone for robust cell growth, containing antibiotic selection) at 32°C, 300rpm in a shaking incubator with 80% humidity for 2 days. After centrifugation and filtration, the clarified culture supernatant containing the protease of interest was used for the assay. The same method was used for the construction of BliD02339 wild-type and variant expression cassettes, subsequent transformation and cultivation of Bacillus subtilis host strains.

[0792] In some cases, AprL variants are produced by integration cassettes targeting the aprE locus of Bacillus subtilis. The DNA pool encoding the target gene is introduced into the expression cassette, and then integrated into the chromosome of a suitable Bacillus subtilis strain using methods known in the art. The DNA fragment contains the following components: BPN' terminator (SEQ ID NO:6), chloramphenicol resistance marker (SEQ ID NO:165) from Staphylococcus aureus, Bacillus subtilis aprE promoter (SEQ ID NO:3), Bacillus subtilis aprE signal peptide sequence (SEQ ID NO:4) and AprL propeptide (SEQ ID NO:5), which is a part of the expression cassette. Transformed cells are cultivated as described above, and the target protein is separated similarly.

[0793] Example 3

[0794] Performance evaluation of AprL variants in laundry applications

[0795] Variants of the AprL parent protease (SEQ ID NO: 2) prepared as described in Example 2 and listed in Table 3 (hereinafter referred to as "AprL variants") were evaluated for cleaning performance and stability as described in Examples 3-7.

[0796] The AprL variants described in Table 3 were tested in the laundry cleaning performance assay described in Example 1 using EMPA-116 BMI soil and the following laundry detergents: Super Kirkland HDD, Color Omo HDD, SurfExcel, Kirkland Ultra Clean HDL, Omo Klein & Clark Teague HDL, and Blue Moon HDL. Table 4 lists the Table 3 AprL variants with a PI>1.1 relative to the AprL parent.

[0797]

[0798]

[0799]

[0800] Example 4

[0801] Performance evaluation of AprL variants in dishwashing applications

[0802] In the ADW cleaning performance assay described in Example 1, PAS-38 egg yolk soil and the following ADW detergents were used: GSM-B pH 10, GSM-B pH 9, All-in-one Quantum and All-in-one testing of the AprL variants described in Table 3. Table 5 lists the AprL variants (listed in Table 3) that had a PI > 1.01 relative to the AprL parent in at least one of the ADW detergents. Table 6 lists the AprL variants of Table 3 that had a PI > 1.01 relative to the AprL parent in all of the ADW detergents.

[0803]

[0804]

[0805]

[0806] Example 5

[0807] AprL variants with improved stability

[0808] The AprL variants described in Table 3 were tested in the stability assays described in Example 1 in the following detergents and buffers: LAS-EDTA buffer at 53.5°C, 10% Super Kirkland HDL pH 8.2 at 56°C, and Omoklein & Clarkteague HDL pH 8.2 at 51°C. Table 7 lists the AprL variants in Table 3 that have (i) a stability PI>1.1 relative to the AprL parent in at least one of the stability assays described in Example 1, and (ii) a cleaning PI>1.01 in the laundry and ADW cleaning performance assays described in Example 1. Table 8 shows the AprL variants in Table 3 that have a PI>1.1 relative to the AprL parent in at least one of the laundry (HDL or HDD), ADW, or stability assays described in Example 1.

[0809]

[0810]

[0811]

[0812] Example 6

[0813] Performance evaluation of additional AprL variants

[0814] In the protease activity assay described in Example 1 (AAPF or DMC assay), in the laundry cleaning performance assay described in Example 1 (using EMPA-116 BMI soil and All HDL and Omoklein & Clarks HDL detergents), in the ADW cleaning performance assay described in Example 1 (using PAS-38 egg yolk soil and GSM-B pH 10 and The AprL variants described in Table 3 were tested in a 400% HPLC-MS / ...

[0815]

[0816]

[0817] Example 7

[0818] Cleaning performance of AprL variants in boron-free detergents

[0819] The cleaning performance of purified AprL variants: M123I-Q136R, M123I-Q136R-G165Q, V045R-Q136R-G165Q-S258E, V045R-M123I-Q136R-G165Q-S258E, and V045R-M123I-Q136R were tested in the following detergents, which do not contain detectable amounts of boron or boron-containing compounds: All 3X, Dirt Lift and Persil Abiotic. These AprL variants were tested in the laundry cleaning performance assay described in Example 1. The results are shown in Table 13 as PI calculated relative to the AprL parent. These proteases were tested at a dose of 2.5 ppm. The amount of boron present in the boron-free detergents of this example was quantified by elemental analysis, and in all cases only trace amounts (less than 5 mg) of boron / kg detergent were detected.

[0820]

[0821] Example 8

[0822] Cleaning performance and detergent stability of AprL and BliD02339 protease variants

[0823] The AprL variants listed in Table 14 were produced as described in Example 2. The variants of Table 14 were tested for cleaning performance and stability in one or more of the cleaning performance or stability assays described in Example 1.

[0824] The AprL variants listed in Table 14 were tested for laundry cleaning performance in Test HDL 1 and Persil Non-Bio detergents (Table 1.1, assay conditions 8 and 10) against EMPA-116 microswatches. Enzyme stability was tested in 10% Test HDL 1 detergent at 52.5°C and / or 54°C (Table 2, assay condition 5). Table 15 shows the results as PI values ​​when compared to the AprL parent, where ND means not determined.

[0825] Table 17 lists the performance of AprL variants tested according to the laundry cleaning performance and stability assays described in Example 1: (i) cleaning performance against EMPA-116, CFT CS-38 and CFT-C10 microswatches in Persil Non-Bio, Test HDL 1 and Test HDL 2 detergents, and (ii) stability in 10% PNB at 63°C for 15-20 min and / or 10% Test HDL 1 at 60°C for 15-20 min. Cleaning performance results are reported as PI values ​​relative to SQCBV35 variant (SEQ ID NO: 63), and stability results are reported as residual activity % of samples after stress relative to non-stress conditions. ND indicates that the value is not determined.

[0826]

[0827]

[0828]

[0829]

[0830]

[0831]

[0832]

[0833]

[0834]

[0835]

[0836]

[0837]

[0838]

[0839] Variants of the BliD02339 parent protease described in Table 18 (SEQ ID NO: 9) (hereinafter "BliD variants") were produced as described in Example 2 and tested for cleaning performance and stability as described in Example 1.

[0840] Table 18 lists BliD variants that showed improvements in cleaning performance relative to the BliD parent for 3 technical soil microswatches: EMPA-116, CFT CS-38, and CFT-C10 when tested in Persil non-biological and test HDL 2 laundry detergents, and also showed improved stability when tested in 10% Persil non-biological (PNB) laundry detergent (stressed at 63°C for 15-20 min). The cleaning performance and stability results are listed in Table 19. The cleaning performance results are reported as PI values ​​relative to the BliD parent (SEQ ID NO: 9), and the stability results are reported as the remaining activity % of the sample after stress relative to the non-stressed condition. ND indicates that the value is not determined.

[0841]

[0842]

[0843]

[0844]

[0845] Example 9

[0846] Comparison of AprL protease and related molecules

[0847] Identification of homologous proteases

[0848] Proteins related to AprL protease (SEQ ID NO: 2) were identified by BLAST search (Altschul et al., Nucleic Acids Res, 25: 3389-402, 1997) against the NCBI non-redundant protein database, and a subset is shown in Table 20. A similar search was run on the Genome Quest Patent database, with the search parameters set to default values, and the subset is shown in Table 21. The mature amino acid sequence (274 amino acids) of AprL protease (SEQ ID NO: 2) was used as the query sequence for both searches. The percent identity (PID) of the two search sets is defined as the number of identical residues divided by the number of aligned residues in the pairwise alignment. The column labeled "Sequence Length" corresponds to the length (in amino acids) of the protein identified by the accession number in the column labeled "Accession Number", while "Alignment Length" refers to the length (in amino acids) of the sequence used for alignment and PID calculation.

[0849]

[0850]

[0851]

[0852]

[0853]

[0854] Alignment of subtilisin sequences with AprL

[0855] The amino acid sequences of mature AprL (SEQ ID NO: 2) and BliD02339 (SEQ ID NO: 9) proteins were aligned with the mature sequences of various subtilisins in Tables 20 and 21 using CLUSTALW software (Thompson et al., Nucleic Acids Research, 22: 4673-4680, 1994) with default parameters. This alignment is shown in FIG1 , wherein a box is drawn around one or more amino acid motifs selected from SEQ ID NO: 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, and 179.

[0856] Example 10

[0857] Phylogenetic tree of subtilisins including AprL and BliD02339

[0858] AprL protein (SEQ ID NO: 2), BliD02339 protein (SEQ ID NO: 9), AprL protease variants: SQM-30 (SEQ ID NO: 36), SQCBV 20 (SEQ ID NO: 57), SQCBV 35 (SEQ ID NO: 63), SQCBV37 (SEQ ID NO: 65), SQCBV188 (SEQ ID NO: 105), SQCBV269 (SEQ ID NO: 140), SQBLV 134 (SEQ ID NO: 160), SQCBV328 (SEQ ID NO: 268), SQCBV419 (SEQ ID NO: 302), SQCBV555 (SEQ ID NO: 358), SQCBV567 (SEQ ID NO: 370), and SQCBV582 (SEQ ID NO: 382). The phylogenetic tree of the mature sequence of 1,2-dihydropyruvate (DTP) 1,6 ... Figure 2 Based on the distribution of the phylogenetic tree, some subtilisins such as Figure 2 The indicated strains were grouped to form the AprL clade.

[0859] Example 11

[0860] Additional AprL variants

[0861] The AprL variants listed in Table 22 were generated as described in Example 2.

[0862]

[0863]

[0864]

[0865]

[0866]

[0867]

[0868]

[0869]

[0870]

[0871] Example 12

[0872] Laundry cleaning performance and detergent stability of additional AprL variants

[0873] The AprL variants listed in Table 22 and SQCBV35 were tested for laundry cleaning performance and stability as described in Example 1. BMI (EMPA-116) microswatches in Persil Non-Bio, Blue Moon, and Liby detergents were used (Table 2, assay conditions 6, 10, and 16). Enzyme stability was tested at 62°C-65°C in 10% Persil Non-Bio, Blue Moon, and Liby detergents (Table 2).

[0874] Table 23 shows the cleaning performance results in Baoying non-biological, which is the PI value compared to the SQCBV35 variant (SEQ ID NO: 63). Table 23 also shows the stability results at 62°C for 15min in 10% PNB, 64°C for 15min in 10% Blue Moon, and 65°C for 15min in 10% Libai, reported as the remaining activity of the sample after stress relative to unstressed conditions. Table 24 shows the cleaning performance results in Baoying non-biological (PNB), Blue Moon and Libai detergents, which is the performance index (PI) value compared to the SQCBV35 variant (SEQ ID NO: 63). Table 24 also shows the stability results at 62°C for 15min in 10% PNB detergent, 64°C for 15min in 10% Blue Moon detergent, and 65°C for 15min in 10% Libai detergent, reported as the remaining activity of the sample after stress relative to unstressed conditions. Table 25 shows the results of cleaning performance in Persil non-biological detergent using EMPA-116, CS-38 and CFT-10 stains, as PI values ​​compared to the SQCBV35 variant (SEQID NO: 63). Table 25 also shows the stability results at 66°C for 20 min in 10% PNB, 68°C for 20 min in 10% Blue Moon, and 69°C for 20 min in 10% Libai, reported as the residual activity of the sample after stress relative to unstressed conditions. %. Table 26 provides the results of clothing cleaning performance and stability of various AprL variants in Libai, Blue Moon and PNB detergents. We describe the stability conditions in Table 2 of Example 1. Table 27 provides the performance of various AprL variants in the mature clothing cleaning assay (performed as described in Example 1: Mature clothing cleaning assay (A)). The mature cleaning performance is reported as the % cleaning activity after incubation of the sample at 37°C for 3 weeks in 100% Blue Moon detergent.

[0875]

[0876]

[0877]

[0878]

[0879]

[0880]

[0881]

[0882]

[0883]

[0884]

[0885]

[0886] Example 13

[0887] ADW cleaning performance and detergent stability of additional AprL variants

[0888] The cleaning performance of AprL protease variants, AprL wt and SQCBV35 listed in Table 22 is measured in tableware-based applications (ADW) using various detergent formulations listed in Table 1.1 and pre-rinsed or unrinsed pre-perforated egg yolk micro-samples (PAS-38) before determination. In order to prepare the pre-rinsed PAS-38 sample, 180 μl of 10mM CAPS buffer pH 11 is added to the MTP containing micro-sample, and the plate is sealed and incubated in iEMS incubator at 60 ° C and 1100rpm for 30min. The buffer is then removed, and the sample is rinsed with deionized water and air dried. In order to measure the enzyme cleanliness in the ADW formula, 175 μL detergent solution (3g / L detergent prepared with 374ppm water hardness) is added to each hole of the MTP, and then 5 μL enzymes are added. The MTP is sealed and incubated in iEMS incubator at 40 ° C and 1100rpm for 30min. After incubation, 100 μL of washing solution from each well is transferred to a new MTP, and the absorbance of the supernatant at 405nm is measured on a SpectraMax microplate reader. Net absorbance is obtained by subtracting the value of the blank control (no enzyme) from each sample in the GSM-B pH 10.5 formula. When using a custom liquid ADW detergent formula (at pH 7 and 9), all supernatants are removed from the MTP wells after enzyme incubation and transferred to another MTP to measure the absorbance at 405nm. The sample in the MTP is rinsed with deionized water, and 180 μL of 10mM CAPS buffer pH 11 is then added to each well, and the plate is incubated for another 10min at 40°C and 1100rpm in an iEMS incubator. After the last incubation, 100 μL of the resulting washing solution is transferred to a new MTP, and the absorbance is measured at 405nm. To determine the overall cleaning performance (at pH 7 and 9) in a custom liquid ADW detergent formulation, the two absorbance measurements taken were added and this total value was used to calculate the performance index (PI) for each enzyme.

[0889] In all cases, the PI for each condition and protease variant was calculated by dividing the total absorbance minus the blank by the total absorbance of the parent protease (tested at the same concentration). The value of the parent protease was determined from the standard curve of the parent protease, which was included in the test and fitted to a Langmuir fit. Table 28 shows the ADW performance results of the AprL variants (using rinsed or unrinsed samples) tested in GSM-B pH 10.5 or custom liquid ADW detergent formulations (adjusted to pH 7 and pH 9). The results are reported as PI values ​​compared to the AprL wild-type protease ("AprL wt").

[0890]

[0891]

Claims

1. A subtilisin protease variant, comprising an amino acid sequence comprising two, three, or four or more variations at positions corresponding to SEQ ID NO: 2, wherein the positions are selected from: (i)1,3,9,10,15,22,24,26,27,28,29,30,31,35,37,40,43,45,48,52,68,71,76,77,7 8, 86, 87, 95, 96, 98, 99, 100, 101, 102, 103, 105, 108, 111, 114, 115, 117, 119, 123, 125, 1 26, 127, 128, 129, 130, 136, 143, 146, 147, 151, 155, 158, 160, 161, 165, 184, 187, 193, 202, 203, 204, 210, 211, 216, 217, 234, 238, 239, 242, 243, 250, 255, 258, 259, 260, 264, and 274; (ii)1, 3, 9, 10, 15, 22, 24, 26, 28, 29, 30, 31, 35, 37, 40, 45, 48, 52, 68, 71, 76, 77, 78, 8 6, 87, 95, 96, 98, 99, 100, 101, 102, 105, 108, 111, 114, 115, 117, 119, 123, 125, 126, 12 7, 128, 129, 130, 136, 143, 146, 147, 151, 155, 158, 160, 161, 165, 184, 187, 193, 202, 203, 204, 210, 211, 216, 217, 234, 238, 239, 242, 243, 250, 255, 258, 259, 260, 264, and 274; (iii)1, 3, 9, 10, 24, 26, 29, 30, 31, 35, 37, 40, 45, 48, 52, 68, 71, 77, 78, 86, 87 ,95,96,98,99,100,101,102,105,108,111,115,117,119,123,127,128,129 , 130, 136, 143, 146, 147, 151, 155, 158, 160, 161, 165, 184, 187, 193, 202, 203, 204, 210, 211, 216, 217, 234, 238, 239, 242, 243, 250, 258, 259, 260, 264, and 274; (iv) 3, 9, 10, 35, 68, 77, 78, 86, 87, 99, 101, 115, 123, 127, 128, 165, 184, 187, 202, 203, 210, 217, 242, 250, 258 and 264; or (v) 3, 68, 77, 78, 123, 127, 128, 165, 184, 202, 210, 217 and 258; Provided that one or more of the two, three or four or more variations are artificial mutations or substitutions; wherein the variant has at least 85% but less than 100% amino acid sequence identity with an AprL-clade subtilisin comprising motif A and / or motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14; and The amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:

2.

2. The subtilisin protease variant of claim 1, wherein the variant comprises an amino acid sequence comprising two, three or four or more amino acid substitutions at two, three or four or more positions corresponding to SEQ ID NO: 2, wherein the substitutions are selected from: (i)1Q, 3Q / V, 9E, 10M, 15I, 22Y, 24N / Q, 26Q, 27R, 28A, 29S, 30T, 31I, 35A, 37 T, 40E, 43A, 45I / Q / R, 48I / T / V, 52R, 68S, 71A, 76K, 77D / E / H / N / Q / S, 78I, 86 H / N / R, 87S / T, 95A / F / Q, 96S, 98H / R, 99A / E / Q / S, 100N, 101A, 102D / T, 103I, 105N, 108H / K, 111Q, 114N, 115F, 117S, 119Q, 123I, 125N, 126Q, 127E / F / Q / S / T / V, 128P / R / S, 129H / R, 130S, 136N / R, 143Q / V, 146A / S, 147L, 151G, 155E / G / N, 158D / N / Q, 160C / D / K, 161D / Q, 165A / E / Q / S, 184E / Q, 187E / P, 193D / Q, 2 02V, 203E / N / Q, 204I, 210E / H / I / P, 211S, 216Q, 217S, 234I / W, 238Q / R, 239K / S / T, 242G / N, 243I, 250G, 255R, 258D / E / P, 259E / P, 260W, 264H / Q / S and 274A; (ii) 1Q, 3Q / V, 9E, 10M, 15I, 22Y, 24N / Q, 26Q, 28A, 29S, 30T, 31I, 35A, 37T, 40E, 45I / Q / R, 48I / T / V, 52R, 68S, 71A, 76K, 77D / E / H / N / S, 78I, 86H / N / R, 87S / T, 95A / F / Q, 96S, 98K / R, 99A / E / Q / S, 100N, 101A, 102D / T, 105N, 108H / K, 111Q, 114N, 115F, 117S, 119Q, 123I, 125N, 126Q, 127E / F / Q / S / T / V, 128P / R / S, 129H / R, 130S, 136N, 143Q / V, 146A / S, 147L, 151G, 155E / G / N, 158D / N / Q, 160D / K, 161D / Q, 165A / E / Q / S, 184E / Q, 187E / P, 193D / Q, 194E, 202V, 203E / N / Q, 204I, 210I / P, 211S, 216Q, 217S, 234I / W, 238Q / R, 239K / S / T, 242G / N, 243I, 250G, 255R, 258D / P, 259E / P, 260W, 264H / Q / S and 274A; (iii) 1Q, 3V, 9E, 10M, 24Q, 26Q, 29S, 30T, 31I, 35A, 37T, 40E, 45I / Q / R, 48T / V, 52R, 68S, 71A, 77E / N, 78I, 86H / R, 87S, 95A / F, 96S, 98R, 99A / E / Q / S, 100N, 101A, 102D / T, 105N, 108H, 111Q, 115F, 117S, 119Q, 123I, 127E / Q / S / T / V, 128P / R, 129H / R, 130S, 136N, 143Q / V, 146A, 147L, 151G, 155G / N, 158D / N / Q, 160D / K, 161D / Q, 165A / E / Q, 184E / Q, 187E / P, 193Q, 202V, 203E / N / Q, 204I, 210I / P, 211S, 216Q, 217S, 234W, 238Q, 239S, 242G / N, 243I, 250G, 258D / P, 259E / P, 260W, 264H / Q / S and 274A; (iv) 3V, 9E, 10M, 35A, 68S, 77N, 78I, 86H, 87S, 99Q / S, 101A, 115F, 123I, 127S / T, 128P / R, 165E / Q, 184Q, 187E, 202V, 203E, 210P, 217S, 242G, 250G, 258D / P and 264H; or (v) 3V, 68S, 77N, 78I, 123I, 127S, 128P, 165Q, 184Q, 202V, 210P, 217S and 258D / P.

3. The subtilisin protease variant of claim 1 or 2, wherein the variant comprises an amino acid sequence comprising two, three or four or more amino acid substitutions selected from: (i)A / G1Q, T3Q / V, P / S9E, L / Q10M, K15I, K22Y, A24N / Q, V26Q, K27R, G / V28A, A / G29S, I / V30T, I / L31I, I35A, A / S37T, P / S / T40E, N / K43A, S / V45I / Q / R, A48I / T / V, A / S52R, A68S, V71A, N76K, S / T77D / E / H / N / Q / S, T78I, N / S86H / N / R, V87S / T, L95A / F / Q, N96S, S98H / R, G99A / E / Q / S, S100N, G101A, S / T102D / T, Y10 3I, A / G105N, S108H / K, E111Q, T114N, A / Q / S / T115F, G / N117S, D119Q, M123I, L125N, G126Q, G127E / F / Q / S / T / V, A / P / S128P / R / S, S129H / R, G / V130S, Q136N / R, A / S143Q / V, A / I / V146A / S, V147L, A / S151G, S1 55E / G / N, S158D / N / Q, N / S / Y160C / D / K, A / S / Q / T161D / Q, G165A / E / Q / S, N / Q184E / Q, S187E / P, A / P / S193 D / Q, A / S202V, A / G / S203E / N / Q, V204I, S / T210E / H / I / P, N211S, L216Q, N217S, L234I / W, P238Q / R, A / N / T239K / S / T, A242G / N, S243I, S250G, N / Y255R, D / S258D / E / P, S259E / P, F260W, K / N / R264H / Q / S and Q274A; (ii) A1Q, T3Q / V, P9E, L10M, K15I, K22Y, A24N / Q, V26Q, K27R, V28A, A29S, V30T, L31I, I35A, A37T, P40E, N43A, V45I / Q / R, A48I / T / V, A52R, A68S, V71A, N76K, S77D / E / H / N / Q / S, T78I, S86H / N / R, V87S / T, L95A / F / Q, N96S, S98H / R, G99A / E / Q / S, S100N, G101A, S102D / T, Y103I, G105N, S108H / K, E111Q, T114N, T115F, G117S, D119Q, M123I, L125N, G126Q, G127E / F / Q / S / T / V, A128P / R / S, S129H / R, G130S, Q136N / R, A143Q / V, V146A / S, V147L, A151G, S155E / G / N, S158D / N / Q, N160C / D / K, T161D / Q, G165A / E / Q / S, N184E / Q, S187E / P, A193D / Q, A202V, G203E / N / Q, V204I, T210E / H / I / P, N211S, L216Q, N217S, L234I / W, P238Q / R, N239K / S / T, A242G / N, S243I, S250G, Y255R, S258D / E / P, S259E / P, F260W, K264H / Q / S, and Q274A; (iii) A / G1Q, T3Q / V, P / S9E, L / Q10M, K15I, K22Y, A24N / Q, V26Q, G / V28A, A / G29S, I / V30T, I / L31I, I35A, A / S37T, P / S / T40E, S / V45I / Q / R, A48I / T / V, A / S52R, A68S, V71A, N76K, S / T77D / E / H / N / S, T78I, N / S86H / N / R, V87S / T, L95A / F / Q, N96S, S98R, G99A / E / Q / S, S100N, G101A, S / T102D / T, A / G105N, S108H / K, E111Q, T114N, A / Q / S / T115F, G / N117S, D119Q, M123I, L125N, G126Q, G127E / F / Q / S / T / V, A / P / S128P / R / S, S129H / R, G / V130S, Q136N, A / S143Q / V, A / I / V146A / S, V147L, A / S151G, S155E / G / N, S158D / N / Q, N / S / Y160D / K, A / S / Q / T161D / Q, G165A / E / Q / S, N / Q184E / Q, S187E / P, A / P / S193D / Q, A / S202V, A / G / S203E / N / Q, V204I, S / T210I / P, N211S, L216Q, N217S, L234I / W, P238Q / R, A / N / T239K / S / T, A242G / N, S243I, S250G, N / Y255R, D / S258D / P, S259E / P, F260W, K / N / R264H / Q / S, and Q274A; (iv) A1Q, T3Q / V, P9E, L10M, K15I, K22Y, A24N / Q, V26Q, V28A, A29S, V30T, L31I, I35A, A37T, P40E, V45I / Q / R, A48I / T / V, A52R, A68S, V71A, N76K, T77D / E / H / N / S, T78I, S86H / N / R, V87S / T, L95A / F / Q, N96S, S98R, G99A / E / Q / S, S100N, G101A, S102D / T, G105N, S108H / K, E111Q, T114N, T115F, G117S, D119Q, M123I, L125N, G126Q, G127E / F / Q / S / T / V, A128P / R / S, S129H / R, G130S, Q136N, A143Q / V, V146A / S, V147L, A151G, S155E / G / N, S158D / N / Q, N160D / K, T161D / Q, G165A / E / Q / S, N184E / Q, S187E / P, A193D / Q, A202V, G203E / N / Q, V204I, T210I / P, N211S, L216Q, N217S, L234I / W, P238Q / R, N239K / S / T, A242G / N, S243I, S250G, Y255R, S258D / P, S259E / P, F260W, K264H / Q / S, and Q274A; (v)A / G1Q, T3V, P / S9E, L / Q10M, A24Q, V26Q, A / G29S, I / V30T, I / L31I, I35A, A / S37T, P / S / T40E, S / V45I / Q / R, A48T / V, A / S52R, A68S, V71A, S / T77E / N, T78I, N / S86H / R, V87S, L95A / F, N96S, S98R, G99A / E / Q / S, S100N, G101A, S / T102D / T, A / G105N, S108H, E111Q, A / Q / S / T115F, G / N117S, D119Q, M123I, G127E / Q / S / T / V, A / P / S128P / R, S129H / R, G / V130S, Q136N, A / S143Q / V, A / I / V146A, V147L, A / S151G, S155G / N, S158D / N / Q, N / S / Y160D / K, A / S / Q / T161D / Q, G165A / E / Q, N184E / Q, S187E / P, A / P / S193Q, A / S202V, A / G / S203E / N / Q, V204I, S / T210I / P, N211S, L216Q, N217S, L234W, P238Q, A / N / T239S, A242G / N, S243I, S250G, D / S258D / P, S259E / P, F260W, K / N / R264H / Q / S, and Q274A; (vi) A1Q, T3V, P9E, L10M, A24Q, V26Q, A29S, V30T, L31I, I35A, A37T, P40E, V45I / Q / R, A48T / V, A52R, A68S, V71A, T77E / N, T78I, S86H / R, V87S, L95A / F, N96S, S98R, G99A / E / Q / S, S100N, G101A, S102D / T, G105N, S108H, E111Q, T115F, G117S, D119Q, M123I, G127E / Q / S / T / V, A128P / R, S129H / R, G130S, Q136N, A143Q / V, V146A, V147L, A151G, S155G / N, S158D / N / Q, N160D / K, T161D / Q, G165A / E / Q, N184E / Q, S187E / P, A193Q, A202V, G203E / N / Q, V204I, T210I / P, N211S, L216Q, N217S, L234W, P238Q, N239S, A242G / N, S243I, S250G, S258D / P, S259E / P, F260W, K264H / Q / S, and Q274A; (vii) T3V, P / S9E, L / Q10M, I35A, A68S, S / T77N, T78I, N / S86H, V87S, G99Q / S, G101A, A / Q / S / T115F, M123I, G127S / T, A / P / S128P / R, G165E / Q, N184Q, S187E, A / S202V, A / G / S203E, S / T210P, N217S, A242G, S250G, D / S258D / P, and K / N / R264H; (viii) T3V, P9E, L10M, I35A, A68S, T77N, T78I, S86H, V87S, G99Q / S, G101A, T115F, M123I, G127S / T, A128P / R, G165E / Q, N184Q, S187E, A202V, G203E, T210P, N217S, A242G, S250G, S258D / P, and K264H; (ix) T3V, A68S, S / T77N, T78I, M123I, G127S, A / S128P, G165Q, N184Q, A / S202V, S / T210P, N217S, and D / S258D / P; or (x) T3V, A68S, T77N, T78I, M123I, G127S, A128P, G165Q, N184Q, A202V, T210P, N217S, and S258D / P.

4. The subtilisin protease variant of claim 1, wherein: (i) the variation at the position corresponding to SEQ ID NO: 2 is not an insertion or a deletion; (ii) when the amino acid sequence of the variant comprises a variation at one or more corresponding SEQ ID NO:2 positions selected from positions 37, 187, 193 and 258, the variation is not proline; (iii) when the amino acid sequence of the variant comprises a variation at two or more corresponding SEQ ID NO:2 positions selected from positions 26, 38, 234 and 250, the variation is not 26R, 38S, 234R, or 250E / N; (iv) when the amino acid sequence of the variant comprises a variation at position 128, the variation at the position corresponding to SEQ ID NO: 2 is not at position 130 or 193; and / or (v) the variant does not consist of the amino acid sequence shown in SEQ ID NO: 5 in WO 2016074925 or SEQ ID NO: 110 in JP 201300714.

5. A subtilisin protease variant as claimed in any preceding claim, provided that: (i) when the amino acid sequence of the variant comprises a substitution at one or more of positions 37, 187, 193 and 258, the substitution is not proline; (ii) when the amino acid sequence of the variant comprises a substitution at two or more of positions 26, 38, 234 and 250, the substitution is not 26R, 38S, 234R or 250E / N; (iii) when the amino acid sequence of the variant comprises a substitution at position 128, the substitution is not at position 130 or 193; and / or (iv) the variant does not consist of the amino acid sequence shown in SEQ ID NO: 5 in WO 2016074925 or SEQ ID NO: 110 in JP 201300714.

6. A subtilisin protease variant according to any preceding claim, wherein the variant comprises an amino acid sequence comprising one or more amino acid substitution groups selected from: (i)T77N-G165Q、T77N-N217S、G165Q-N217S、A68S-T77N、N184Q-N217S、A68S-G165Q、G165Q-N184Q、T77N-A128P、T77N-N184Q、A68S-N217S、A128P-N217S、A128P-G165Q、A68S-A128P、A68S-N184Q、A128P-N184Q、T3V-N217S、A202V-N217S、T3V-T77N、T3V-N184Q、N184Q-A202V、G165Q-A202V、M123I-G165Q、T3V-G165Q、T3V-A128P、T3V-A68S、T77N-A202V、M123I-N217S、A68S-A202V、M123I-N184Q、A128P-A202V、T77N-M123I、T3V-A202V、N217S-S258P、A68S-M123I、N184Q-S258P、G165Q-S258P、M123I-A128P、T77N-S258P、T3V-M123I、A128P-S258P、A202V-S258P、A68S-S258P、T3V-S258P、M123I-A202V、M123I-S258P、A68S-G127S、G127S-A128P、T77N-G127S、G127S-A202V、G127S-N184Q、G127S-N217S、G127S-G165Q、T3V-G127S、G127S-S258P、M123I-G127S、T77N-T210P、G165Q-T210P、T210P-N217S、A128P-T210P、A68S-T210P、N184Q-T210P、T210P-S258P、A202V-T210P、M123I-T210P、T3V-T210P、G127S-T210P、G127T-N184Q、G127T-N217S、V87S-G165Q、M123I-Q136R、A68S-G127T、G127T-A128P、T77N-V87S、G127T-G165Q、T77N-G127T、T78I-A202V、A68S-S258D、G165Q-S258D、T77N-S258D、T77N-T78I、T78I-G165Q、N184Q-S258D、N217S-S258D、P9E-M123I、A128P-A242G、A202V-S258D、A68S-T78I、G165Q-A242G、T77N-A242G、T78I-A128P、T78I-G127S、P9E-G165Q、Q136R-G165Q、T78I-N184Q、V87S-A128P、V87S-N217S、A68S-V87S、G127T-A202V、I35A-T77N、T3V-T78I、T78I-N217S、A68S-A242G、A68S-G99S、G99S-A128P、G99S-G165Q、I35A-G165Q、L10M-A202V、T3V-G127T、T77N-G99S、T78I-S258P、V45R-M123I、V87S-M123I、M123I-N239S、P9E-N184Q、S155G-G165Q、T78I-M123I、V45R-Q136R、V87S-A202V、A128P-S258D、I35A-A202V、M123I-G127T、M123I-S258D、N184Q-N239S、P9E-N217S、Q136R-S258E、S86H-S258P、T3V-A242G、T3V-S258D、G99S-N184Q、G99S-N217S、I35A-V87S、N217S-A242G、P9E-Q136R、G127T-S258P、I35A-A128P、I35A-A68S、I35A-N217S、L10M-M123I、P9E-S258P、P9E-T210P、P9E-T77N、S86H-A202V、S86H-G165Q、S86H-N184Q、T77N-S155G、T77N-V147L、T78I-T210P、V87S-S155G、A202V-A242G、A242G-S258P、A68S-G165E、G127S-A242G、G165E-N184Q、G165E-N217S、M123I-S258E、N184Q-A242G、S86H-T115F、T3V-G99S、V147L-G165Q、A128R-G165Q、A128R-S258D、A68S-A128R、A68S-V147L、G127S-S258D、G165E-A202V、G165Q-S258E、G165S-T210P、G203E-T210P、G99S-G127S、G99S-M123I、G99S-T210P、L10M-A128P、L10M-A68S、L10M-N184Q、L10M-N217S、L10M-S258P、L10M-T77N、M123I-A242G、M123I-G165E、M123I-S187P、N239S-S258P、P9E-A128P、P9E-A202V、P9E-A68S、P9E-V45R、Q136R-N184Q、Q136R-N239S、S86H-N217S、T115F-S258P、T210P-A242G、T3V-G165E、T3V-L10M、T3V-P9E、T77N-A128R、T77N-G165E、T78I-A242G、V45R-G165Q、V87T-M123I、V87T-N184Q、V87T-N239S、V87T-Q136R、A68S-G101A、A68S-V71A、G101A-A128P、G101A-G165Q、G101A-S258P、G165A-N184Q、G165E-S258P、G99S-S258P、I35A-M123I、K15I-G165S、K15I-T210P、L10M-G127S、L10M-G165Q、Q136R-S187P、S187E-A202V、S187P-S258P、S250G-S258P、T115F-A202V、T115F-G165Q、T3V-G101A、T3V-I35A、T3V-V87S、T77N-G101A、T77N-G203E、T78I-S258D、V71A-G165Q、V71A-T77N、V87S-S258P、V87S-V147L、A128P-G165E、A128P-S155G、A128P-V147L、A128R-A202V、A128R-N184Q、A128R-N217S、A1Q-S86H、A68S-G99Q、A68S-S155G、G101A-A202V、G101A-G127S、G101A-N184Q、G101A-N217S、G165Q-G203E、G165Q-N239S、G165S-N239S、G99Q-A128P、G99Q-G165Q、G99Q-N184Q、G99Q-N217S、G99S-A242G、I35A-N184Q、I35A-T78I、I35A-V147L、L10M-T210P、L10M-T78I、M123I-G165S、N184Q-G203E、N184Q-S187P、N43A-M123I、P9E-G127S、P9E-S258E、Q136R-S258P、T115F-N217S、T3V-S86H、T77H-M123I、T77H-N184Q、T77H-N239S、T77H-Q136R、T77H-V87T、T77N-G99Q、T77N-S250G、T78I-G99S、T78I-V87S、V146S-G165S、V146S-N239S、V146S-T210P、V45R-S258E、V87S-A242G、V87S-N184Q、A128S-T210P、A143V-A202V、A143V-G165Q、A143V-S258P、A1Q-N184Q、A1Q-S258P、A68S-G203E、A68S-S187E、A68S-T114N、G101A-M123I、G101A-T210P、G165A-N217S、G165Q-S187P、G99Q-A202V、G99S-A202V、I35A-A242G、I35A-S258P、L10M-I35A、L10M-V87S、M123I-A128R、M123I-V146S、N184Q-S187E、N217S-L234W、N239S-S250G、N43A-Q136R、N43A-V45R、P9E-N239S、P9E-V87T、S155G-N217S、S187E-N217S、S86H-A143V、S86H-Q274A、S86H-S250G、T114N-A128P、T114N-G165Q、T114N-N184Q、T114N-N217S、T115F-A143V、T115F-N184Q、T210P-N239S、T210P-S250G、T3V-A128R、T3V-G99Q、T77H-G165Q、T77N-S187E、T77N-T114N、V147L-N217S、V45R-N184Q、V71A-N184Q、V71A-N217S、V71A-T210P、T77N-G165Q-N217S、A68S-T77N-G165Q、T77N-N184Q-N217S、A68S-T77N-N217S、T77N-A128P-G165Q、T77N-A128P-N217S、A68S-T77N-A128P、G165Q-N184Q-N217S、T77N-G165Q-N184Q、A68S-A128P-G165Q、A68S-G165Q-N217S、A128P-N184Q-N217S、A128P-G165Q-N217S、A68S-A128P-N217S、A68S-N184Q-N217S、A68S-T77N-N184Q、T77N-A128P-N184Q、A68S-G165Q-N184Q、A128P-G165Q-N184Q、A68S-A128P-N184Q、T3V-N184Q-N217S、T3V-T77N-N217S、N184Q-A202V-N217S、T3V-T77N-N184Q、T3V-A128P-N217S、T3V-T77N-A128P、G165Q-A202V-N217S、T3V-A128P-N184Q、T77N-A202V-N217S、T3V-G165Q-N217S、T3V-T77N-G165Q、T3V-A68S-N217S、T3V-A68S-T77N、A68S-A202V-N217S、T3V-G165Q-N184Q、A128P-A202V-N217S、G165Q-N184Q-A202V、T77N-G165Q-A202V、T77N-N184Q-A202V、T3V-A128P-G165Q、T3V-A68S-A128P、T3V-A68S-N184Q、A128P-N184Q-A202V、A68S-N184Q-A202V、A68S-T77N-A202V、T3V-A68S-G165Q、T77N-A128P-A202V、A128P-G165Q-A202V、A68S-A128P-A202V、A68S-G165Q-A202V、M123I-G165Q-N217S、M123I-N184Q-N217S、T77N-M123I-G165Q、T77N-M123I-N217S、T3V-A202V-N217S、M123I-G165Q-N184Q、T77N-M123I-N184Q、T3V-N184Q-A202V、A68S-M123I-N217S、T3V-T77N-A202V、T3V-A128P-A202V、T3V-G165Q-A202V、M123I-A128P-N217S、T3V-A68S-A202V、A68S-M123I-N184Q、A68S-T77N-M123I、M123I-A128P-N184Q、T3V-M123I-N217S、T77N-M123I-A128P、A68S-M123I-G165Q、M123I-A128P-G165Q、A68S-M123I-A128P、N184Q-N217S-S258P、T3V-M123I-N184Q、T3V-M123I-G165Q、T3V-T77N-M123I、T77N-N217S-S258P、G165Q-N217S-S258P、T3V-A68S-M123I、T3V-M123I-A128P、T77N-N184Q-S258P、A128P-N184Q-S258P、A128P-N217S-S258P、T77N-A128P-S258P、T77N-G165Q-S258P、A202V-N217S-S258P、G165Q-N184Q-S258P、N184Q-A202V-S258P、A68S-A128P-S258P、A68S-N184Q-S258P、A68S-N217S-S258P、A68S-T77N-S258P、M123I-A202V-N217S、T3V-N217S-S258P、A128P-G165Q-S258P、G165Q-A202V-S258P、T3V-A128P-S258P、T3V-N184Q-S258P、A68S-G165Q-S258P、T3V-M123I-A202V、T3V-T77N-S258P、A128P-A202V-S258P、M123I-N184Q-A202V、T77N-A202V-S258P、A68S-A202V-S258P、M123I-G165Q-A202V、T77N-M123I-A202V、T3V-A68S-S258P、A68S-M123I-A202V、M123I-A128P-A202V、T3V-A202V-S258P、T3V-G165Q-S258P、A68S-G127S-A128P、A68S-T77N-G127S、T77N-G127S-A128P、A68S-G127S-A202V、A68S-G127S-N184Q、A68S-G127S-N217S、G127S-A128P-A202V、G127S-A128P-N184Q、G127S-A128P-N217S、A68S-G127S-G165Q、G127S-A128P-G165Q、G127S-A202V-N217S、G127S-N184Q-A202V、G127S-N184Q-N217S、M123I-N217S-S258P、T77N-G127S-A202V、T77N-G127S-N184Q、T77N-G127S-N217S、M123I-N184Q-S258P、T77N-G127S-G165Q、T3V-A68S-G127S、T3V-G127S-A128P、G127S-G165Q-A202V、G127S-G165Q-N184Q、G127S-G165Q-N217S、M123I-A202V-S258P、M123I-G165Q-S258P、T3V-M123I-S258P、T3V-T77N-G127S、A68S-G127S-S258P、G127S-A128P-S258P、A68S-M123I-S258P、M123I-A128P-S258P、T3V-G127S-A202V、T3V-G127S-N184Q、T3V-G127S-N217S、T77N-M123I-S258P、T3V-G127S-G165Q、T77N-G127S-S258P、G127S-A202V-S258P、G127S-N184Q-S258P、G127S-N217S-S258P、T3V-G127S-S258P、G127S-G165Q-S258P、A68S-M123I-G127S、M123I-G127S-A128P、M123I-G127S-A202V、M123I-G127S-N184Q、M123I-G127S-N217S、T77N-M123I-G127S、T3V-M123I-G127S、M123I-G127S-G165Q、M123I-G127S-S258P、T77N-G165Q-T210P、A68S-T77N-T210P、A68S-G165Q-T210P、A68S-A128P-T210P、T77N-A128P-T210P、T77N-T210P-N217S、A68S-N184Q-T210P、N184Q-T210P-N217S、T77N-N184Q-T210P、A128P-G165Q-T210P、A68S-T210P-N217S、G165Q-N184Q-T210P、G165Q-T210P-N217S、A128P-N184Q-T210P、A128P-T210P-N217S、T77N-T210P-S258P、G165Q-T210P-S258P、T3V-A128P-T210P、T3V-A68S-T210P、T3V-T77N-T210P、A128P-T210P-S258P、A202V-T210P-N217S、A68S-T210P-S258P、N184Q-A202V-T210P、T210P-N217S-S258P、T3V-G165Q-T210P、T77N-A202V-T210P、A202V-T210P-S258P、G165Q-A202V-T210P, T3V-N184Q-T210P, A128P-A202V-T210P, A68S-A202V-T210P, N184Q-T210P-S258P, T3V-A2 02V-T210P, T3V-T210P-N217S, T3V-T210P-S258P, M123I-T210P-N217S, T77N-M123I-T210P, A68S-M123I-T210P , M123I-A128P-T210P, M123I-N184Q-T210P, A68S-G127S-T210P, G127S-A128P-T210P, T77N-G127S-T210P, G127 S-T210P-S258P, M123I-G165Q-T210P, T3V-G127S-T210P, G127S-G165Q-T210P, T3V-M123I-T210P, G127S-A202V -T210P, G127S-N184Q-T210P, G127S-T210P-N217S, G127T-N184Q-N217S, M123I-A202V-T210P, M123I-T210P-S2 58P, A68S-G127T-N184Q, A68S-G127T-N217S, G127T-A128P-N184Q, G127T-A128P-N217S, T77N-V87S-G165Q, A68 S-G127T-A128P, G127T-G165Q-N184Q, G127T-G165Q-N217S, T77N-G127T-N184Q, T77N-G127T-N217S, A68S-G165Q-S258D, A68S-T77N-G127T, A68S-T77N-S258D, G127T-A128P-G165Q, T77N-G127T-A128P, and T77N-G165Q-S258D; or, (ii)N184Q-N217S、T77N-N217S、T77N-N184Q、T77N-G165Q、G165Q-N184Q、G165Q-N217S、A128P-N217S、A68S-T77N、T77N-A128P、A68S-N217S、A128P-N184Q、A68S-N184Q、A68S-G165Q、A128P-G165Q、A68S-A128P、A202V-N217S、N184Q-A202V、G165Q-A202V、T3V-N217S、T3V-N184Q、T3V-T77N、T77N-A202V、A68S-A202V、T3V-A128P、T3V-G165Q、A128P-A202V、T3V-A68S、T3V-A202V、N184Q-S258P、N217S-S258P、M123I-N217S、M123I-N184Q、T3V-M123I、T77N-S258P、A128P-S258P、G165Q-S258P、T77N-M123I、A202V-S258P、M123I-G165Q、A68S-M123I、A68S-S258P、M123I-A128P、M123I-A202V、T3V-S258P、A68S-G127S、G127S-A128P、T77N-G127S、G127S-A202V、G127S-N184Q、G127S-N217S、G127S-G165Q、M123I-S258P、T3V-G127S、G127S-S258P、M123I-G127S、T77N-T210P、A128P-T210P、A202V-T210P、A68S-T210P、G165Q-T210P、T210P-S258P、T3V-T210P、N184Q-T210P、T210P-N217S、G127S-T210P、M123I-T210P、T78I-A202V、A68S-S258D、G165Q-S258D、T77N-S258D、T77N-T78I、T78I-G165Q、G127T-N184Q、G127T-N217S、N184Q-S258D、N217S-S258D、A202V-S258D、A68S-T78I、T78I-A128P、T78I-G127S、A68S-G127T、T78I-N184Q、G127T-A128P、G127T-A202V、T3V-T78I、T78I-N217S、G127T-G165Q、L10M-A202V、T77N-G127T、T78I-S258P、T3V-G127T、T78I-M123I、V87S-A202V、A128P-A242G、A128P-S258D、G165Q-A242G、I35A-A202V、M123I-S258D、T3V-A242G、T3V-S258D、T77N-A242G、S86H-S258P、G127T-S258P、P9E-T77N、S86H-A202V、S86H-G165Q、S86H-N184Q、T78I-T210P、A202V-A242G、A242G-S258P、A68S-A242G、A68S-G165E、G127S-A242G、G165E-N184Q、G165E-N217S、I35A-V87S、L10M-M123I、M123I-G127T、N184Q-A242G、N217S-A242G、A128R-G165Q、A128R-S258D、A68S-A128R、A68S-G99S、G127S-S258D、G165E-A202V、G99S-A128P、G99S-G127S、G99S-G165Q、I35A-G165Q、I35A-T77N、L10M-A128P、L10M-A68S、L10M-N184Q、L10M-N217S、L10M-S258P、L10M-T77N、M123I-A242G、P9E-A128P、P9E-A202V、P9E-A68S、P9E-G165Q、P9E-N184Q、P9E-N217S、P9E-S258P、S86H-T115F、T210P-A242G、T3V-G165E、T3V-G99S、T3V-L10M、T3V-P9E、T77N-A128R、T77N-G165E、T77N-G99S、T77N-V87S、T78I-A242G、V87S-A128P、V87S-G165Q、V87S-M123I、V87S-N217S、A68S-G101A、G101A-A128P、G101A-G165Q、G101A-S258P、G165E-S258P、G99S-S258P、I35A-M123I、I35A-N217S、L10M-G127S、L10M-G165Q、M123I-G165E、P9E-T210P、S187E-A202V、S86H-N217S、T115F-A202V、T115F-G165Q、T115F-S258P、T3V-G101A、T3V-V87S、T77N-G101A、T77N-G203E、T78I-S258D、A128P-G165E、A128R-A202V、A128R-N184Q、A128R-N217S、A68S-G99Q、G101A-A202V、G101A-G127S、G101A-N184Q、G101A-N217S、G165Q-G203E、G99Q-A128P、G99Q-G165Q、G99Q-N184Q、G99Q-N217S、G99S-A242G、G99S-T210P、I35A-A128P、I35A-T78I、L10M-T210P、L10M-T78I、P9E-G127S、P9E-M123I、T3V-I35A、T77N-G99Q、T78I-G99S、T78I-V87S、V87S-N184Q、A143V-A202V、A143V-G165Q、A143V-S258P、A1Q-N184Q、A1Q-S86H、A68S-G203E、A68S-S187E、A68S-V87S、G101A-M123I、G101A-T210P、G99Q-A202V、G99S-A202V、G99S-M123I、G99S-N184Q、G99S-N217S、I35A-N184Q、L10M-I35A、L10M-V87S、M123I-A128R、N184Q-G203E、N184Q-S187E、S187E-N217S、S250G-S258P、S86H-A143V、T115F-A143V、T115F-N184Q、T115F-N217S、T3V-A128R、T3V-G99Q、T3V-S86H、T77N-S187E、T77N-N184Q-N217S、A128P-N184Q-N217S、T77N-A128P-N217S、A68S-N184Q-N217S、A68S-T77N-N217S、G165Q-N184Q-N217S、T77N-A128P-N184Q、T77N-G165Q-N217S、T77N-G165Q-N184Q、A68S-T77N-G165Q、A68S-T77N-N184Q、T77N-A128P-G165Q、A128P-G165Q-N217S、A68S-A128P-N217S、A68S-T77N-A128P、A68S-G165Q-N217S、A128P-G165Q-N184Q、A68S-A128P-N184Q、A68S-A128P-G165Q、A68S-G165Q-N184Q、N184Q-A202V-N217S、G165Q-A202V-N217S、T77N-A202V-N217S、T3V-N184Q-N217S、A68S-A202V-N217S、T3V-T77N-N217S、A128P-A202V-N217S、G165Q-N184Q-A202V、T77N-N184Q-A202V、T3V-A128P-N217S、T3V-T77N-A128P、T3V-T77N-N184Q、T77N-G165Q-A202V、A128P-N184Q-A202V、A68S-N184Q-A202V、T3V-A128P-N184Q、A68S-T77N-A202V、T77N-A128P-A202V、T3V-G165Q-N217S、T3V-T77N-G165Q、T3V-A68S-N217S、T3V-A68S-T77N、A128P-G165Q-A202V、A68S-A128P-A202V、T3V-G165Q-N184Q、A68S-G165Q-A202V、T3V-A128P-G165Q、T3V-A68S-A128P、T3V-A68S-N184Q、T3V-A68S-G165Q、T3V-A202V-N217S、T3V-N184Q-A202V、T3V-T77N-A202V、T3V-A128P-A202V、T3V-G165Q-A202V、T3V-A68S-A202V、N184Q-N217S-S258P、T3V-M123I-N217S、M123I-N184Q-N217S、T77N-M123I-N217S、A128P-N184Q-S258P、A128P-N217S-S258P、A68S-M123I-N217S、M123I-G165Q-N217S、T77N-A128P-S258P、T77N-M123I-N184Q、T77N-N184Q-S258P、M123I-A128P-N217S、T3V-M123I-N184Q、T77N-N217S-S258P、A202V-N217S-S258P、A68S-M123I-N184Q、M123I-A128P-N184Q、N184Q-A202V-S258P、T3V-M123I-G165Q,T3V-T77N-M123I、T77N-M123I-G165Q、A68S-A128P-S258P、A68S-T77N-M123I、M123I-G165Q-N184Q、T3V-A68S-M123I、A68S-N184Q-S258P、G165Q-N184Q-S258P、G165Q-N217S-S258P、M123I-A202V-N217S、T3V-M123I-A128P、T77N-M123I-A128P、A128P-G165Q-S258P、A68S-N217S-S258P、M123I-A128P-G165Q、T3V-N217S-S258P、T77N-G165Q-S258P、A68S-M123I-A128P、A68S-M123I-G165Q、A68S-T77N-S258P、T3V-A128P-S258P、T3V-N184Q-S258P、G165Q-A202V-S258P、T3V-M123I-A202V、A128P-A202V-S258P、M123I-N184Q-A202V、T3V-T77N-S258P、A68S-A202V-S258P、A68S-G165Q-S258P、M123I-G165Q-A202V、T77N-A202V-S258P、T77N-M123I-A202V、A68S-M123I-A202V、M123I-A128P-A202V、T3V-A202V-S258P、T3V-A68S-S258P、T3V-G165Q-S258P、A68S-G127S-A128P、A68S-T77N-G127S、T77N-G127S-A128P、A68S-G127S-A202V、A68S-G127S-N184Q、A68S-G127S-N217S、G127S-A128P-A202V、G127S-A128P-N184Q、G127S-A128P-N217S、A68S-G127S-G165Q、G127S-A128P-G165Q、G127S-A202V-N217S、G127S-N184Q-A202V、G127S-N184Q-N217S、T77N-G127S-A202V、T77N-G127S-N184Q、T77N-G127S-N217S、T77N-G127S-G165Q、T3V-A68S-G127S、T3V-G127S-A128P、G127S-G165Q-A202V、G127S-G165Q-N184Q、G127S-G165Q-N217S、M123I-A202V-S258P、M123I-N217S-S258P、M123I-N184Q-S258P、T3V-M123I-S258P、T3V-T77N-G127S、A68S-G127S-S258P、G127S-A128P-S258P、M123I-A128P-S258P、T3V-G127S-A202V、T3V-G127S-N184Q、T3V-G127S-N217S、A68S-M123I-S258P、T3V-G127S-G165Q、T77N-G127S-S258P、M123I-G165Q-S258P、T77N-M123I-S258P、G127S-A202V-S258P、G127S-N184Q-S258P、G127S-N217S-S258P、T3V-G127S-S258P、G127S-G165Q-S258P、A68S-M123I-G127S、M123I-G127S-A128P、M123I-G127S-A202V、M123I-G127S-N184Q、M123I-G127S-N217S、T77N-M123I-G127S、T3V-M123I-G127S、M123I-G127S-G165Q、M123I-G127S-S258P、A68S-A128P-T210P、A68S-T77N-T210P、T77N-A128P-T210P、N184Q-A202V-T210P、T3V-A128P-T210P、T3V-A68S-T210P、T3V-T77N-T210P、T77N-G165Q-T210P、T77N-T210P-S258P、A128P-G165Q-T210P、A128P-T210P-S258P、A202V-T210P-N217S、A68S-G165Q-T210P、A68S-T210P-S258P、N184Q-T210P-N217S、T3V-G165Q-T210P、T77N-A202V-T210P、A128P-A202V-T210P、A128P-N184Q-T210P、A128P-T210P-N217S、A202V-T210P-S258P、A68S-A202V-T210P、A68S-N184Q-T210P、A68S-T210P-N217S、G165Q-A202V-T210P、G165Q-T210P-S258P、T3V-A202V-T210P、T3V-N184Q-T210P、T3V-T210P-S258P、T77N-N184Q-T210P、T77N-T210P-N217S、G165Q-N184Q-T210P、N184Q-T210P-S258P、T210P-N217S-S258P、T3V-T210P-N217S、G165Q-T210P-N217S、A68S-G127S-T210P、G127S-A128P-T210P、T77N-G127S-T210P、G127S-T210P-S258P、T3V-G127S-T210P、G127S-G165Q-T210P、A68S-M123I-T210P、G127S-A202V-T210P、G127S-N184Q-T210P、G127S-T210P-N217S、M123I-A128P-T210P、M123I-A202V-T210P、M123I-N184Q-T210P、M123I-T210P-N217S、T3V-M123I-T210P、T77N-M123I-T210P、M123I-G165Q-T210P、M123I-T210P-S258P、A68S-G165Q-S258D、A68S-T77N-S258D、T77N-G165Q-S258D、T77N-T78I-G165Q、A68S-N184Q-S258D、A68S-N217S-S258D、G127T-N184Q-N217S、G165Q-N184Q-S258D、G165Q-N217S-S258D、M123I-G127S-T210P、T77N-N184Q-S258D、T77N-N217S-S258D、A68S-T77N-T78I、A68S-T78I-A128P、A68S-T78I-G127S、A68S-T78I-G165Q、T77N-T78I-A128P、T77N-T78I-G127S、T78I-A128P-G165Q、T78I-G127S-A128P、T78I-G127S-G165Q、A68S-G127T-N184Q、A68S-G127T-N217S、N184Q-N217S-S258D、T77N-T78I-N184Q、T78I-G165Q-N184Q、A202V-N217S-S258D、A68S-A202V-S258D、G127T-A128P-N184Q、G127T-A128P-N217S、G127T-A202V-N217S、G127T-N184Q-A202V、G165Q-A202V-S258D、T3V-T77N-T78I、T3V-T78I-G165Q、T77N-A202V-S258D、T77N-T78I-A202V、T77N-T78I-N217S、T78I-G165Q-A202V、T78I-G165Q-N217S、T78I-N184Q-A202V、T78I-N184Q-N217S、A68S-G127T-A128P、A68S-T78I-A202V、A68S-T78I-N184Q、A68S-T78I-N217S、G127T-G165Q-N184Q、G127T-G165Q-N217S、T3V-A68S-T78I、T3V-T78I-A128P、T3V-T78I-G127S、T77N-G127T-N184Q、T77N-G127T-N217S、T77N-T78I-S258P、T78I-A128P-A202V、T78I-A128P-N184Q、T78I-A128P-N217S、T78I-A202V-N217S、T78I-G127S-A202V、T78I-G127S-N184Q、T78I-G127S-N217S、T78I-G165Q-S258P、A68S-G127T-A202V、A68S-T77N-G127T、G127T-A128P-G165Q、N184Q-A202V-S258D、T3V-G127T-N184Q、T3V-G127T-N217S、T77N-G127T-A128P、T78I-M123I-A202V、A128P-G165Q-A242G、A128P-G165Q-S258D、A128P-N184Q-S258D、A128P-N217S-S258D、A68S-A128P-S258D、A68S-G127T-G165Q、A68S-T78I-S258P、G127T-A128P-A202V、T3V-A128P-A242G、T3V-A68S-G127T、T3V-A68S-S258D、T3V-G127T-A202V、T3V-G165Q-A242G、T3V-G165Q-S258D、T3V-T77N-A242G、T3V-T77N-S258D、T3V-T78I-N184Q、T3V-T78I-N217S、T3V-T78I-S258P、T77N-A128P-A242G、T77N-A128P-S258D、T77N-G127T-G165Q、T77N-G165Q-A242G、T78I-A128P-S258P、T78I-G127S-S258P, A68S-M123I-S258D, G127T-G165Q-A202V, M123I-G165Q-S258D , T3V-G127T-A128P, T3V-N217S-S258D, T3V-T78I-A202V, T77N-G127T-A202V, T77N -M123I-S258D, T77N-T78I-M123I, T78I-M123I-G165Q, T78I-M123I-N184Q, T78I-N 184Q-S258P, A68S-T78I-M123I, A68S-T78I-T210P, G127T-N184Q-S258P, G127T-N2 17S-S258P, M123I-A202V-S258D, M123I-N217S-S258D, T3V-A202V-S258D, T3V-M12 3I-S258D, T3V-N184Q-S258D, T3V-T77N-G127T, T3V-T78I-T210P, T77N-T78I-T210 P, T78I-A128P-T210P, T78I-A202V-S258P, T78I-G127S-T210P, T78I-G165Q-T210P, T78I-M123I-A128P, T78I-M123I-G127S, T78I-M123I-N217S, and T78I-N217S-S258P.

7. A subtilisin protease variant as claimed in any preceding claim, wherein the variant comprises an amino acid sequence comprising one or more substitution groups selected from the group consisting of: M123I-Q136R; P9E-M123I; V45R-M123I; G127T-G165S-T210E; M123I-G165S-N239S; M123I-Q136R-G165Q; M123I-Q136R-S258E; M123I-V146S-T210E; P9E-V45R-M123I; Q136R-G165Q-S258E; V45R-M123I-Q136R; G127T-V146S-G165S-N239S; L10M-K27R-S98H-M123I;M123I-Q136R-G165Q-S258E; M123I-V146S-G165S-T210P;M123I-V146S-T210P-S250G; P9E-M123I-Q136R-G165Q;P9E-Q136R-G165Q-S258E; P9E-V45R-M123I-Q136R;V45R-M123I-Q136R-G165Q; V45R-M123I-Q136R-S258E;V45R-Q136R-G165Q-S258E; K27R-N43A-S98H-M123I-T210H;P9E-M123I-Q136R-G165Q-S258E; P9E-V45R-M123I-Q136R-G165Q;P9E-V45R-M123I-Q136R-S258E; V45R-M123I-Q136R-G165Q-S258E;Y103I-G127T-V146S-G165S-N239S; T77H-V87T-M123I-Q136R-N184Q-N239S; V26R-V45R-S98R-E111Q-M123I-Q136R; T77H-V87T-M123I-Q136R-G165Q-N184Q-N239S; T77S-V87S-M123I-Q136R-G165Q-S187P-S258P; T77S-V87S-M123I-Q136R-N160C-S187P-S258P; T77H-V87T-M123I-Q136R-N184Q-S187P-N239S-S258P; T3Q-K22Y-A24N-V45I-A48I-T77N-N217S-L234I-Y255R; P9E-T77Q-V87T-M123I-Q136R-G165Q-N184Q-N217S-N239S-S258E; N43A-V45R-T77H-V87T-M123I-Q136R-G165A-N184Q-S187P-N239S-S258P; P9E-N43A-V45R-T77S-V87S-M123I-Q136R-G165Q-S187P-N217S-S258P; P9E-N43A-V45R-T77H-V87T-M123I-Q136R-G165Q-N184Q-S187P-N239S-S258P; P9E-N43A-V45R-T77Q-V87T-M123I-Q136R-G165Q-N184Q-N217S-N239S-S258E; P9E-N43A-V45R-T77H-V87T-M123I-Q136R-G165A-N184Q-S187P-N217S-N239S-S258E;T77N-N217S-L234W;T77N-N217S、T77N-G126Q-G165Q-T210P-N217S-L234W-S258P、T77N-G127F-G165Q-A202V-T210P-N217S-L234W-S258P; A1Q-T3V-V28A-I35A-A68S-T77N-S86H-T115F-N217S-N239S-S250G-S258P-Q274A;T77N-M123I-T210P-N239S-S250G-S258P; V45I-A68S-T77N-M123I-L125N-G165Q-N217S-S258P; A68S-V71A-T77N-L95A-G165Q-N184Q-T210P-S258P; M123I-T210P-N217S-S258P;G127F-G165Q-N217S-S258P; T77N-G165Q-N217S-S258P;T77S-G165A-N184Q-T210P-N217S; A68S-T77N-A128P-G165Q-N184Q-N217S;V71A-T77S-N184Q-S258P; T77N-M123I-G165Q-N184Q-N217S;V71A-L95A-A128S-T210P; V71A-M123I-A128S-T210P;V71A-M123I-A128S-G165E; T77S-A128P-N184Q-N217S-F260W;T77H-A128S-G165Q-T210P; T77D-S86R-S155E-G165A-N184Q; A68S-V71A-T77N-A128S-G165Q-T210P-N217S; V28A-I35A-A68S-T77N-A128S-G165Q-N184Q-G203N-T210P; A128P-T210P;T210P-S258P;A128P-T210P-S259P;A128P-S187P-T210P; A128P-S187P-T210P-S258P;P9E-T77S-V87T-T210P-N239K; K15I-L95Q-M123I-G165S-T210P-N217S; K15I-M123I-V146S-G165S-T210P-N239S; K15I-N76K-M123I-V146S-G165S-T210P-N239S; K15I-V146S-G165S-T210P-N239S; P009E-V146S-N184Q-G203E-T210P-P238R-N239S; M123I-G165S-N217S;K15I-L95Q-S108K-G165S-G203E-T210P-N217S; K15I-L95Q-S108K-G165S-G203E-T210P; P9E-K15I-G165S-A193D-G203E-T210P;T77N-M123I-G165Q-N217S; T77H-G165Q;T77N-G165Q-N217S;A68S-V71A-T77N-G165Q-N217S; T77N-V87S-M123I-S155G-G165Q-N217S;T77H-V87S-S155G-G165Q; T77N-V87S-S155G-G165Q-N217S;T77N-M123I-G165Q;T77N-G165Q; A68S-V71A-T77N-G165Q;T77N-V87S-M123I-S155G-G165Q; I35A-A68S-T77N-V87S-V147L-G165Q; A68S-T77N-V87S-V147L-S155E-G165Q; T77N-V87S-M123I-V147L-S155G-G165Q; A68S-T77N-M123I-G165Q-N184Q-N217S; T77N-M123I-A128P-G165Q-N184Q-N217S; A68S-V71A-T77N-S100N-A128P-G165Q-N184Q-T210P-N217S-N239T;T77N-A128P-G165Q-N184Q-N217S; A68S-A128P-G165Q-N184Q-N217S;A68S-T77N-G165Q-N184Q-N217S;A68S-T77N-A128P-N184Q-N217S;A68S-T77N-A128P-G165Q-N217S; A68S-T77N-A128P-G165Q-N184Q;M123I-G165Q-N184Q-N217S;T77N-G165Q-N184Q-N217S;T77N-M123I-N184Q-N217S; T77N-M123I-G165Q-N184Q; T3V-A68S-T77N-A128P-G165Q-N184Q-N217S; T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-N217S; T3V-T77N-M123I-G165Q-N184Q-N217S; A68S-T77N-S108H-A128P-G165Q-N184Q-N217S; A68S-T77N-G99S-A128P-G165Q-N184Q-N217S; A68S-T77N-T114N-A128P-G165Q-N184Q-N217S; A68S-T77N-M123I-A128P-G165Q-N184Q-N217S; A68S-T77N-G127T-A128P-G165Q-N184Q-N217S; A68S-T77N-A128P-G165Q-N184Q-T210P-N217S; A68S-T77N-G99S-M123I-A128P-G165Q-N184Q-N217S; A68S-T77N-G99S-G127T-A128P-G165Q-N184Q-N217S; A68S-T77N-G99S-A128P-G165Q-N184Q-T210P-N217S; A68S-T77N-T114N-M123I-A128P-G165Q-N184Q-N217S; A68S-T77N-T114N-G127T-A128P-G165Q-N184Q-N217S; A68S-T77N-T114N-A128P-G165Q-N184Q-T210P-N217S; A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-N217S; A68S-T77N-M123I-A128P-G165Q-N184Q-T210P-N217S; A68S-T77N-G127T-A128P-G165Q-N184Q-T210P-N217S; A68S-T77N-G99S-M123I-A128P-G165Q-N184Q-T210P-N217S; A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-T210P-N217S; T3V-A68S-T77N-G99S-M123I-A128P-G165Q-N184Q-N217S; T3V-A68S-T77N-T114N-M123I-A128P-G165Q-N184Q-N217S; T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-N217S; T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-T210P-N217S; S86H-T115F-A143V-G165Q-A202V-S258P; S86H-L95A-N184Q-A202V-T210P-N217S-L234W-S250G-S258P; V30T-I35A-T77N-G165Q-A202V-T210P-S250G-S258P; A1Q-T3V-I35A-T77N-T78I-S86H-T115F-G165Q-N184Q-N217S-L234W-S258P; A1Q-A37T-T77N-T78I-S86H-M123I-G165Q-N184Q-A202V-N239S-S250G-S258P-Q274A;P9E-T77N-N96S-G165Q-G203E-S250G; P9E-T77N-N96S-G165E-G203E-T210P-S250G; A1Q-T3V-S86H-G130S-S155G-G165Q-N184Q-A202V-G203N-T210P-Q274A;A1Q-T3V-S86H-G165Q-N184Q-N239S-S250G-S258P; A1Q-T77N-S86H-V147L-A151G-N184Q-N217S-S258P; S86H-T115F-S155G-G165Q-N184Q-A202V-Q274A; A68S-S86H-T115F-N184Q-A202V-G203N-N239S-S250G-S258P-Q274A; A68S-T77N-V87S-A128P-S155G-G165Q-N217S; A68S-T77N-A128P-G165Q;A68S-T77N-V87S-A128P-S155G-G165Q; I35A-A68S-T77N-V87S-A128P-V147L-G165Q-N217S; A68S-T77N-V87S-A128P-S155G-G165Q-N217S-A242G; A68S-T77N-V87S-A128P-V147L-S155E-G165Q; A68S-T77N-V87S-A128P-S155E-G165Q-N217S; A68S-T77N-S86N-V87S-A128P-S155G-G165Q-N217S-A242G; I35A-A68S-T77N-A128P-V147L-G165Q-N217S; I35A-A68S-T77N-A128P-S155G-G165Q-A242G; I35A-A68S-T77N-A128P-V147L-G165Q-A242G; A68S-T77N-V87S-A128P-S155G-G165Q-A242G; A68S-T77N-N86S-G165Q-K194E-S211N-N217S; A68S-T77N-N86S-V87S-S155G-G165Q-K194E-S211N-N217S; A68S-T77N-N86S-G165Q-K194E-S211N; A68S-T77N-N86S-V87S-S155G-G165Q-K194E-S211N; I35A-A68S-T77N-N86S-V87S-V147L-G165Q-K194E-S211N-N217S; A68S-T77N-N86S-V87S-S155G-G165Q-K194E-S211N-N217S-A242G; A68S-T77N-N86S-V87S-V147L-S155E-G165Q-K194E-S211N; A68S-T77N-N86S-V087S-S155E-G165Q-K194E-S211N-N217S; A68S-T77N-V87S-S155G-G165Q-K194E-S211N-N217S-A242G; I35A-A68S-T77N-N86S-V147L-G165Q-K194E-S211N-N217S; I035A-A068S-T077N-N086S-S155G-G165Q-K194E-S211N-A242G; I35A-A68S-T77N-N86S-V147L-G165Q-K194E-S211N-A242G; A68S-T77N-N86S-V87S-S155G-G165Q-K194E-S211N-A242G; A68S-T77N-G127T-A128P-G165Q-N184Q-A202V-N217S; T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-N217S; A68S-T77N-G127T-A128P-G165Q-N184Q-N217S-S258P; T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-N217S-S258P; A68S-T77N-G127T-A128P-G165Q-N184Q-G203Q-N217S; T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-G203Q-N217S; A68S-T77N-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-N217S-S258P; A68S-T77N-A128P-G165Q-N184Q-G203Q-N217S-S258P; T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-G203Q-N217S-S258P; S86H-T115F-G127T-A143V-G165Q-N184Q-A202V-N217S-S258P; T3V-S86H-T115F-M123I-A143V-G165Q-A202V-N217S-S258P; A68S-T77N-A128P-S129H-G165Q-N184Q-A202V-N217S; T3V-A68S-T77N-M123I-A128P-S129H-G165Q-N184Q-A202V-N217S; S86H-T115F-S129H-A143V-G165Q-N184Q-A202V-N217S-S258P; T3V-S86H-T115F-M123I-S129H-A143V-G165Q-A202V-N217S-S258P; A68S-T77N-A128P-S155N-G165Q-N184Q-A202V-N217S; T3V-A68S-T77N-M123I-A128P-S155N-G165Q-N184Q-A202V-N217S; I35A-A68S-T77N-V87S-A128P-V147L-G165Q-A202V-N217S; T3V-I35A-A68S-T77N-V87S-M123I-A128P-V147L-G165Q-A202V-N217S;A68S-T77N-A128P-Q136N-S155N-G165Q-N184Q-N217S-A242N; T3V-A68S-T77N-M123I-A128P-Q136N-S155N-G165Q-N184Q-N217S-A242N;A68S-V71A-T77N-A128P-G165Q-N184Q-A202V-N217S; T3V-A68S-V71A-T77N-M123I-A128P-G165Q-N184Q-A202V-N217S; A68S-T77N-A128P-G165Q-N184Q-S187P-A202V-N217S; T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-S187P-A202V-N217S; A68S-T77N-A128R-G165Q-N184Q-A202V-N217S-S258D; T3V-A68S-T77N-M123I-A128R-G165Q-N184Q-A202V-N217S-S258D; A68S-T77N-A128P-S129R-G165Q-N184Q-N217S-S258D;T3V-A68S-T77N-M123I-A128P-S129R-G165Q-N184Q-N217S-S258D;A68S-T77N-A128R-G165Q-N184Q-A202V-N217S-S258D-K264H; T3V-A68S-T77N-M123I-A128R-G165Q-N184Q-A202V-N217S-S258D-K264H;A68S-T77N-A128R-G165Q-N184Q-G203E-S258D; T3V-A68S-T77N-M123I-A128R-G165Q-N184Q-G203E-S258D; T3V-A68S-T77N-M123I-A128R-G165Q-N184E-A202V-N217S-S258D; T3V-A52R-A68S-T77N-M123I-A128R-G165Q-N184E-A202V-N217S-S258D; T3V-T77N-S100N-G105N-D119Q-A128P-S158N-N160D-T161Q-G165A-N184Q-N217S-S258P-K264S; T3V-T77N-S100N-G105N-A128P-S158D-T161Q-G165A-N184Q-N217S-S258P; T3V-T77N-S100N-G105N-A128P-S158N-N160D-T161Q-G165A-N184Q-N217S-S258P; A24Q-V45I-A68S-T77N-S98R-E111Q-M123I-S158Q-G165E-N184Q-S187E-V204I-T210I-N217S-S259E; P9E-A52R-S86R-E111Q-S129R-S158Q-G165E-V204I-T210I-S258D-S259E-K264Q; T3V-A68S-T77N-G127T-A128P-G165Q-N184Q-A202V-N217S; T3V-I35A-T77N-V87S-M123I-A128P-G165Q-N184Q-A202V-N217S-A242G;T3V-A68S-T77N-G127E-A128P-G165Q-N184Q-A202V-N217S; T3V-A68S-T77N-M123I-G127V-A128P-G165Q-N184Q-N217S; T3V-A68S-T77N-M123I-A128P-A151G-G165Q-N184Q-N217S; T3V-A68S-T77N-G127T-A128P-A151G-G165Q-N184Q-N217S; T3V-T77N-M123I-A128P-A151G-G165Q-N184Q-A202V-N217S; T3V-A68S-T77N-L95F-A128P-G165Q-N184Q-A202V-N217S; T3V-A68S-T77N-L95A-M123I-A128P-G165Q-N184Q-N217S; T3V-A68S-T77N-L95F-M123I-A128P-G165Q-N184Q-N217S; T3V-A68S-T77N-G99A-A128P-G165Q-N184Q-A202V-N217S; T3V-A68S-T77N-G99E-A128P-G165Q-N184Q-A202V-N217S; T3V-A68S-T77N-G99Q-A128P-G165Q-N184Q-A202V-N217S; T3V-A68S-T77N-G99A-M123I-A128P-G165Q-N184Q-N217S; T3V-A68S-T77N-S108H-A128P-G165Q-N184Q-N217S; T3V-I35A-T77N-V87S-M123I-A128P-V146A-G165Q-N184Q-A202V-N217S-A242G; T3V-A68S-T77N-G127T-A128P-G165E-N184Q-A202V-N217S; T3V-A68S-T77N-M123I-A128P-V146A-G165Q-N184Q-N217S; T3V-A68S-T77N-M123I-A128P-T161D-G165Q-N184Q-A202V-N217S; T3V-V45R-A68S-T77N-S102D-A128P-T161D-G165Q-N184Q-A202V-N217S; T3V-A68S-T77N-G127T-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127V-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-I35A-T77N-V87S-G127T-A128P-G165Q-N184Q-A202V-N217S-A242G-S258P; T3V-T77N-M123I-G127Q-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-T77N-M123I-G127V-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-T77N-M123I-G127E-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-A202V-N217S; T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-A202V-T210P-N217S; T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-A68S-T77N-M123I-G127T-A128P-G165Q-N184Q-A202V-N217S-S258D;L10M-I35A-T78I-V87S-M123I-A202V-S258D-F260W; L10M-I35A-T78I-V87S-M123I-A202V-F260W-K264H; L10M-I35A-T78I-V87S-A202V-S258D; L10M-I35A-T78I-V87S-S187E-A202V;L10M-T78I-V87S-S187E-A202V;L10M-I35A-T78I-S187E-A202V; T3V-L10M-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-I35A-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-V87S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165E-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-L216Q-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-A242G-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P-F260W; A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-L31I-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; A68S-T77N-G127T-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D; A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D; T3V-A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D; T3V-A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D; A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258D;T3V-A68S-T77N-S108H-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-G99Q-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-G99Q-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-A68S-T77N-G127S-A128P-G165E-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-P9E-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-S187E-A202V-T210P-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-G203E-T210P-N217S-S258P; T3V-L10M-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-P9E-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-S187E-A202V-N217S-S258P; T3V-P9E-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-S187E-A202V-T210P-N217S-S258P; T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-G203E-T210P-N217S-S258P; T3V-A68S-T77N-G101A-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-L10M-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-P9E-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-S187E-A202V-T210P-N217S-S258P; T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-G203E-T210P-N217S-S258P; T3V-A68S-T77N-G101A-M123I-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-L10M-A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-L10M-A68S-T77N-G101A-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-P9E-L10M-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-P9E-A68S-T77N-G99Q-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S243I-S258P; T3V-A68S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-S258P; T3V-A68S-T77E-M123I-G127T-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77E-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P-K264H; T3V-A68S-T77N-S102T-M123I-G127S-A128P-G165Q-N184Q-A202V-N211S-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N211S-N217S-S258P; T3V-A68S-T77N-S102T-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P-S259P; T3V-A68S-T77N-M123I-G127S-A128P-T161Q-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-S158Q-G165Q-N184Q-A202V-N217S-S258P; T3V-V45Q-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A193Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-A143Q-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-G117S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A48V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A48T-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A29S-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S; T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-N217S-S258P;T3V-A68S-T77N-A128P-G165Q-N184Q-A202V-N217S-S258P; A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S; A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-G99Q-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-T78I-V87S-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-A242G-S258P; T3V-V26Q-A68S-T77N-M123I-G127S-A128P-G165E-N184Q-A202V-T210P-N217S-S258P; T3V-P9E-L10M-A68S-T77N-M123I-G127S-A128P-G165E-N184Q-A202V-T210P-N217S-S258P; T3V-P9E-L10M-A68S-T77N-G101A-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-T78I-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-A68S-T77N-T78I-M123I-G127S-A128P-N160K-G165Q-N184Q-A202V-T210P-N217S-A242G-S258P; T3V-A68S-T77N-V87S-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P; T3V-P40E-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77E-M123I-G127T-A128P-G165E-N184Q-A202V-N217S-P238Q-S258P; T3V-A68S-T77N-G99S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77E-M123I-G127T-G165E-N184Q-A202V-N217S-S258P;A68S-T77N-A128P-G165Q-N184Q-N217S-S258P; T3V-A68S-T77N-G101A-S102T-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S; T3V-A 68S-T77N-T78I-G99S-G127S-A128P-G165Q-T210P-A242G-S258P-K264H; T3V-A68S-T77N-T78I-G99S-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-A242G-S258P; T3V-A68S-T77N-M123I-G127T-G165E-N184Q-A202V-N217S-P238Q-S258P; T3V-A68S-T77N-T78I-G99S-G127S-A128P-G165Q-T210P-A242G-S258P; T3V-A68S-T77N-T78I-G99S-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-A242G-S258D; I35A-M123I-G127T-T210P-S243I; T3V-A68S-T77N-G99S-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P-F260W; T3V-A68S-T77N-T78I-G99S-G101A-G127S-A128P-G165Q-T210P-A242G-S258P; A68S-T77N-G99Q-A128P-G165Q-N184Q-N217S; A68S-T77N-A128P-G165Q-N184Q-A202V-N217S; A68S-T77N-A128P-G165Q-N184Q-N217S-S258D; T3V-A68S-T77N-T78I-G99S-G101A-M123I-G127S-A128P-G165Q-N184Q-A202V-T210P-N217S-A242G-S258P; and V45R-S98R.

8. A subtilisin protease variant as claimed in any preceding claim, wherein the variant further comprises: i. Motif A, motif B, or a combination thereof; ii. Motif A and motif B; iii. Motif 1 between Asp32 and His63; iv. Motif 6 or Motif 7 between Lys236 and Arg246; or v. Motif 1 between Asp32 and His63 and motif 6 or motif 7 between Lys236 and Arg246; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14; and wherein the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:

2.

9. A subtilisin protease variant as described in any preceding claim, wherein the variant comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% amino acid sequence identity to the amino acid sequence of the AprL-clade subtilisin protease identified in Figure 1.

10. An isolated subtilisin protease variant as claimed in any preceding claim, wherein the variant comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2 or 9.

11. A subtilisin protease variant as claimed in any preceding claim, wherein the variant is selected from a parent having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with an AprL-clade subtilisin protease comprising (i) motif A, motif B or a combination thereof; or (ii) motif A and motif B; wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; and wherein motif B is selected from motif 5, motif 6, motif 7, motif 8, motif 9, motif 10, motif 11, motif 12, motif 13 and motif 14.

12. A subtilisin protease variant as claimed in any preceding claim, wherein the variant is selected from a parent comprising the amino acid sequence of SEQ ID NO: 2 or 9.

13. A subtilisin protease variant according to any preceding claim, wherein the variant has protease activity and / or is isolated.

14. A subtilisin protease variant as claimed in any preceding claim, wherein the variant has one or more improved properties when compared to a reference subtilisin protease; wherein the improved properties are selected from improved protease activity, improved detergent cleaning performance, improved detergent thermostability, and improved detergent maturation laundry cleaning; and wherein the detergent is optionally a boron-free composition.

15. The subtilisin variant of any preceding claim, wherein the reference subtilisin comprises the amino acid sequence of SEQ ID NO: 2, 9 or 63.

16. The subtilisin protease variant of claim 13 or 14, wherein the improved property is (i) improved protease activity, wherein the variant has a PI>1 against N-suc-AAPF-pNA or dimethylcasein substrate; (ii) improved detergent cleaning performance, wherein the variant has a BMI, POM, egg and / or egg yolk cleaning PI>1; (iii) improved detergent thermostability, wherein the variant has a stability PI>1 or a residual activity % ≥5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more after 15-20 minutes at a stress temperature of 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or more; and / or (iv) improved detergent mature laundry cleaning, wherein the variant has (a) a mature laundry cleaning PI>1, (b) a residual cleaning activity of>10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more after 24 hours at 55°C, and / or (c) a residual cleaning activity of>10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more after 3 days or 3 weeks at 37°C.

17. The subtilisin protease variant of claim 16, wherein the (i) protease activity is measured according to the protease activity assay described in Example 1; (ii) Detergent Cleaning Performance The cleaning performance in the laundry (HDL and HDD) and ADW detergent assays described in Example 1 was measured; (iii) Detergent thermal stability is measured according to the general sample setup for stability determination described in Example 1; and / or (iv) Detergent Mature Clothes Cleaning was measured according to the Mature Clothes Cleaning assay described in Example 1.

18. A composition comprising one or more subtilisin variants according to any preceding claim.

19. The composition of claim 18, wherein the composition is a detergent composition.

20. The composition of claim 19, wherein the detergent composition is selected from the group consisting of laundry detergents, fabric softening detergents, dishwashing detergents and hard surface cleaning detergents.

21. The composition of any one of claims 18-20, wherein the composition further comprises one or more calcium ions and / or zinc ions; one or more enzyme stabilizers; from about 0.001% to about 1.0% by weight of the subtilisin variant; one or more bleaching agents; one or more auxiliary materials; and / or one or more additional enzymes or enzyme derivatives selected from the group consisting of: Acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, DNA enzyme or nuclease, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exomannanase, galactanase, glucoamylase, hemicellulase, hyaluronidase, keratinase, laccase, lactase, ligninase , lipases, lipoxygenases, lysozymes, mannanases, oxidases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonases, proteases, pullulanases, reductases, rhamnogalacturonases, β-glucanases, tannases, transglutaminases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, metalloproteases, additional serine proteases, and combinations thereof.

22. The composition of any one of claims 18-21, wherein the composition contains phosphate or is phosphate-free and / or contains boron or is boron-free.

23. The composition of any one of claims 18-22, wherein the composition is a granule, powder, solid, stick, liquid, tablet, gel, paste or unit dose composition.

24. A method of cleaning comprising contacting a surface or article in need of cleaning with a subtilisin variant as described in any one of claims 1-17 or a composition as described in any one of claims 18-22; and optionally further comprising a step of rinsing the surface or article after contacting the surface or article with the variant or composition, wherein optionally the article is a dishware or fabric.

25. A polynucleotide comprising a nucleic acid sequence encoding a subtilisin variant as described in any one of claims 1-17, wherein the polynucleotide is optionally isolated.

26. An expression vector or expression cassette comprising the polynucleotide according to claim 25.

27. The expression vector or expression cassette of claim 26, wherein the nucleic acid sequence is operably linked to a promoter.

28. A recombinant host cell comprising the vector or cassette of claim 26 or claim 27 or the polynucleotide of claim 25.

29. A composition comprising a subtilisin variant as described in any one of claims 1-17, wherein the composition is a disinfectant composition, an industrial or institutional cleaning composition, a medical device cleaning composition, an animal feed composition, a contact lens cleaning composition, a wound cleaning composition, or a textile processing composition, a leather processing composition, or a feather processing composition.

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