Protease variants and uses thereof

Abstract

Disclosed herein are one or more subtilisin variants, including one or more subtilisin variants with improved stability and / or stain removal properties compared to one or more reference subtilisins, nucleic acids encoding same, and compositions and methods related to their production and use.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to and claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 343,618, filed May 31, 2016, which is incorporated herein by reference in its entirety.

[0002] Reference to Electronically Submitted Sequence Listing The contents of the Sequence Listing (Name: NB40785WOPCT_ST25; Size: 882 KB; Creation Date: May 26, 2017), which was submitted electronically with the application as an ASCII text file, constitutes part of the application and is hereby incorporated by reference in its entirety.

[0003] Disclosed herein are one or more subtilisin variants, including one or more subtilisin variants having improved stability and / or stain removal properties compared to one or more reference subtilisins, nucleic acids encoding same, and compositions and methods related to their production and use. [Background technology]

[0004] Serine proteases are enzymes (EC No. 3.4.21) that have an active site serine that initiates the hydrolysis of peptide bonds in proteins. Serine proteases comprise a diverse class of enzymes with a wide range of specificities and biological functions, but are further divided into chymotrypsin-like (trypsin-like) and subtilisin-like based on their structure. Prototypical subtilisin (EC No. 3.4.21.62) was first obtained from Bacillus subtilis. Subtilisin and its homologs are members of the S8 peptidase family of the MEROPS classification scheme. Members of the S8 family have a catalytic triad with the amino acid sequence Asp, His, and Ser arranged in that order. Although many useful mutant proteases have been developed for cleaning applications, there is still a need for improved protease mutants. Summary of the Invention [Means for solving the problem]

[0005] One embodiment is one or more subtilisin variants comprising an amino acid sequence comprising two, three or four or more mutations at positions corresponding to SEQ ID NO:2, said positions being at or near the positions of: (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, 1 14, 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; (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; said variants being selected from the group consisting of an AprL-Clade subtilisin enzyme containing motif A and / or motif B. and wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2.

[0006] Yet another embodiment is 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, with the proviso that one or more of said two, three or four or more substitutions are artificial substitutions, comprising: (i) 1Q, 3Q / V, 9E, 10M, 15I, 22Y, 24N / Q, 26Q, 27R, 28A, 29S, 30T, 31I, 35A, 37T, 40E, 43A, 45I / Q / R, 48I / 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, 10 3I, 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, 1 65A / E / Q / S, 184E / Q, 187E / P, 193D / Q, 202V, 203E / N / Q, 204I, 210E / H / I / P, 211S, 216Q, 217S, 234 I / 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および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および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; said variants being selected from motif A and / or motif B. and wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4; 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 corresponding to the amino acid sequence of SEQ ID NO:2.

[0007] 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 of the subtilisin variants described herein comprise motif A, motif B, or a combination thereof. In still other embodiments, one or more of the 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 yet a further embodiment, 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.

[0008] Some embodiments relate to compositions comprising one or more subtilisin variants described herein. Further embodiments relate to cleaning methods 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]

[0009] [Figure 1A-1D] 1A-1D show the CLUSTAL W sequence alignment of AprL, Blid02339 and other AprL-clade enzymes. [Diagram 2] FIG. 2 shows a phylogenetic tree of subtilisins including AprL, Blid02339, other members of the AprL-clade, and other subtilisins including the mutants described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Unless otherwise indicated herein, one or more subtilisin variants described herein can be made 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. Terms and abbreviations not defined shall have their ordinary meanings as used in the art. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. Any definitions provided herein should be interpreted in the context of the specification as a whole. As used herein, the singular forms "a," "an," and "the" include the plural unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acid sequences are written left to right in a 5' to 3' orientation, and amino acid sequences are written left to right in an amino to carboxy orientation. All numerical ranges provided herein include all narrower numerical ranges that fall within such broader numerical ranges, as if all narrower numerical ranges were expressly set forth herein.

[0011] The term "about" as used herein in connection 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" refers to a pH value of 5.5 to 6.5, unless the pH value is otherwise specifically defined.

[0012] Naming of an amino acid substitution or mutation in one or more of the subtilisin variants described herein uses one or more of the following: position; position: amino acid substitution or mutation; or starting amino acid: position: amino acid substitution. Reference to a "position" (i.e., 5, 8, 17, 22, etc.) includes the starting amino acid that may be present at such position, and mutations or substitutions from the starting amino acid that may be present at such position. Reference to a "position: amino acid substitution and / or mutation" (i.e., 1S / T / G, 3G, 17T, etc.) includes the starting amino acid that may be present at such position, and one or more amino acids that may be changed from the starting amino acid and / or that may replace such starting amino acid. The starting amino acid is excluded from the amino acid substitution if the replacing amino acid and the starting amino acid are the same. Reference to a starting amino acid or amino acid substitution or mutation may also be expressed as several starting, replacing or mutating amino acids separated by a slash (" / "). For example, D275S / K indicates a substitution at position 275 with serine (S) or lysine (K), and P / S197K indicates that the initiating amino acid proline (P) or serine (S) at position 197 is substituted with lysine (K).

[0013] The positions of the amino acid residues in a given amino acid sequence are numbered relative to the amino acid sequence of SEQ ID NO:2. That is, the amino acid sequence of the AprL-clade enzyme as set forth in SEQ ID NO:2 serves as a reference sequence. For example, the amino acid sequence of the AprL-clade subtilisin enzyme or one or more subtilisin variants described herein is aligned with the amino acid sequence of SEQ ID NO:2 using an alignment algorithm described herein, and each amino acid residue in the given amino acid sequence that is aligned (preferably optimally aligned) with an amino acid residue in SEQ ID NO:2 is conveniently numbered with reference to the position number of the corresponding amino acid residue. For example, a sequence alignment algorithm as described herein identifies where insertions or deletions have occurred in the subject sequence compared to the query sequence.

[0014] The term "variant" with respect to a polypeptide amino acid sequence refers to a polypeptide amino acid sequence that differs from a particular wild-type, parent, or reference polypeptide amino acid sequence by containing one or more artificially made substitutions, insertions, or deletions of amino acids. Similarly, the term "variant" with respect to a polynucleotide nucleic acid sequence refers to a polynucleotide nucleic acid sequence that differs from a particular wild-type, parent, or reference polynucleotide by containing one or more artificially made substitutions, insertions, or deletions of nucleic acids. The identity of the wild-type, parent, or reference polypeptide amino acid sequence or polynucleotide nucleic acid sequence will be clear from the context.

[0015] The term "mutation", for example, when used in the phrase "two, three or more amino acid mutations relative to SEQ ID NO:2" or "two, three or more mutations at corresponding positions in SEQ ID NO:2", encompasses each 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 desired mutant is aligned with SEQ ID NO:2 and each position of the mutant is compared to SEQ ID NO:2 to identify the 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 mutation.

[0016] The terms "protease" and "proteinase" refer to enzymes capable of degrading proteins and peptides. Proteases have the ability to perform "proteolysis" by hydrolyzing the peptide bonds that link the amino acids together in the peptide or polypeptide chain that forms the protein. This activity of proteases as protein digestive enzymes is called "proteolytic activity". There are many well-known procedures for measuring proteolytic activity. For example, proteolytic activity can be determined by comparative assays that analyze the ability of each protease to hydrolyze an appropriate substrate. Examples of substrates useful in the analysis of protease or proteolytic activity include, but are not limited to, dimethylcasein (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, e.g., WO 99 / 34011 and U.S. Pat. No. 6,376,450). The pNA peptidyl assay (see, e.g., Del Mar et al., Anal Biochem, 99:316-320, 1979) can also be used to determine active enzyme concentration. This assay measures the rate of release of p-nitroaniline when the enzyme hydrolyzes a soluble synthetic substrate, e.g., succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (suc-AAPF-pNA). The rate of production of yellow color from the hydrolysis reaction is measured spectrophotometrically at 410 nm, and the rate of production is proportional to the active enzyme concentration. In addition, measurement of absorbance at 280 nanometers (nm) can also be used to measure the total protein concentration in purified protein samples. Activity on substrate / protein concentration indicates enzyme specific activity.

[0017] The phrases "substantially boron-free composition" or "substantially boron-free detergent" refer to a composition or detergent, respectively, that contains trace amounts of boron, e.g., perhaps less than about 1000 ppm (1 mg / kg or liter equals 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 of other composition or detergent ingredients.

[0018] As used herein, the term "Bacillus" includes all species within the genus "Bacillus" known to those of skill in the art, including, but not limited to, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alcalophilus, B. argininos ... kalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. gibsonii and B. thuringiensis. It should be understood that the genus Bacillus is continually being reorganized taxonomically. Thus, the genus is intended to include organisms such as reclassified species, for example, but not limited to, B. stearothermophilus, now referred to as "Geobacillus stearothermophilus," or Bacillus polymyxa, now referred to as Paenibacillus polymyxa.The production of resistant endospores under stressful environmental conditions is considered to be the defining characteristic of the genus Bacillus, but this characteristic has also been implicated in the recently named genera Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, and others. The same also applies to the genera Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus and Virgibacillus.

[0019] As used herein, the term "mutation" refers to an artificial modification made to a reference amino acid or nucleic acid sequence. This term is intended to encompass artificial substitutions, insertions and deletions.

[0020] As used herein, the term "vector" refers to a nucleic acid construct used to introduce or transfer a nucleic acid into a target cell or tissue. Typically, a vector is used to introduce foreign DNA into a cell or tissue. Vectors include plasmids, cloning vectors, bacteriophages, viruses (e.g., viral vectors), cosmids, expression vectors, shuttle vectors, and the like. Typically, a vector contains an origin of replication, a multiple cloning site, and a selection marker. The process of inserting a vector into a target cell is generally referred to as transformation. In some embodiments, the invention includes a vector comprising a DNA sequence encoding a serine protease polypeptide (e.g., a precursor or mature serine protease polypeptide) operably linked to a suitable prosequence (e.g., a secretory signal peptide sequence, etc.) capable of expressing the DNA sequence in a suitable host and causing folding and translocation of the recombinant polypeptide chain.

[0021] As used herein with respect to introducing a nucleic acid sequence into a cell, the term "introduced" refers to any suitable method for transferring a nucleic acid sequence into a cell. Such methods include, but are not limited to, protoplast fusion, transfection, transformation, electroporation, conjugation and transduction. Transformation refers to the genetic modification of a cell resulting from the uptake, optional integration into the genome, and expression of genetic material (e.g., DNA).

[0022] "Expression cassette" or "expression vector" refers to a nucleic acid construct or vector, recombinantly or synthetically produced, for the expression of a nucleic acid of interest (e.g., a foreign nucleic acid or a transgene) in a target cell. The nucleic acid of interest usually expresses a protein of interest. An expression vector or expression cassette usually includes a promoter nucleotide sequence that drives or promotes the expression of the foreign nucleic acid. An expression vector or expression cassette also usually includes other specific nucleic acid elements that allow the transcription of a specific nucleic acid in the target cell. A recombinant expression cassette can be incorporated into a plasmid, a chromosome, a mitochondrial DNA, a plastid DNA, a virus, or a nucleic acid fragment. Some expression vectors have the ability to integrate and express heterologous DNA fragments into a host cell or the genome of a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of an appropriate expression vector for expressing a protein from a nucleic acid sequence incorporated into the expression vector is within the knowledge of one of ordinary skill in the art.

[0023] As used herein, a nucleic acid is "operably linked" to another nucleic acid sequence when it is placed in a functional relationship with the other nucleic acid sequence. For example, a promoter or enhancer is operably linked to a nucleotide coding sequence if the promoter acts to transcribe the coding sequence. A ribosome binding site may be operably linked to a coding sequence if it is located in such a way that it promotes transcription of the coding sequence. Usually, "operably linked" DNA sequences are contiguous. However, enhancers need not be contiguous. Linking is accomplished by ligation at appropriate restriction enzyme recognition sites. If no such sites exist, synthetic polynucleotide adaptors or linkers can be used according to conventional methods.

[0024] As used herein, the term "gene" refers to a polynucleotide (e.g., a DNA segment) that encodes a polypeptide and includes regions preceding and following the coding region. In some cases, genes contain intervening sequences (introns) between individual coding segments (exons).

[0025] As used herein, "recombinant," when used in reference to a cell, generally means 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 a gene that is not present in the same form in the native (non-recombinant) form of the cell, or the recombinant cell may contain a native gene (present in the native form of the cell) that has been modified and reintroduced into the cell. A recombinant cell may contain a nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell. Such modifications include those obtained by gene replacement, site-specific mutagenesis, and related techniques known to those of skill in the art. Recombinant DNA techniques include techniques for producing recombinant DNA in vitro and for transferring recombinant DNA into a cell where it can be expressed or propagated, thereby producing a recombinant polypeptide. "Recombination" and "recombining" of polynucleotides or nucleic acids generally refer to the assembly or joining of two or more nucleic acids or polynucleotide chains or fragments to produce a new polynucleotide or nucleic acid.

[0026] A nucleic acid or polynucleotide is said to "encode" a polypeptide if, in its natural state or when manipulated by methods known to those of skill in the art, it can be transcribed and / or translated to produce a polypeptide or a fragment thereof. The antisense strand of such a nucleic acid is also said to encode a sequence.

[0027] The terms "host strain" and "host cell" refer to a suitable host for an expression vector containing a DNA sequence of interest.

[0028] A "protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. The terms "protein" and "polypeptide" are used interchangeably herein. For amino acids, one-letter or three-letter codes 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 of the 20 amino acids. It is also understood that a polypeptide can be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0029] "Prosequence" or "propeptide sequence" refers to amino acid sequences between the signal peptide sequence and the mature protease sequence necessary for proper folding and secretion of the protease; these are sometimes called intramolecular chaperones. Cleavage of the prosequence or propeptide sequence generates the mature active protease. Bacterial serine proteases are often referred to as proenzymes.

[0030] The terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that may be involved in the secretion or direct transport of a mature or precursor form of a protein. A signal sequence is usually located at the N-terminus of a precursor or mature protein sequence. A signal sequence may be endogenous or exogenous. A signal sequence is usually not present in a mature protein. A signal sequence is usually cleaved from a protein by a signal peptidase after the protein has been transported.

[0031] The term "mature" form of a protein, polypeptide, or peptide refers to the functional form of the protein, polypeptide, or peptide that does not include signal peptide and propeptide sequences.

[0032] The term "precursor" form of a protein or peptide refers to the mature form of the protein having a prosequence operably linked to the amino- or carboxy-terminus of the protein. A precursor may also have a "signal" sequence operably linked to the amino-terminus of the prosequence. A precursor may also have additional polypeptides associated with post-translational activity (e.g., polypeptides which are cleaved from it to leave the mature form of the protein or peptide).

[0033] The term "wild type" in relation to an amino acid sequence or a nucleic acid sequence indicates that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "natural" refers to something that is found in nature (e.g., a protein or polynucleotide sequence). Conversely, the term "non-natural" refers to something that is not found in nature (e.g., recombinant polynucleotide and protein sequences that are produced in a laboratory or that are produced by modification of a wild type sequence).

[0034] As used herein in reference to an amino acid residue position, "corresponding to" or "corresponding to" or "corresponding" 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 equivalent to the recited residue in a protein or peptide. As used herein, a "corresponding region" generally refers to an analogous position in a related or reference protein.

[0035] The terms "derived from" and "obtained from" refer not only to proteins produced or producible by the strain of organism under discussion, but also to proteins encoded by DNA sequences isolated from such strains and proteins produced in host organisms containing such DNA sequences. Additionally, the terms refer to proteins encoded by DNA sequences of synthetic and / or cDNA origin and having the identifying characteristics of the protein under discussion. By way of example, "protease derived from Bacillus" refers to enzymes with proteolytic activity naturally produced by Bacillus, and serine proteases such as those produced by Bacillus sources, but which are produced by other host cells transformed with nucleic acids encoding the serine proteases through the use of genetic engineering techniques.

[0036] The term "identical" in the context of two polynucleotide or polypeptide sequences refers to the nucleic acids or amino acids in the sequences that are the same when the two sequences are aligned for maximum correspondence, as measured by a sequence comparison algorithm or sequence analysis algorithm.

[0037] "% identity" or "percent identity" or "PID" refers to the identity of protein sequences. Percent identity can be determined using standard techniques known in the art. The percent amino acid identity shared by sequences of interest can be determined by aligning sequences and directly comparing sequence information, for example, using programs such as BLAST, MUSCLE or CLUSTAL. The BLAST algorithm is described, for example, in Altschul et al., J Mol Biol, 215:403-410 (1990) and Karlin et al., Proc Natl Acad Sci USA, 90:5873-5787 (1993). Percent (%) amino acid sequence identity value is determined by dividing the number of matched identical residues by the total number of residues of 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.

[0038] As used herein, "homologous protein" or "homologous protease" refers to proteins that have clear similarities in primary, secondary and / or tertiary structure. Protein homology can refer to similarity in linear amino acid sequence when proteins are aligned. Homology can be determined by amino acid sequence alignment, for example, using programs such as BLAST, MUSCLE or CLUSTAL. Homology searches of protein sequences can be performed using BLASTP and PSI-BLAST of NCBI BLAST with a threshold of 0.001 (E-value cutoff). (Altschul et al., "Gapped BLAST and PSI BLAST a new generation of protein database search programs", Nucleic Acids Res, Set 1;25(17):3389-402(1997)). BLAST programs use several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most related sequences with respect to biological similarity, but is not recommended for query sequences with fewer than 20 residues (Altschul et al., Nucleic Acids Res, 25:3389-3402, 1997, and Schaffer et al., Nucleic Acids Res, 29:2994-3005, 2001). Examples of default BLAST parameters for searching nucleic acid sequences include: Neighboring words threshold=11; E-value cutoff=10; Scoring Matrix=NUC.3.1 (match=1, mismatch=-3); Gap Opening=5; and Gap Extension=2.Examples of default BLAST parameters for searching amino acid sequences include: Word size=3; E-value cutoff=10; Scoring Matrix=BLOSUM62; Gap Opening=11; and Gap Extension=1. This information can be used to group protein sequences and / or build a phylogenetic tree from them. Amino acid sequences can be input into programs such as Vector NTI Advance suite, and guide trees can be created using the Neighbor-Joining (NJ) method (Saito and Nei, Mol Biol Evol, 4:406-425, 1987). Tree construction can be calculated using Kimura's sequence distance correction, ignoring positions containing gaps. Programs such as AlignX can show the calculated distance values ​​in parentheses following the molecule names shown on the phylogenetic tree.

[0039] Understanding the homology between molecules can reveal information about the evolutionary history and function of a molecule, and if a newly sequenced protein is homologous to a previously characterized protein, it strongly indicates the biochemical function of the novel protein. The most fundamental relationship between two entities is homology, and two molecules are said to be homologous if they are derived from a common ancestor. Homologous molecules or homologs can be divided into two classes: paralogs and orthologs. Paralogs are homologs that exist within one species. Paralogs often differ in their detailed biochemical function. Orthologs are homologs that exist between different species and have very similar or identical functions. Protein superfamilies are the largest classifications (clades) of proteins for which their common ancestry can be inferred. Usually, this common ancestry is based on sequence alignment and mechanistic similarity. Superfamilies usually contain several protein families that show sequence similarity within the family. The term "protein clan" is commonly used for protease superfamilies, based on the MEROPS protease classification system.

[0040] 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 the following parameters: Gap opening penalty=10.0; Gap extension penalty=0.05; Protein weight matrix=BLOSUM series; DNA weight matrix=IUB; Delay divergent sequences %=40; Gap separation distance=8; DNA transitions weight=0.50; List of hydrophilic residues=GPSNDQEKR; Use negative matrix=OFF; Toggle Residue specific penalties=ON; Toggle hydrophilic penalties=ON; and Toggle end gap separation penalty. penalty = OFF. The CLUSTAL algorithm includes deletions that occur at either end. For example, a variant with a 5 amino acid deletion at either end of a 500 amino acid polypeptide (or within the polypeptide) has a percent sequence identity of 99% (495 / 500 identical residues x 100) to the "reference" polypeptide. Such a variant would be encompassed by variants with "at least 99% sequence identity" to the polypeptide.

[0041] Nucleic acids and polynucleotides are "isolated" when they are at least partially or completely separated from other components, such as, but not limited to, other proteins, nucleic acids, cells, etc. Similarly, polypeptides, proteins, or peptides are "isolated" when they are at least partially or completely separated from other components, such as, but not limited to, other proteins, nucleic acids, cells, etc. On a molar basis, an isolated species is more abundant than other species in a composition. For example, an isolated species may comprise 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% (on a molar basis) of all macromolecular species present. The species of interest is preferably purified to substantial homogeneity (i.e., no contaminating species are detectable in the composition by conventional detection methods). Purity and homogeneity can be determined by a number of techniques well known in the art, such as by subjecting nucleic acid or protein samples to agarose or polyacrylamide gel electrophoresis, respectively, followed by visualization by staining. If necessary, high resolution techniques, such as high performance liquid chromatography (HPLC) or similar means, can be used to purify the material.

[0042] The term "purified" as applied to a nucleic acid or polypeptide generally refers to a nucleic acid or polypeptide that is essentially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a distinct band in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is "purified". A purified nucleic acid or polypeptide is at least about 50% pure, and typically is 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% (e.g., weight % on a molar basis) or more pure. In this context, a composition is enriched for a molecule if the concentration of the molecule is substantially increased following application of a purification or concentration technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, or other particular material or component being present in a composition in a relative or absolute concentration that is higher than in the starting composition.

[0043] As used herein, the term "functional assay" refers to an assay that provides an indication of the activity of a protein. In some embodiments, the term refers to an assay system that analyzes the ability of a protein to function in its normal capacity. For example, in the case of a protease, a functional assay involves determining the effect of the protease on degrading a proteinaceous substrate.

[0044] The term "cleaning activity" refers to the cleaning performance achieved by a serine protease polypeptide or reference subtilisin under conditions used in the proteolysis, hydrolysis, cleaning, or other processes disclosed. In some embodiments, the cleaning performance of a serine protease or reference subtilisin can be determined by using various assays for cleaning one or more various enzyme-sensitive stains on an article or surface (e.g., stains resulting from food, grass, blood, ink, milk, oil, and / or egg proteins). The cleaning performance of one or more subtilisin variants or reference subtilisins described herein can be measured by subjecting the stains on an article or surface to standard cleaning conditions and evaluating the extent to which the stain is removed using various chromatographic, spectrophotometric, or other quantitative methods. Exemplary cleaning assays and methods are known in the art and include, but are not limited to, those described in WO 99 / 34011 and U.S. Pat. No. 6,605,458, as well as the cleaning assays and methods included in the Examples set forth below.

[0045] The term "cleaning effective amount" of one or more subtilisin variants, or reference subtilisins, described herein refers to the amount of protease that will achieve a desired level of enzymatic activity in a particular cleaning composition. Such effective amounts are readily ascertained by one of skill in the art and are based on many factors, such as the particular protease being used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, tablet, bar) composition is desired.

[0046] The term "cleaning adjunct material" refers to any liquid, solid, or gaseous material contained in a cleaning composition other than one or more subtilisin variants or recombinant polypeptides or active fragments thereof described herein. In some embodiments, the cleaning compositions of the present disclosure include one or more cleaning adjunct materials. Each cleaning adjunct material is typically selected depending on the particular type and form of the cleaning composition (e.g., liquid, granular, powder, bar, paste, spray, tablet, gel, foam, or other composition). Each cleaning adjunct material is preferably compatible with the protease enzyme used in the composition.

[0047] Cleaning compositions and cleaning formulations include any composition suitable for cleaning, bleaching, disinfecting and / or sanitizing any object, item, and / or surface. Such compositions and formulations include, but are not limited to, for example, liquid and / or solid compositions, such as cleaning or detergent compositions (e.g., liquid, tablet, gel, bar, granular, and / or solid laundry cleaning or detergent compositions, and tightly woven fabric detergent compositions; hard surface cleaning compositions and formulations, such as glass, wood, ceramic, and metal countertops and windows; carpet cleaners; oven cleaners; fabric deodorants; fabric softeners; and textiles, laundry enhancing cleaning or detergent compositions, laundry additive cleaning compositions, and laundry pre-spotter cleaning compositions; dishwashing compositions, including hand wash or manual dishwashing compositions (e.g., "hand wash" or "manual" dishwashing detergents), and automatic dishwashing compositions (e.g., automatic dishwashing detergents). Single unit dosage forms, such as pills, tablets, gelcaps, or other single unit dosage forms, such as pre-measured powders or liquids, are also used in the present invention.

[0048] A cleaning composition or cleaning formulation, as used herein, unless otherwise indicated, refers to a granular or powdered all-purpose or heavy-duty detergent, in particular a cleaning detergent; a liquid, granular, gel, solid, tablet, paste or unit dose all-purpose detergent, in particular a so-called heavy-duty liquid (HDL) type detergent or heavy-duty powder (HDD) type detergent; a liquid detergent for tightly woven fabrics; a hand washing or manual dishwashing detergent, for example a high foaming detergent; a household or commercial hand washing or manual dishwashing detergent, an automatic dishwashing detergent, or Dish or tableware detergents (including a variety of tablet, powder, solid, granular, liquid, gel and rinse aid types); liquid cleaners and disinfectants for humans and other animals, such as antibacterial hand wash types, wash bars, mouthwashes, denture cleaners, car cleaners, carpet cleaners, bathroom cleaners, hair shampoos and / or hair rinses; shower gels, foam baths and metal cleaners; and cleaning aids such as bleach additives and "stain sticks" or pre-treats. In some embodiments, the granular compositions are in "compact" form, and in some embodiments, the liquid compositions are in "concentrated" form.

[0049] As used herein, "fabric cleaning compositions" include hand and machine laundry detergent compositions, including laundry additive compositions, as well as compositions suitable for use in soaking and / or pre-treating stained fabrics (e.g., cloth, linen, and other textile materials).

[0050] As used herein, "non-fabric cleaning compositions" include, but are not limited to, non-textile (i.e., non-fabric) surface cleaning compositions, including, for example, hand or manual or automatic dishwashing detergent compositions, oral cleaning compositions, denture cleaning compositions, contact lens cleaning compositions, wound debridement compositions, and personal cleaning compositions.

[0051] As used herein, the term "detergent composition" or "detergent formulation" is used in reference to a composition intended for use in a cleaning medium to clean soiled or stained objects, including certain fabric and / or non-fabric objects or articles. In some embodiments, the detergent of the present disclosure comprises one or more subtilisin variants described herein, and further comprises one or more surfactants, transferases, hydrolases, oxidoreductases, builders (e.g., builder salts), bleaching agents, bleach activators, bluing agents, fluorescent dyes, anti-caking agents, masking agents, enzyme activators, antioxidants, and / or solubilizers. In some cases, the builder salts are mixtures of silicates and phosphates, preferably with more silicates (e.g., sodium metasilicate) than phosphates (e.g., sodium tripolyphosphate). Some embodiments relate to cleaning or detergent compositions that do not include any phosphates (e.g., phosphates or phosphate builders).

[0052] As used herein, the term "bleaching" refers to the treatment of a material (e.g., fabric, laundry, pulp, etc.) or surface for a sufficient period of time and / or under appropriate pH and / or temperature conditions to lighten (i.e., whiten) and / or clean the material. Examples of chemicals suitable for bleaching include, but are not limited to, ClO2, H2O2, peracid, NO2, etc.

[0053] As used herein, the "cleaning performance" of a protease (e.g., one or more subtilisin variants described herein, or a recombinant polypeptide or active fragment thereof) refers to the cleaning contribution of one or more subtilisin variants described herein that add additional cleaning performance to a detergent compared to a detergent that does not have one or more subtilisin variants described herein added to the composition. Cleaning performance is compared under relevant cleaning conditions. In some test systems, other relevant factors such as detergent composition, foam concentration, water hardness, cleaner, time, pH and / or temperature can be adjusted in such a way as to mimic conditions typical of household applications in a particular market segment (e.g., hand washing or manual dish washing, automatic dish washing, dish washing, tableware washing, fabric washing, etc.).

[0054] The term "relevant washing conditions" is used herein to indicate the conditions of actual household use in the manual dishwashing, automatic dishwashing or laundry detergent market segments, in particular washing temperature, time, washer, suds concentration, detergent type and water hardness.

[0055] As used herein, the term "sanitization" refers to the removal of contaminants from a surface and the inhibition or killing of microorganisms on the surface of an article.

[0056] The "compact" form of the cleaning compositions herein is best reflected by their density and, in terms of composition, by the amount of inorganic filler salt. Inorganic filler salts are conventional components of detergent compositions in powder form. In conventional detergent compositions, the filler salt is present in substantial amounts, typically about 17 to about 35% by weight of the total composition. In contrast, in compact compositions, the filler salt is present in an amount not exceeding about 15% by weight of the total composition. In some embodiments, the filler salt is present in an amount not exceeding about 10% by weight of the composition, more preferably not exceeding about 5% by weight. In some embodiments, the inorganic filler salt is selected from sulfates and chlorides of alkali and alkaline earth metals. In some embodiments, the filler salt is sodium sulfate.

[0057] Disclosed herein is one or more subtilisin variants useful for cleaning applications and cleaning methods, as well as various industrial applications. Disclosed herein is one or more isolated, recombinant, substantially pure, or non-naturally occurring subtilisin variants. In some embodiments, one or more subtilisin variants described herein can be incorporated into cleaning compositions useful for cleaning applications and methods for cleaning articles or surfaces in need of cleaning.

[0058] In one embodiment, (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, 143, 146, 147, 151, 155, 165, 183, 184, 187, 193, 202, 203, 210, 217, 2 34, 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) one or more subtilisin variants comprising two, three or four or more mutations relative to SEQ ID NO:2 at positions selected from 3, 68, 77, 86, 99, 115, 123, 127, 128, 165, 184, 202, 210, 217 and 258, with the proviso that one or more of said two, three or four or more mutations is a non-naturally occurring amino acid, said variants having 85% or greater amino acid sequence identity to an AprL-Clade subtilisin enzyme comprising motif A and / or motif B, wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4, and 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2. Another embodiment relates to one or more subtilisin variants comprising an amino acid sequence comprising two, three or four or more mutations at positions corresponding to SEQ ID NO:2, said positions being selected from the group consisting of: (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, 100, 102, 104, 106, 108, 109, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 224, 26, 27, 28, 29, 30, 31, 35, 37, 40, 43, 45, 48, 52, 68, 71, 76, 77, 78, 86, 87, 95, 96, 98, 100, 102, 104, 106, 108, 109, 220, 221, 222, 224, 26, 27, 28, 29, 30, 31, 35, 37, 40, 43, 45, 48, 52, 68, 71, 76, 77, 78, 86, 87, 95, 96, 98, 100, 10 , 99, 100, 101, 102, 103, 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;(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, 1 15, 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, 21 7, 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, 1 93, 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 wherein the variants have at least 85% but less than 100% amino acid sequence identity to an AprL-Clade subtilisin enzyme comprising motif A and / or motif B, wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4, and 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2;

[0059] These are (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、120A、123I、125N、1 26Q, 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、193D、202V、203 E / N、210E / H / P、217S、234I / W、238R / T、239K / S / T、240N、242G、247W 50G、251E、255R、258E / H / P、259P、260Wおよび274A / F;(ii)1Q、3Q / V、9E、1 5I, 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、108H / K、114 N、115F、120A、123I、125N、126Q、127F / T、128P / S、129T、130S、143V、1 46A / S, 147L, 151G, 155A / E / G, 165A / E / Q / S, 183G, 184Q, 187P, 193D 02V、203E / N、210P、217S、234I / W、238R / T、239K / S / T、240N、242G、247 W, 250G, 255R, 258P, 259P, 260W, 274A;(iii)1Q, 3V, 15I, 35A, 68S; 71A, 77N, 86H, 87S, 95A, 99S, 115F, 123I, 127T, 128P / S, 147L, 155G 65Q / S, 184Q, 202V, 210P, 217S, 234W, 239S, 242G, 250G, 258P, 274A;or (iv) one or more subtilisin variants comprising an amino acid sequence comprising two, three or four or more mutations relative to SEQ ID NO:2 selected from 3V, 68S, 77N, 86H, 99S, 115F, 123I, 127T, 128P, 165Q, 184Q, 202V, 210P, 217S and 258P, with the proviso that one or more of said two, three or four or more mutations are non-naturally occurring amino acids, said variants having 85% or greater amino acid sequence identity to an AprL-Clade subtilisin enzyme comprising motif A and / or motif B, wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4, and 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2;

[0060] They are (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, N7 6. S / T77, I / T78, N / S86, V87, A / I91, L95, N96, S98, G99, S100, S / T102, Y1 03, 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, G 165, G / K / N / S183, N / Q184, S187, A / P / S193, K194, A / S / V202, G / S203, S / T 210, S211, N217, L234, P238, A / N / T239, A242, D / N / T247, S250, R / S251, N / Y255, D / S258, S259, F260, Q274;(ii)A / G1, T3, P9, K15, K22, A24, G / V2 8. I / V30, I35, A / S / T37, V45, A48, A68, V71, L74, N76, S / T77, I / T78, N / S8 6. 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 / S193, K194, A / S / V202, G / S203, S / T2 10, S211, N217, L234, P238, A / N / T239, A242, D / N / T247, S250, N / Y255, D / S258, S259, F260, Q274;(iii)A / G1, T3, K15, I35, A68, V71, S / T77, N / S8 6. V87, L95, G99, A / Q / T115, M123, G127, A / P / T128, V147, S155, G165, N18 4, A / S / V202, S / T210, N217, L234, A / N / T239, A242, S250, D / S258, Q274;or (iv) one or more subtilisin variants comprising an amino acid sequence comprising two, three or four or more amino acid substitutions selected from T3, A68, S / T77, N / S86, G99, A / Q / T115, M123, G127, A / P / T128, G165, N184, A / S / V202, S / T210, N217 and D / S258, with the proviso that one or more of said two, three or four or more substitutions are non-naturally occurring amino acids, said variants comprising having 85% or greater amino acid sequence identity to an AprL-Clade subtilisin enzyme comprising motif A and / or motif B, wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4, and 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2. Yet another embodiment is directed to (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, L1 25, G126, G127, A / P128, S129, G / V130, Q136, A143, A / I / V146, V147, A / S151, S155, N / S160, G165, K / S183, N184, S187, A / S193, K1 94, 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 / V146, V147, A / S151, S155, G165, K / S183, N184, S18 7, 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;(ii i) A / G1, T3, K15, I35, A68, V71, T77, N / S86, V87, L95, G99, Q / T115, M123, G127, A / P128, V147, S155, G165, N184, A / V202, S / T210, N217, L23 or (iv) T3, A68, T77, N / S86, G99, Q / T115, M123, G127, A / P128, G165, N184, A / V202, S / T210, N217 and D / S258, with the proviso that one or more of said two, three or four or more substitutions are non-naturally occurring amino acids. wherein the variants have 85% or greater amino acid sequence identity to an AprL-Clade subtilisin enzyme comprising motif A and / or motif B, wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4, and 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2;

[0061] They are (i)1Q, 3Q / V, 9E / T, 10M, 15I / V, 17H, 19E, 22Y, 24N, 25D 6R, 27R, 28A, 30T, 35A, 37T, 38G, 43A, 45I / R, 48I, 68S, 71A, 74G, 76K 7D / H / N / Q / S、78I、86H / N / R / S、87S / T、91G、95A / Q、96S、98H / K / R、99L / S 、100N / R、102R、103I、108H / K、111Q、114N、115F、120A、123I、125N、126 Q、127F / T、128P / S、129T、130S、136A / R、143V、146A / S、147L、151G、155 A / E / G、160C / R、165A / E / Q / S、183A / G、184Q、187P、193D、194E、202V、20 3E / N、210E / H / P、211N、217S、234I / W、238R / T、239K / S / T、242G、247W、2 50G、251E、255R、258E / H / P、259P、260Wおよび274A / F;(ii)1Q、3Q / V、9E、1 5I、22Y、24N、28A、30T、35A、37T、45I、48I、68S、71A、74G、76K、77D / H / N / S、78I、86H / N / R / S、87S / T、95A / Q、96S、98K、99L / S、100N、108H / K、114 N、115F、120A、123I、125N、126Q、127F / T、128P / S、129T、130S、143V、14 6A / S、147L、151G、155A / E / G、165A / E / Q / S、183G、184Q、187P、193D、194 E, 202V, 203E / N, 210P, 211N, 217S, 234I / W, 238R / T, 239K / S / T, 242G 47W, 250G, 255R, 258P, 259P, 260W, 274A;(iii)1Q, 3V, 15I, 35A, 68S 71A, 77N, 86H, 87S, 95A, 99S, 115F, 123I, 127T, 128P / S, 147L, 155G 65Q / S, 184Q, 202V, 210P, 217S, 234W, 239S, 242G, 250G, 258P, 274A;or (iv) one or more subtilisin variants comprising an amino acid sequence comprising two, three or four or more amino acid substitutions selected from 3V, 68S, 77N, 86H, 99S, 115F, 123I, 127T, 128P, 165Q, 184Q, 202V, 210P, 217S and 258P, with the proviso that one or more of said two, three or four or more substitutions are non-naturally occurring amino acids, said variants having 85% or greater amino acid sequence identity to an AprL-Clade subtilisin enzyme comprising motif A and / or motif B, wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4, and 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2;

[0062] Yet 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, with the proviso that one or more of said two, three or four or more substitutions are artificial substitutions, comprising: (i) 1Q, 3Q / V, 9E, 10M, 15I, 22 Y, 24N / Q, 26Q, 27R, 28A, 29S, 30T, 31I, 35A, 37T, 40E, 43A, 45I / Q / R, 48I / 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, 1 03I, 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, 1 65A / E / Q / S, 184E / Q, 187E / P, 193D / Q, 202V, 203E / N / Q, 204I, 210E / H / I / P, 211S, 216Q, 217S, 234 I / 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および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および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, wherein said variant is selected from motif A and / or has at least 85% but less than 100% amino acid sequence identity to an AprL-Clade subtilisin enzyme comprising motif B, wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4, and 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2;

[0063] They are (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, V8 7S / T、A / I91G、L95A / Q、N96S、S98H / K / R、G99L / S、S100N / R、S / T102R、Y103I、S 108H / 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 / S1 51G、S155A / E / G、N / S / Y160C / R、G165A / E / Q / S、G / K / N / S183A / G、N184Q、S187 P、A / P / S193D、K194E、A / S202V、G / S203E / N、S / T210E / H / P、S211N、N217S、L23 4I / W、P238R / T、A / N / T239K / S / T、A242G、D / N / T247W、S250G、R / S251E、N / Y25 5R、D / S258E / H / P、S259P、F260WおよびQ274A / F;(ii)A / G1Q、T3Q / V、P9E、K15I、K 22Y、A24N、G / V28A、I / V30T、I35A、A / S37T、V45I、A48I、A68S、V71A、L74G、N7 6K、S / T77D / H / N / S、T78I、N / S86H / 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、S129T、G / V130S、A / S143V、A / I / V146A / S、V147L、A / S151G、S 155A / E / G、G165A / E / Q / S、K / S183G、N184Q、S187P、A / P / S193D、K194E、A / S20 2V、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, A68 S, 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 / T115F, M123I, G127T, A / T128P, G165Q, N184Q, A / S202V, S / T210P, N21 and D / S258P, with the proviso that one or more of said two, three or four or more substitutions are non-naturally occurring amino acids, said variants having 85% or greater amino acid sequence identity to an AprL-Clade subtilisin enzyme comprising motif A and / or motif B, wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4, and 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2. Still yet further embodiments are directed to: (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 / S151 G、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、S2 59P、F260Wおよび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, T78 I、N / S86H / N / R / S、V87S / T、L95A / Q、N96S、S98K、G99L / S、S100N、S108H / K、T1 14N、Q / T115F、V120A、M123I、L125N、G126Q、G127F / T、A / P128P / S、S129T、G / V 130S、A143V、A / I / V146A / S、V147L、A / S151G、S155A / E / G、G165A / E / Q / S、K / S 183G, N184Q, S187P, A / S193D, K194E, A202V, G / S203E / N, S / T210P, S211N, N2 17S、L234I / W、P238R / T、N / T239K / S / T、A242G、N / T247W、S250G、N / Y255R、D / S258P、S259P、F260WおよびQ274A;(iii)A / G1Q、T3V、K15I、I35A、A68S、V71A、T7 7N、N / S86H、V87S、L95A、G99S、Q / T115F、M123I、G127T、A / P128P / S、V147L、S 155G, G165Q / S, N184Q, A202V, S / T210P, N217S, L234W, N / T239S, A242G, S250 G, D / S258P, Q274A, and (iv)T3V, A68S, T77N, N / S86H, G99S, Q / T115F, M123 I, G127T, A128P, G165Q, N184Q, A202V, S / T210P, N217S, D / S258P, etc.and wherein one or more subtilisin variants comprising an amino acid sequence comprising two, three or four or more amino acid substitutions, with the proviso that one or more of said two, three or four or more substitutions are non-naturally occurring amino acids, said variants having 85% or greater amino acid sequence identity to an AprL-Clade subtilisin enzyme comprising motif A and / or motif B, wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4, and 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2.

[0064] These are (i)A / G1Q, T3Q / V, P / S9E, L / Q10M, K1 5I、K22Y、A24N / Q、V26Q、K27R、G / V28A、A / G29S、I / V3 0T、I / L31I、I35A、A / S37T、P / S / T40E、N / K43A、S / V45I / Q / R、A48I / T / V、A / S52R、A68S、V71A、N76K、S / T77 D / 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 / T102 D / T、Y103I、A / G105N、S108H / K、E111Q、T114N、A / Q / S / T115F、G / N117S、D119Q、M123I、L125N、G126Q、G127 E / 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 / S19 3D / 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および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および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および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および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、V 87S, L95A / F, N96S, S98R, G99A / E / Q / S, S100N, G101A, S / T102D / T, A / G105N, S108H, E 111Q, 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 / T239 S、A242G / N、S243I、S250G、D / S258D / P、S259E / P、F260W、K / N / R264H / Q / S、およびQ274A;(vi)A1Q, T3V, P9E, L10M, A24Q, V26Q, A29S, V30T, L31I, I35A, A37T, P 40E、V45I / Q / R、A48T / V、A52R、A68S、V71A、T77E / N、T78I、S86H / R、V87 S、L95A / F、N96S、S98R、G99A / E / Q / S、S100N、G101A、S102D / T、G105N、S 108H, E111Q, T115F, G117S, D119Q, M123I, G127E / Q / S / T / V, A128P / R. S129H / R、G130S、Q136N、A143Q / V、V146A、V147L、A151G、S155G / N、S15 8D / N / Q、N160D / K、T161D / Q、G165A / E / Q、N184E / Q、S187E / P、A193Q、A2 02V、G203E / N / Q、V204I、T210I / P、N211S、L216Q、N217S、L234W、P238Q N239S, A242G / N, S243I, S250G, S258D / P, S259E / P, F260W, K264H / Q / SおよびQ274A;(vii)T3V、P / S9E、L / Q10M、I35A、A68S、S / T77N、T78I、N / S 86H、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、A2 42G、S250G、D / S258D / PおよびK / N / R264H;(viii)T3V、P9E、L10M、I35A、A 68S, 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, K264H; (ix)T3V, A68S, S / T77N, T78I, M123I, G12 7S, A / S128P, G165Q, N184Q, A / S202V, S / T210P, N217S, D / S258D / P;or (x) one or more subtilisin variants comprising an amino acid sequence comprising two, three or more amino acid substitutions selected from T3V, A68S, T77N, T78I, M123I, G127S, A128P, G165Q, N184Q, A202V, T210P, N217S and S258D / P, with the proviso that one or more of said two, three or more substitutions are artificial, said variants comprising motif A and / or motif B. wherein the variant has at least 85% and less than 100% amino acid sequence identity to an AprL-Clade subtilisin enzyme, including motif A selected from motif 1, motif 2, motif 3 and motif 4, and motif B 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 corresponding to the amino acid sequence of SEQ ID NO:2;

[0065] Further embodiments include (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,A6 8S-A202V,M123I-N184Q,A128P-A202V,T77N-M123I,T3V-A202V,N217S-S258P,A68S-M123I,N184Q-S258P,G165Q-S258P,M123 I-A128P,T77N-S258P,T3V-M123I,A128P-S258P,A202V-S258P,A68S-S258P,T3V-S258P,M123I-A202V,M123I-S258P,A68S-G1 27S,G127S-A128P,T77N-G127S,G127S-A202V,G127S-N184Q,G127S-N217S,G127S-G165Q,T3V-G127S,G127S-S258P,M123I-G1 27S,T77N-T210P,G165Q-T210P,T210P-N217S,A128P-T210P,A68S-T210P,N184Q-T210P,T210P-S258P,A202V-T210P,M123I-T 210P,T3V-T210P,G127S-T210P,G127T-N184Q,G127T-N217S,V87S-G165Q,M123I-Q136R,A68S-G127T,G127T-A128P,T77N-V87 S,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,<h2 style=";text-align:left;direction:ltr">I35A-T78I,I3,<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">5A-V147L, L10M-T210P, L10M-T78I, M123I-G165S, N184Q-G203E, N184Q-S187P, N43A-M123I, P9E-G127S, P9E-S258E, Q136R-S258P, T115F-N217S, T3 V-S86H,T77H-M123I,T77H-N184Q,T77H-N239S,T77H-Q136R,T77H-V87T, T77N-G99Q,T77N-S250G,T78I-G99S,T78I-V87S,V146S-G165S,V146S-N23 9S,V146S-T210P,V45R-S258E,V87S-A242G,V87S-N184Q,A128S-T210P,A 143V-A202V,A143V-G165Q,A143V-S258P,A1Q-N184Q,A1Q-S258P,A68S-G2 03E,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-N2 17S,S187E-N217S,S86H-A143V,S86H-Q274A,S86H-S250G,T114N-A128P, T114N-G165Q,T114N-N184Q,T114N-N217S,T115F-A143V,T115F-N184Q,T2 10P-N239S,T210P-S250G,T3V-A128R,T3V-G99Q,T77H-G165Q,T77N-S187 E,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,T, 77N-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,A, 128P-T210P-S258P,A202V-T210P-N217S,A68S-T210P-S258P,N184Q-A202V-T210P,T210P-N217S-S258P,T3V-G165Q-T210P,T77N-A 202V-T210P,A202V-T210P-S258P,G165Q-A202V-T210P,T3V-N184Q-T210P,A128P-A202V-T210P,A68S-A202V-T210P,N184Q-T210P-S 258P,T3V-A202V-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,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-N217S,M123I-A202V-T210P,M123I-T210P-S258P,A68S-G127T-N184Q,A68S-G127T-N217S,G127T-A128P-N184Q,G127 T-A128P-N217S,T77N-V87S-G165Q,A68S-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,T, 3V-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-N217 S,I35A-V87S,L10M-M123I,M123I-G127T,N184Q-A242G,N217S-A242G,A128 R-G165Q,A128R-S258D,A68S-A128R,A68S-G99S,G127S-S258D,G165E-A20 2V,G99S-A128P,G99S-G127S,G99S-G165Q,I35A-G165Q,I35A-T77N,L10M-A 128P,L10M-A68S,L10M-N184Q,L10M-N217S,L10M-S258P,L10M-T77N,M123 I-A242G,P9E-A128P,P9E-A202V,P9E-A68S,P9E-G165Q,P9E-N184Q,P9E-N2 17S,P9E-S258P,S86H-T115F,T210P-A242G,T3V-G165E,T3V-G99S,T3V-L1 0M,T3V-P9E,T77N-A128R,T77N-G165E,T77N-G99S,T77N-V87S,T78I-A242 G,V87S-A128P,V87S-G165Q,V87S-M123I,V87S-N217S,A68S-G101A,G101A -A128P,G101A-G165Q,G101A-S258P,G165E-S258P,G99S-S258P,I35A-M123 I,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-A 202V,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,A128, P-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,<h2 style=";text-align:left;direction:ltr">T3V-G127S-N184Q,T3,<h2 style=";text-align:left;direction:ltr"> V-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-A128, P,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-T78 I-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,T77N-T78I-T210P,T78I-A128P -T210P,T78I-A202V-S258P,T78I-G127S-T210P,T78I-G165Q-T210P,T78I-M123I-A128P,T78I-M123I-G127S, One or more subtilisin variants comprising an amino acid sequence comprising a set of one or more amino acid substitutions selected from T78I-M123I-N217S, and T78I-N217S-S258P, with the proviso that one or more of said two, three or more substitutions are artificial, said variants having at least 85% but less than 100% amino acid sequence identity to an AprL-Clade subtilisin enzyme comprising motif A and / or motif B, wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4, and 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2.

[0066] Another embodiment is 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-Q13 6R-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-M12 3I-Q136R-G165Q;P9E-V45R-M123I-Q136R-S258E;V45R-M123I-Q136R-G16 5Q-S258E;Y103I-G127T-V146S-G165S-N239S;T77H-V87T-M123I-Q136R-N 184Q-N239S;V26R-V45R-S98R-E111Q-M123I-Q136R;T77H-V87T-M123I-Q1 36R-G165Q-N184Q-N239S;T77S-V87S-M123I-Q136R-G165Q-S187P-S258P;T 77S-V87S-M123I-Q136R-N160C-S187P-S258P;T77H-V87T-M123I-Q136R-N 184Q-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; 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style=";text-align:left;direction:ltr">T3V-A68S-T77N-M123I-A128P-G16;<h2 style=";text-align:left;direction:ltr"> 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8P-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-F 260W;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 and V45R-S98R, with the proviso that one or more of said two, three or more substitutions are artificial, and said variants are selected from the group consisting of an Aproxidase containing motif A and / or motif B, having at least 85% but less than 100% amino acid sequence identity to the L-Clade subtilisin enzyme, motif A being selected from motif 1, motif 2, motif 3 and motif 4, motif B being 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2.

[0067] Another embodiment is 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-Q13 6R-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-M12 3I-Q136R-G165Q;P9E-V45R-M123I-Q136R-S258E;V45R-M123I-Q136R-G16 5Q-S258E;Y103I-G127T-V146S-G165S-N239S;T77H-V87T-M123I-Q136R-N 184Q-N239S;V26R-V45R-S98R-E111Q-M123I-Q136R;T77H-V87T-M123I-Q1 36R-G165Q-N184Q-N239S;T77S-V87S-M123I-Q136R-G165Q-S187P-S258P;T 77S-V87S-M123I-Q136R-N160C-S187P-S258P;T77H-V87T-M123I-Q136R-N 184Q-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-S; 258P;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;<h2 style=";text-align:left;direction:ltr">T77N-M123I-N184Q-N217S;T77N-M1;<h2 style=";text-align:left;direction:ltr"> 23I-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;<h2 style=";text-align:left;direction:ltr">I35A-A68S-T77N-A128P-;<h2 style=";text-align:left;direction:ltr"> V147L-G165Q-N217S;I35A-A68S-T77N-A128P-S155G-G165Q-A242G;I35A-A68S-T77N-A128P-V147L-G165Q-A242G;A68S-T77N-V87S-A128P-S155G-G165Q-A242G;A068S-T077N-N086S-G165Q-K194E-S211N-N217S;A068S-T077N-N086S-V087S-S155G-G165Q-K194E-S211N-N217S;A068S-T077N-N086S-G165Q-K194E-S211N;A068S-T077N-N086S-V087S-S155G-G165Q-K194E-S211N;I035A-A068S-T077N-N086S-V087S-V147L-G165Q-K194E-S211N-N217S;A068S-T077N-N086S-V087S-S155G-G165Q-K194E-S211N-N217S-A242G;A068S-T077N-N086S-V087S-V147L-S155E-G165Q-K194E-S211N;A068S-T077N-N086S-V087S-S155E-G165Q-K194E-S211N-N217S;A068S-T077N-V087S-S155G-G165Q-K194E-S211N-N217S-A242G;I035A-A068S-T077N-N086S-V147L-G165Q-K194E-S211N-N217S;I035A-A068S-T077N-N086S-S155G-G165Q-K194E-S211N-A242G;0I35A-A068S-T077N-N086S-V147L-G165Q-K194E-S211N-A242G;and A068S-T077N-N086S-V087S-S155G-G165Q-K194E-S211N-A242G, with the proviso that one or more of said two, three or four or more mutations are non-naturally occurring amino acids, said variants having at least 85% or more amino acid sequence identity to an AprL-Clade subtilisin enzyme comprising motif A and / or motif B, wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4, and 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2;

[0068] Still further embodiments include: 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-G20 3Q-N217S;T3V-A68S-T77N-M123I-A128P-G165Q-N184Q-G203Q-N217S;A6 8S-T77N-A128P-G165Q-N184Q-A202V-N217S-S258P;T3V-A68S-T77N-M12 3I-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-S258 P;A68S-T77N-A128P-S129H-G165Q-N184Q-A202V-N217S;T3V-A68S-T77N -M123I-A128P-S129H-G165Q-N184Q-A202V-N217S;S86H-T115F-S129H-A 143V-G165Q-N184Q-A202V-N217S-S258P;T3V-S86H-T115F-M123I-S129H -A143V-G165Q-A202V-N217S-S258P;A68S-T77N-A128P-S155N-G165Q-N1 84Q-A202V-N217S;T3V-A68S-T77N-M123I-A128P-S155N-G165Q-N184Q-A 202V-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;T; 3V-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-L3 1I-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;A6 8S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P;A68S-T77N-G1 27S-A128P-G165Q-N184Q-A202V-T210P-N217S-S258P;A68S-T77N-G127T-A 128P-G165Q-N184Q-A202V-N217S-S258P;T3V-A68S-T77N-T78I-M123I-G1 27S-A128P-G165Q-N184Q-A202V-N217S-S258D;A68S-T77N-T78I-M123I-G1 27S-A128P-G165Q-N184Q-A202V-N217S-S258D;T3V-A68S-T77N-T78I-G12 7S-A128P-G165Q-N184Q-A202V-N217S-S258P;A68S-T77N-T78I-G127S-A12 8P-G165Q-N184Q-A202V-N217S-S258P;T3V-A68S-T77N-G127S-A128P-G16 5Q-N184Q-A202V-N217S-S258D;A68S-T77N-G127S-A128P-G165Q-N184Q-A2 02V-N217S-S258D;T3V-A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A20 2V-N217S-S258D;A68S-T77N-T78I-G127S-A128P-G165Q-N184Q-A202V-N21 7S-S258D;T3V-A68S-T77N-S108H-G127S-A128P-G165Q-N184Q-A202V-N217 S-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-N2;17S-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-G; 165Q-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-A68S-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;and V45R-S98R, with the proviso that one or more of said two, three or more substitutions are artificial, and said variants comprise one or more subtilisin variants comprising an amino acid sequence comprising a set of one or more amino acid substitutions selected from motif A and / or motif B. and wherein motif A is selected from motif 1, motif 2, motif 3 and motif 4, and 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 variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2;

[0069] In further embodiments, one or more of the subtilisin variants described herein are 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, 220E, 220F, 220G, 220H, 220I ... , 217D, 221S, 231V, 234L, 235H, 244R, 251K, and 273A, and / or one or more insertions selected from a D inserted at position 36, an E inserted at position 96, and a D inserted between positions 98 and 99, wherein the amino acid positions of the variants are numbered corresponding to the amino acid sequence of SEQ ID NO:2.

[0070] In one 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 to an AprL-Clade subtilisin enzyme comprising motif A, motif B or a combination thereof. In another embodiment, 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 to an AprL-Clade subtilisin enzyme 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 to an AprL-Clade subtilisin enzyme comprising motif A and motif B. In yet another embodiment, 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 to an AprL-Clade subtilisin enzyme comprising motif A and motif B. The AprL-clade subtilisin enzymes are described in more detail in PCT Application No. PCT / US16 / 32514, filed May 13, 2016. In some embodiments, the AprL-Clade subtilisin enzyme is selected from one of the enzymes shown in Figure 1. In another embodiment, the AprL-Clade subtilisin enzyme comprises the amino acid sequence of SEQ ID NOs: 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 yet another embodiment, the AprL-Clade subtilisin enzyme comprises the amino acid sequence of SEQ ID NO:2, 9, 189, 198, 202, 203 or 205.In yet another embodiment, the AprL-Clade subtilisin enzyme comprises the amino acid sequence of SEQ ID NO:2 or 9.

[0071] 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 yet further 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 yet other embodiments, the AprL-clade enzyme comprises motif 1 between Asp32 and His63, where the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2. In still yet other embodiments, the AprL-clade enzyme comprises motif 6 or motif 7 between Lys236 and Arg246. 2. In yet still further embodiments, the AprL-clade enzyme comprises (i) motif 1 between Asp32 and His63, and (ii) motif 6 between Lys236 and Arg246, wherein the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2. In yet other embodiments, one or more subtilisin variants described herein comprise motif 1 between Asp32 and His63, wherein the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2. In yet still further embodiments, one or more subtilisin variants described herein comprise motif 6 or motif 7 between Lys236 and Arg246, wherein the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2. In yet another embodiment, one or more subtilisin variants described herein comprise (i) motif 1 between Asp32 and His63, and (ii) motif 6 or motif 7 between Lys236 and Arg246, wherein the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2.However, the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2.

[0072] 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 ("motif 2"), with the amino acid positions numbered corresponding to the amino acid sequence of SEQ ID NO: 2. In further embodiments, the AprL-clade enzyme comprises a FVX1GEX2X3YNT (SEQ ID NO: 168) motif ("motif 3"), with the amino acid positions numbered corresponding to the amino acid sequence of SEQ ID NO: 2. In yet further embodiments, the AprL-clade enzyme comprises a F50V51X152G53E54X355Y56N57T58 (SEQ ID NO: 169) motif ("motif 4"), with the amino acid positions numbered corresponding to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the AprL-clade enzyme comprises a loop region between residues 52-60, wherein the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2.

[0073] In other embodiments, the AprL-clade enzyme comprises a LSX4S (SEQ ID NO: 170) motif ("motif 5"), where X4 is A or G. In yet 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 ("motif 8"), where the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 2. In further embodiments, the AprL-clade enzyme comprises a L240S241G242S243 (SEQ ID NO: 174) motif ("motif 9"), where the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 2. In yet further embodiments, the AprL-clade enzyme comprises a KXXXLSX4SQX5R (SEQ ID NO: 175) motif ("motif 10"), where X is any amino acid, X4 is A or G, and X5 is I or V. In yet further embodiments, the AprL-clade enzyme comprises a KXXXLSASQX5R (SEQ ID NO: 176) motif ("motif 11"), where X is any amino acid, and X5 is I or V. In yet further embodiments, the AprL-clade enzyme comprises a KXXXLSGSQX5R (SEQ ID NO: 177) motif ("motif 12"), where X is any amino acid, and X5 is I or V. In yet further embodiments, the AprL-clade enzyme comprises a K236XXXLSASQX5R246 (SEQ ID NO: 178) motif ("motif 13"), where X is any amino acid, X5 is I or V, and the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 2. In still yet further embodiments, the AprL-clade enzyme comprises a K236XXXLSGSQX5R246 (SEQ ID NO: 179) motif ("motif 14"), where X is any amino acid and X5 is I or V, and the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2.

[0074] 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 ("motif 2"), where the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 2. In further embodiments, one or more subtilisin variants described herein comprise a FVX1GEX2X3YNT (SEQ ID NO: 168) motif ("motif 3"), where X1 is A or S, X2 is none or any amino acid, and X3 is A or S. In yet further embodiments, one or more subtilisin variants described herein comprise a F50V51X152G53E54X355Y56N57T58 (SEQ ID NO: 169) motif ("motif 4"). wherein X1 is A or S and X3 is A or S, and wherein the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2. In some embodiments, one or more of the subtilisin variants described herein comprises a loop region between residues 52-60, and wherein the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2.

[0075] In other embodiments, one or more subtilisin variants described herein comprise a LSX4S (SEQ ID NO: 170) motif ("motif 5"), where X4 is A or G. In yet 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 ("motif 8"), where the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 2. In further embodiments, one or more subtilisin variants described herein comprise a L240S241G242S243 (SEQ ID NO: 174) motif ("motif 9"), where the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 2. In still further embodiments, one or more subtilisin variants described herein comprise a KXXXLSX4SQX5R (SEQ ID NO: 175) motif ("motif 10"), where X is any amino acid, X4 is A or G, and X5 is I or V. In still further embodiments, one or more subtilisin variants described herein comprise a KXXXLSASQX5R (SEQ ID NO: 176) motif ("motif 11"), where X is any amino acid, and X5 is I or V. In still yet further embodiments, one or more subtilisin variants described herein comprise a KXXXLSGSQX5R (SEQ ID NO: 177) motif ("motif 12"), where X is any amino acid, and X5 is I or V. In still further embodiments, one or more subtilisin variants described herein comprise a K236XXXLSASQX5R246 (SEQ ID NO: 178) motif ("motif 13"). wherein X is any amino acid, X5 is I or V, and the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2.In still yet further embodiments, one or more of the subtilisin variants described herein comprises a K236XXXLSGSQX5R246 (SEQ ID NO: 179) motif ("motif 14"), where X is any amino acid and X5 is I or V, and where the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2.

[0076] In another embodiment, one or more of the subtilisin variants described herein comprise two, three or four or more mutations as described herein relative to SEQ ID NO:2, with the proviso that (i) said mutation relative to SEQ ID NO:2 is not an insertion or deletion, (ii) if the amino acid sequence of said variant comprises a mutation relative to SEQ ID NO:2 at one or more of positions 37, 187, 193 and 258, said mutation is not a proline, (iii) if the amino acid sequence of said variant comprises a mutation relative to SEQ ID NO:2 at two or more of positions 26, 38, 234 and 250, said mutation is not 26R, 38S, 234R or 250E / N, and / or (iv) if the amino acid sequence of said variant comprises a mutation at position 128, said mutation relative to SEQ ID NO:2 is not at positions 130 or 193, with the proviso that the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2.

[0077] In yet another embodiment, one or more of the subtilisin variants described herein comprise two, three or four or more mutations as described herein at a position corresponding to SEQ ID NO:2, including (i) that the mutation at a position corresponding to SEQ ID NO:2 is not an insertion or deletion; (ii) that the amino acid sequence of the variant comprises a mutation at one or more positions corresponding to SEQ ID NO:2 selected from positions 37, 187, 193 and 258, which mutation is not a proline; (iii) that the amino acid sequence of the variant comprises a mutation at one or more positions selected from positions 26, 38, 234 and 250, which mutation is not a proline. (iv) if the amino acid sequence of the mutant contains a mutation at position 128, said mutation at a position corresponding to SEQ ID NO:2 is not at position 130 or 193; and / or (v) the mutant does not consist of the amino acid sequence set forth as SEQ ID NO: 5 in WO2016074925 or SEQ ID NO: 110 in JP2013-00714A.

[0078] In yet another embodiment, one or more of the subtilisin variants described herein comprise two, three or four or more mutations as described herein, with the proviso that (i) if the amino acid sequence of said variant comprises a substitution at one or more of positions 37, 187, 193 and 258, said substitution is not a proline, (ii) if the amino acid sequence of said variant comprises a substitution at two or more of positions 26, 38, 234 and 250, said substitution is not 26R, 38S, 234R or 250E / N, and / or (iii) if the amino acid sequence of said variant comprises a substitution at position 128, said substitution is not at positions 130 or 193, with the proviso that the amino acid positions are numbered corresponding to the amino acid sequence of SEQ ID NO:2.

[0079] In yet another embodiment, one or more of the subtilisin variants described herein comprise two, three or four or more mutations as described herein, with the proviso that (i) if the amino acid sequence of said variant comprises a substitution at one or more of positions 37, 187, 193 and 258, said substitution is not proline; (ii) if the amino acid sequence of said variant comprises a substitution at two or more of positions 26, 38, 234 and 250, said substitution is not 26R, 38S, 234R or 250E / N; (iii) if the amino acid sequence of said variant comprises a substitution at position 128, said substitution is not at position 130 or 193; and / or (iv) said variant does not consist of the amino acid sequence set forth as SEQ ID NO: 5 in WO2016074925 or SEQ ID NO: 110 in JP2013-00714A.

[0080] In one embodiment, one or more of the subtilisin 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 enzyme identified in FIG. In some embodiments, one or more subtilisin 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 SEQ ID NO:9. In other embodiments, one or more subtilisin variants described herein comprise an amino acid sequence that has 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 enzyme identified in Figure 1 or (ii) SEQ ID NO: 2 or 9. In another embodiment, one or more subtilisin variants described herein comprise an amino acid sequence that has at least 89% but less than 100% amino acid sequence identity to the amino acid sequence of the AprL-Clade subtilisin enzyme identified in Figure 1. In another embodiment, one or more subtilisin variants described herein comprise an amino acid sequence that has 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 that has at least 90% but less than 100% amino acid sequence identity with the amino acid sequence of the AprL-Clade subtilisin enzyme identified in Figure 1. In some embodiments, one or more subtilisin variants described herein comprise an amino acid sequence that has at least 90% but less than 100% amino acid sequence identity with 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 that has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% less than 100% amino acid sequence identity with the amino acid sequence of the AprL-Clade subtilisin enzyme identified in Figure 1. In still yet other embodiments, one or more subtilisin variants described herein comprise an amino acid sequence that has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% less than 100% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 2 or 9. In still other embodiments, one or more of the subtilisin variants described herein are isolated, recombinant, substantially pure, or non-naturally occurring subtilisins having subtilisin activity or casein hydrolyzing activity (e.g., dimethylcasein hydrolyzing activity).

[0081] In one embodiment, one or more of the subtilisin variants described herein have enzymatic activity (e.g., protease activity) and are therefore useful in cleaning applications, including, but not limited to, methods of cleaning dishware, tableware, fabrics, and items having hard surfaces (hard surfaces such as tables, table tops, walls, furniture, floors, ceilings, etc.). Examples of cleaning compositions comprising one or more of the subtilisin variants described herein are described below. The enzymatic activity (e.g., protease enzymatic activity) of one or more of the subtilisin variants described herein can be readily measured by procedures well known to those of skill in the art. The examples provided below describe methods of evaluating enzymatic activity and cleaning performance. The performance of the polypeptide enzymes of the invention in removing stains (e.g., protein stains such as blood / milk / ink, dye / milk / ink or egg yolk), cleaning hard surfaces, or cleaning laundry, dishware or tableware products can be readily determined using procedures well known in the art and / or using procedures described in the Examples.

[0082] In some embodiments, one or more subtilisin variants described herein have one or more improved properties compared to a reference subtilisin, the improved properties being selected from improved protease activity, improved cleaning performance in a detergent, and improved thermal stability in a detergent and improved degraded laundry cleanability in a detergent, optionally with the proviso that the detergent is a boron-free detergent. In other embodiments, one or more subtilisin variants described herein have one or more improved properties compared to a reference subtilisin, the improved properties being selected from improved protease activity, improved cleaning performance in a detergent, improved thermal stability in a detergent, and improved degraded laundry cleanability in a detergent, optionally with the proviso that the detergent is a boron-free detergent, and the reference subtilisin comprises the amino acid sequence of SEQ ID NO: 2, 9 or 63. In yet other embodiments, one or more subtilisin variants described herein have one or more improved properties compared to a reference subtilisin, the improved properties being selected from improved protease activity, improved cleaning performance in a detergent, improved thermal stability in a detergent, and improved degraded laundry cleanability in a detergent, optionally a boron-free detergent, and wherein the reference subtilisin comprises the amino acid sequence of SEQ ID NO: 2 or 9. In one embodiment, one or more subtilisin variants described herein are more stable through longer wash times compared to the reference subtilisin. In another embodiment, one or more subtilisin variants described herein are more stable through shorter, lower temperature wash cycles or longer, higher temperature wash cycles compared to the reference subtilisin. In still yet further embodiments, the one or more improved properties are (i) improved protease activity, wherein the variants have a PI>1 on N-suc-AAPF-pNA or dimethylcasein substrates; (ii) improved cleaning performance in detergent, wherein the variants have a PI>1 in BMI, POM, egg, and / or egg yolk cleaning; (iii) improved thermal stability in detergent, wherein the variants have a stability PI>1; and / or (iv) improved degraded laundry cleanability in detergent, wherein the variants have a degraded laundry cleanability PI>1.In yet further embodiments, the one or more improved properties are (i) improved protease activity, wherein the variants have a PI>1 on N-suc-AAPF-pNA or dimethylcasein substrates, (ii) improved cleaning performance in detergent, wherein the variants have a PI>1 in washing BMI, POM, egg, and / or egg yolk, and (iii) improved thermal stability in detergent, wherein the variants have a stability PI>1 or have a % residual activity>5%, 10%, 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 higher. 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more; and / or (iv) improved degraded laundry cleanliness in detergents, wherein the variants have (a) a degraded laundry cleanliness 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%, 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, optionally in a boron-free composition. In still further embodiments, one or more of the subtilisin variants described herein have improved protease activity, wherein the variants have a PI > 1 on N-suc-AAPF-pNA or dimethylcasein substrate. In still yet further embodiments, one or more subtilisin variants described herein have improved cleaning performance in a detergent, said variants having a PI>1 for BMI, POM and / or egg yolk washing, optionally with the proviso that the detergent is a boron-free composition. In another embodiment, one or more subtilisin variants described herein have improved thermal stability in a detergent, said variants having a stability PI>1.provided that the detergent is optionally a boron-free composition. In yet another embodiment, one or more subtilisin variants described herein have improved thermal stability in detergents, wherein the variants have 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 higher, provided that the detergent is optionally a boron-free composition. In another embodiment, one or more of the subtilisin variants described herein have improved degraded laundry cleanliness in detergent, wherein the variants have (a) a degraded laundry cleanliness 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. provided that the detergent is optionally a boron-free composition. In another embodiment, one or more subtilisin variants described herein have improved degraded laundry cleanliness in a detergent, said variants having a degraded laundry cleanliness PI>1, provided that the detergent is optionally a boron-free composition. In another embodiment, one or more subtilisin variants described herein have improved protease activity, said variants having a PI>1 on an N-suc-AAPF-pNA or dimethylcasein substrate, said PI being measured by the protease activity assay of Example 1.In further embodiments, one or more subtilisin variants described herein have improved detergent cleaning performance, said variants having a PI>1 in BMI, POM, egg, and / or egg yolk cleaning, said PI being measured according to the cleaning performance in laundry (HDL or HDD) detergent and ADW detergent assays of Example 1. In still further embodiments, one or more subtilisin variants described herein have improved detergent thermal stability, said variants having a stability PI>1, said PI being measured according to the general sample set-up for stability assays of Example 1. In still yet further embodiments, one or more of the subtilisin variants described herein have improved thermal stability in detergent, wherein the variants have 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 higher, wherein the residual activity is measured according to the general sample set up for stability assay in Example 1. In still further embodiments, one or more of the subtilisin variants described herein have improved degraded laundry cleanliness in detergent, wherein the variants have a degraded laundry cleanliness PI> 1, wherein the PI is measured according to the degraded laundry cleanliness assay in Example 1. In still yet further embodiments, one or more of the subtilisin variants described herein have improved degraded laundry cleaning properties in detergent, wherein said variants have 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 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, wherein said residual cleaning activity is measured according to the degraded laundry cleaning assay of Example 1.

[0083] In some embodiments, one or more subtilisin variants described herein exhibit cleaning performance in cleaning compositions. Cleaning compositions often contain ingredients that are detrimental to enzyme stability and performance, and cleaning compositions are harsh environments for enzymes, e.g., serine proteases, to retain function. Thus, it is not trivial to put an enzyme into a cleaning composition and expect enzyme function (e.g., serine protease activity as indicated by cleaning performance). In some embodiments, one or more subtilisin variants described herein exhibit cleaning performance in an automatic dishwasher (ADW) detergent composition. In some embodiments, cleaning performance in an automatic dishwasher (ADW) detergent composition includes cleaning egg yolk stains. In some embodiments, one or more subtilisin variants described herein exhibit cleaning performance in a laundry detergent composition. In some embodiments, cleaning performance in a laundry detergent composition includes blood / milk / ink, egg, egg yolk and / or POM stains. Within each of the cleaning compositions, one or more subtilisin variants described herein exhibit cleaning performance with or without a bleach component. In still yet further embodiments, one or more ADW or laundry detergent compositions described herein comprise one or more subtilisin variants described herein, which variants are stable in the presence of one or more adjunct ingredients and / or one or more additional enzymes, and / or which variants are stable against autoproteolysis.

[0084] Various changes can be made to one or more subtilisin variants described herein, such as one or more conservative or non-conservative amino acid insertions, deletions and / or substitutions (including cases where such changes do not substantially alter the enzymatic activity of the variant). Similarly, the nucleic acids of the invention can be made to one or more substitutions of one or more nucleotides in one or more codons, such that a particular codon encodes the same or different amino acid, resulting in a silent mutation (e.g., where a mutation in a nucleotide does not change the encoded amino acid) or a non-silent mutation, such as 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 truncations of one or more nucleic acids (or codons) in the sequence. Many such changes in the nucleic acid sequence will not substantially alter the enzymatic activity of the resulting encoded polypeptide enzyme compared to the polypeptide enzyme encoded by the original nucleic acid sequence. The nucleic acid sequences described herein can also be modified to include one or more codons that provide optimal expression in an expression system (e.g., a bacterial expression system), while, if desired, the one or more codons still encode the same amino acid.

[0085] Described herein are one or more isolated, non-naturally occurring, or recombinant polynucleotides comprising a nucleic acid sequence encoding one or more subtilisin variants described herein, or a recombinant polypeptide or active fragment thereof. One or more nucleic acids described herein are useful for recombinantly producing (e.g., expressing) one or more subtilisin variants described herein, typically by expression of a plasmid expression vector comprising a sequence encoding one or more subtilisin variants described herein, or fragments thereof. One embodiment provides a nucleic acid encoding one or more subtilisin variants described herein, which variants are in a mature form having proteolytic activity. In some embodiments, one or more subtilisin variants described herein are recombinantly expressed with a homologous propeptide sequence. In other embodiments, one or more subtilisin variants described herein are recombinantly expressed with a heterologous propeptide sequence (e.g., a GG36 propeptide sequence).

[0086] One or more of the nucleic acid sequences described herein can be produced using any suitable synthesis, manipulation, and / or isolation technique, or a combination thereof. For example, one or more of the polynucleotides described herein can be produced by standard nucleic acid synthesis techniques, such as solid-phase synthesis techniques well known to those skilled in the art. In such techniques, fragments of up to 50 or more nucleic acid bases are typically synthesized and then linked (e.g., by enzymatic or chemical ligation) to form the substantially desired contiguous nucleic acid sequence. The synthesis of one or more of the polynucleotides described herein can also be facilitated by suitable methods known in the art. Such methods include, but are not limited to, the classical phosphoramidite method (see, e.g., Beaucage et al. Tetrahedron Letters 22:1859-69 (1981)) or the method described by Matthes et al, EMBO J.3:801-805 (1984), which is commonly practiced in automated synthesis. One or more of the polynucleotides described herein can also be produced by using an automated DNA synthesizer. Custom nucleic acids can be ordered from a variety of commercial sources (e.g., Midland Certified Reagent Company, Great American Gene Company, Operon Technologies Inc., and DNA2.0). Other techniques for synthesizing nucleic acids and related principles are described, for example, in Itakura et al., Ann. Rev. Biochem. 53:323 (1984) and Itakura et al., Science 198:1056 (1984).

[0087] Recombinant DNA techniques useful for modifying nucleic acids, such as restriction endonuclease digestion, ligation, reverse transcription and cDNA production, and polymerase chain reaction (e.g., PCR), are well known in the art. One or more polynucleotides described herein can also be obtained by screening a cDNA library using one or more hybridizable oligonucleotide probes, or by using PCR-amplified polynucleotides encoding one or more AprL-clade variants or recombinant polypeptides or active fragments thereof described herein. Procedures for screening and isolating cDNA clones, and PCR amplification procedures are well known to those skilled in the art and are described in standard texts known to those skilled in the art. One or more polynucleotides described herein can be obtained by modifying a naturally occurring polynucleotide backbone (e.g., encoding one or more subtilisin variants or reference subtilisins described herein), for example, by known mutagenesis procedures (e.g., site-directed mutagenesis, site-saturation mutagenesis, and in vitro recombination). A variety of methods suitable for generating modified polynucleotides as described herein that encode one or more subtilisin variants as described herein are known in the art, including, but not limited to, site-saturation mutagenesis, systematic mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis and directed evolution, as well as a variety of other recombinant methods.

[0088] Further embodiments relate 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; as well as compositions comprising one or more such vectors, nucleic acids, expression vectors, expression cassettes, DNA constructs, cells, cell cultures, or any combination or mixture thereof.

[0089] Some embodiments relate to one or more recombinant cells comprising one or more vectors (e.g., expression vectors or DNA constructs) described herein, which vectors comprise one or more nucleic acids or polynucleotides described herein. Some such recombinant cells are transformed or transfected with such one or more vectors, although other methods are available and known in the art. Such cells are commonly referred to as host cells. Some such cells include bacterial cells, for example, cells of species of the genus Bacillus, such as, but not limited to, B. subtilis cells. Other embodiments relate to recombinant cells (e.g., recombinant host cells) comprising one or more subtilisins described herein.

[0090] In some embodiments, one or more of the vectors described herein are expression vectors or expression cassettes that include one or more of the polynucleotide sequences described herein operably linked to one or more additional nucleic acid segments required for efficient gene expression (e.g., a promoter operably linked to one or more of the polynucleotide sequences described herein). The vectors may include transcription terminators and / or selection genes (e.g., antibiotic resistance genes), which allow for continuous cultural maintenance of plasmid-infected host cells by growth in antimicrobial-containing media.

[0091] Expression vectors may be derived from plasmid 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 replicative plasmids for B. subtilis include those shown 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, DC (1993), pp. 615-624 and p2JM103BBI).

[0092] For expression and production of a protein of interest (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 the cell under conditions appropriate for expression of the variants. 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, while in other embodiments, the plasmid vector containing the polynucleotide sequence encoding one or more subtilisin variants described herein remains in the cell as an autonomous extrachromosomal element. Some embodiments provide both extrachromosomal nucleic acid elements and incoming nucleotide sequences integrated into the host cell genome. The vectors described herein are useful for production of one or more subtilisin variants described herein. In some embodiments, the polynucleotide construct encoding one or more subtilisin variants described herein is present on an integrating vector that allows for integration and, optionally, amplification of the polynucleotide encoding the variant into the host chromosome. Examples of integration sites will be familiar to those of skill in the art. In some embodiments, transcription of a polynucleotide encoding one or more of the subtilisin variants described herein is accomplished by a promoter that is the wild-type promoter of the parent subtilisin, hi some other embodiments, the promoter is heterologous to one or more of the subtilisin variants described herein, but is functional in the host cell.Examples of promoters useful in bacterial host cells include, but are not limited to, amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpaII promoters; the promoter of the B. stearothermophilus maltogenic amylase gene, the B. amyloliquefaciens (BAN) amylase gene; the B. subtilis alkaline protease gene; the B. clausii alkaline protease gene, the B. pumilus xylosidase gene; the B. thuringiensis cryIIIA; and the B. licheniformis alpha-amylase gene. Further promoters include, but are not limited to, the A4 promoter, as well as the PR or PL promoters of phage lambda, and the lac, trp or tac promoters of E. coli.

[0093] One or more subtilisin variants described herein may be produced in suitable microbial host cells, such as bacteria and fungi. In some embodiments, one or more subtilisin variants described herein may be produced by Gram-positive bacteria. In some embodiments, the host cell is a Bacillus species, a Streptomyces species, an Escherichia species, an Aspergillus species, a Trichoderma species, a Pseudomonas species, a Corynebacterium species, a Saccharomyces species, or a Pichia species. In some embodiments, one or more subtilisin variants described herein are produced by a Bacillus species host cell. Exemplary host cells of Bacillus species that may be utilized to produce one or more of the subtilisin variants described herein include, but are not limited to, B. licheniformis, B. lentus, B. subtilis, B. amyloliquefaciens, B. lentus, B. brevis, B. stearothermophilus, and the like. Examples of Bacillus species include B. stearothermophilus, B. alkalophilus, B. coagulans, B. circulans, B. pumilis, B. thuringiensis, B. clausii, and B. megaterium, as well as other organisms in the genus Bacillus. In some embodiments, B. subtilis host cells are used to produce the variants described herein.U.S. Pat. Nos. 5,264,366 and 4,760,025 (Re. Pat. No. 34,606) describe various Bacillus host strains that can be used to produce one or more of the subtilisin variants described herein, although other suitable strains can also be used.

[0094] Some bacterial strains that can be used to produce one or more of the subtilisin variants described herein include non-recombinant (i.e., wild-type) Bacillus strains, as well as variants and / or recombinant strains of various naturally occurring strains. In some embodiments, the host strain is a recombinant strain, and a polynucleotide encoding one or more of the subtilisin variants described herein has been introduced into the host. In some embodiments, the host strain is a B. subtilis host strain, particularly a recombinant B. subtilis host strain. Many B. subtilis strains are known, including, but not limited to, strains 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753-PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP 211 (see, e.g., Hoch et al., Genetics, 2002). 73:215-228 (1973); see also U.S. Patent Nos. 4,450,235, 4,302,544, and EP 0134048. The use of B. 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)).

[0095] In some embodiments, the Bacillus host cell is a species of Bacillus that includes 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, any mutation in the opp operon that results in the same phenotype as a mutation in the oppA gene is contemplated for use in some embodiments of the mutant Bacillus strains described herein. In some embodiments, these mutations occur alone, while in other embodiments, combinations of mutations are present. In some embodiments, the altered Bacillus host cell strains that can be used to produce one or more subtilisin variants described herein are Bacillus host strains that already contain a mutation in one or more of the above genes. Additionally, host cells of Bacillus species that contain mutations and / or deletions of endogenous protease genes are also used. In some embodiments, the Bacillus host cell contains a deletion of the aprE and nprE genes.In other embodiments, the Bacillus spp. host cell comprises a deletion of five protease genes, while in other embodiments, the Bacillus spp. host cell comprises a deletion of nine protease genes (see, e.g., U.S. Patent Application Publication No. 2005 / 0202535).

[0096] A host cell is transformed with one or more nucleic acid sequences encoding one or more subtilisin variants described herein using any suitable method known in the art. Methods for introducing nucleic acids (e.g., DNA) into Bacillus or E. coli cells using a plasmid DNA construct or vector and transforming such plasmid DNA constructs or vectors into such cells are well known. In some embodiments, the plasmid is then isolated from the E. coli cell and transformed into the Bacillus cell. However, it is not necessary to use an intermediate microorganism such as E. coli, and in some embodiments, the DNA construct or vector is directly introduced into the Bacillus host.

[0097] Exemplary methods for introducing one or more of the nucleic acid sequences described herein into Bacillus cells can be found in, for example, Ferrari et al., "Genetics," in Harwood 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). Indeed, methods of transformation, including protoplast transformation and transfection, transduction, and protoplast fusion, are well known and suitable for use herein. Methods for transformation of Bacillus cells known in the art include methods such as plasmid marker rescue transformation, which involves uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (see Contente et al., Plasmid 2:555-571 (1979); Haima et al., Mol. Gen. Genet. 223:185-191 (1990); Weinrauch et al., J. Bacteriol. 154:1077-1087 (1983); and Weinrauch et al., J. Bacteriol. 169:1205-1211 (1987)).In this method, an incoming donor plasmid recombines with homologous regions of a resident "helper" plasmid in a manner analogous to chromosomal transformation.

[0098] In addition to commonly used methods, in some embodiments, a host cell is directly transformed with a DNA construct or vector comprising a nucleic acid encoding one or more subtilisin variants described herein (i.e., no intermediate cells are used to amplify or otherwise process the DNA construct or vector prior to introduction into a host cell). Introduction of a DNA construct or vector described herein into a host cell includes 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, liposomes, and the like. In a further embodiment, the DNA construct or vector is not inserted into a plasmid but is co-transformed with a plasmid. In a further embodiment, the selectable marker is deleted from the altered Bacillus strain by methods known in the art (see, e.g., Stahl et al., J. Bacteriol., 158:411-418 (1984) and Palmeros et al., Gene 247:255-264 (2000)).

[0099] 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 of skill in the art and are well described in the scientific literature. In some embodiments, a culture (e.g., a cell culture) is provided that comprises one or more subtilisin variants or nucleic acid sequences described herein.

[0100] In some embodiments, host cells transformed with one or more polynucleotide sequences encoding one or more subtilisin variants described herein are cultured in an appropriate nutrient medium under conditions that allow expression of the variants, and the resulting variants are then recovered from the culture. In some embodiments, the variants produced by the cells are recovered from the medium by conventional procedures, including, but not limited to, separating the host cells from the medium by centrifugation or filtration, precipitating the protein components of the supernatant or filtrate with a salt (e.g., ammonium sulfate), and chromatographic purification (e.g., ion exchange, gel filtration, affinity, etc.).

[0101] In some embodiments, one or more subtilisin variants produced by the recombinant host cell are secreted into the culture medium. Nucleic acid sequences encoding purification-facilitating domains can be used to facilitate purification of the variants. A vector or DNA construct comprising a polynucleotide sequence encoding one or more subtilisin variants described herein can further comprise a nucleic acid sequence encoding a purification-facilitating domain to facilitate purification of the variants (see, e.g., Kroll et al., DNA Cell Biol. 12:441-53 (1993)). Such purification-facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (see, e.g., Porath, Protein Expr. Purif. 3:263-281 (1992)), protein A domains that allow purification on immobilized immunoglobulins, and domains used in the FLAGS extension / affinity purification system. The incorporation of a cleavable linker sequence, such as factor XA or enterokinase (eg, those available from Invitrogen, San Diego, Calif.) between the purification domain and the heterologous protein is also used to facilitate purification.

[0102] A variety of methods can be used to measure the production levels of one or more mature subtilisin variants described herein. Such methods include, but are not limited to, methods using either polyclonal or monoclonal antibodies, e.g., specific for the protease. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), and fluorescence activated cell sorting (FACS). These and other assays are well known in the art (see, e.g., Maddox et al., J. Exp. Med. 158:1211 (1983)).

[0103] Some other embodiments provide methods of generating or producing one or more of the mature subtilisin variants described herein. The mature subtilisin variants do not include a signal peptide or propeptide sequence. Some methods include generating or producing one or more of the subtilisin variants described herein in a recombinant bacterial host cell, such as, for example, a cell of a Bacillus species (e.g., a B. subtilis cell). Other embodiments provide methods of producing one or more of the subtilisin variants described herein, comprising culturing a recombinant host cell comprising a recombinant expression vector that includes a nucleic acid sequence encoding one or more of the subtilisin variants described herein under conditions conducive to production of the variant. Some such methods further include recovering the variant from the culture.

[0104] Further embodiments provide methods of producing one or more subtilisin variants described herein, comprising: (a) introducing a recombinant expression vector containing a nucleic acid encoding the variant into a population of cells (e.g., bacterial cells, such as B. subtilis cells); and (b) culturing the cells in a medium under conditions conducive to production of the variant encoded by the expression vector. Some such methods further comprise (c) isolating the variant from the cells or the culture medium.

[0105] Unless otherwise stated, all ingredient or composition levels provided herein are adjusted based on the activity level of that ingredient or composition, and do not include impurities such as residual solvents or by-products that may be present in commercial sources. The weight of enzyme ingredients is based on total active protein. All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise indicated. The compositions described herein include cleaning compositions, such as detergent compositions. In the exemplified detergent compositions, enzyme levels represent pure enzyme by weight of the total composition, and detergent ingredients are represented by weight of the total composition unless otherwise specified.

[0106] In one embodiment, one or more subtilisin variants described herein are useful in cleaning applications, such as, but not limited to, cleaning dishware or tableware products, fabrics, medical instruments, and hard surface items (e.g., tables, table tops, walls, furniture products, floor and ceiling hard surfaces). In other embodiments, one or more subtilisin variants described herein are useful in disinfecting applications, such as, but not limited to, disinfecting automatic dishwashers or washing machines.

[0107] Other embodiments relate to compositions comprising one or more subtilisin variants described herein. In some embodiments, the composition is a cleaning composition. In other embodiments, the composition is a detergent composition. In still other embodiments, the composition is selected from laundry detergent compositions, automatic dishwashing (ADW) compositions, hand-washing (manual) dishwashing detergent compositions, hard surface cleaning compositions, eyeglass cleaning compositions, medical equipment cleaning compositions, disinfecting (e.g., malodor or microbial) 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. Still further embodiments relate to fabric cleaning compositions, and other embodiments relate to non-fabric cleaning compositions. In some embodiments, the cleaning composition does not include boron. In other embodiments, the cleaning composition does not include phosphate. In still other embodiments, the composition comprises one or more subtilisin variants described herein and one or more of excipients, adjuncts, and / or additional enzymes.

[0108] In still yet further embodiments, the compositions described herein contain phosphate, are free of phosphate, contain boron, are free of boron, or are a combination thereof. In other embodiments, the compositions are free of boron. In some embodiments, the non-boron-containing compositions are compositions to which no borate stabilizer has been added. In other embodiments, the non-boron-containing compositions are compositions with a boron content of less than 5.5%. In yet further embodiments, the non-boron-containing compositions are compositions with a boron content of less than 4.5%. In yet further embodiments, the non-boron-containing compositions are compositions with a boron content of less than 3.5%. In yet further embodiments, the non-boron-containing compositions are compositions with a boron content of less than 2.5%. In still further embodiments, the non-boron-containing compositions are compositions with a boron content of less than 1.5%. In another embodiment, the non-boron-containing compositions are compositions with a boron content of less than 1.0%. In still further embodiments, the non-boron-containing compositions are compositions with a boron content of less than 0.5%. In yet a further embodiment, the non-boron-containing composition is a composition that is substantially free of boron.

[0109] In another embodiment, one or more compositions described herein have the form of gel, tablet, powder, granule, solid, liquid, unit dose, and combinations thereof. In yet another embodiment, one or more compositions described herein have the form selected from low water compact formulation, low water HDL or UD, or high water formulation or HDL. In some embodiments, the cleaning composition described herein has a unit dose form. In other embodiments, the unit dose form is selected from pill, tablet, capsule, gel capsule, sachet, sachet, multi-compartment sachet, and pre-measured powder or liquid. In some embodiments, the unit dose form is designed to control the release of ingredients in the multi-compartment sachet (or other unit dose form). Suitable unit dose forms and controlled release formulations are described, for example, in EP 2100949; WO 02 / 102955; U.S. Pat. No. 4,765,916; U.S. Pat. No. 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 sachet.

[0110] Examples of laundry detergent compositions include, but are not limited to, for example, liquid and powder laundry detergent compositions. Examples of hard surface cleaning compositions include, but are not limited to, for example, compositions used to clean non-dishware products, non-tableware products, tables, table tops, furniture products, walls, floors, and ceilings hard surfaces. Examples of hard surface cleaning compositions are described, for example, in U.S. Patent Nos. 6,610,642, 6,376,450, and 6,376,450. Examples of personal care compositions include, but are not limited to, compositions used to clean dentures, teeth, hair, contact lenses, and skin. Examples of such oral care composition ingredients include, for example, those described in U.S. Patent No. 6,376,450.

[0111] In some 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 as useful or effective for cleaning surfaces in need of proteinaceous stain removal.

[0112] In some embodiments, adjunct materials are incorporated to, for example, aid or enhance cleaning performance, to treat the substrate being cleaned, or to alter the aesthetics of the cleaning composition, similar to perfumes, colorants, dyes, and the like. Certain embodiments relate to compositions comprising one or more adjunct materials and one or more subtilisin variants described herein. Other embodiments relate to compositions comprising one or more adjunct materials and one or more subtilisin variants described herein, wherein the adjunct materials are selected from the group consisting of bleach catalysts, additional enzymes, enzyme stabilizers (including, for example, enzyme stabilization systems), chelating agents, optical brighteners, soil release polymers, dye transfer agents, dispersants, suds suppressors, dyes, fragrances, colorants, fillers, photoactivators, fluorescent agents, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, shrinkage inhibitors, wrinkle inhibitors, germicides, mildew inhibitors, The present invention relates to a composition selected from colored speckles, silver care agents, tarnish inhibitors, corrosion inhibitors, 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, polymeric dispersants, mud stain removal / anti-redeposition agents, structure elastifiers, fabric softeners, carriers, hydrotropes, processing aids, pigments, and combinations thereof. Examples of auxiliary materials and concentrations of use are described in U.S. Patent Nos. 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 do not mix well with one or more of the subtilisin variants described herein, methods can be used to keep the auxiliary materials and the variants separate (i.e., not in contact with each other) until suitable mixing of the two components is possible. Such separation methods may include any suitable method known in the art (eg, gel caps, encapsulation, tablets, physical separation, etc.).

[0113] Some embodiments relate to a cleaning additive comprising one or more subtilisin variants described herein. In some embodiments, the additive is packaged in a dosage form for addition to a cleaning process. In some embodiments, the additive is packaged in a dosage form for addition to a cleaning process in which a peroxydine source is utilized and high bleaching effect is desired.

[0114] Examples of fillers or carriers for granular compositions include, but are not limited to, various sulfates, carbonates, and silicates; talc, and clay. Examples of 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 contains about 5% to about 90% of such fillers or carriers. Such compositions can contain acidic fillers to lower the pH of the solution resulting from the cleaning method or application.

[0115] 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% by weight, about 0.0001 to about 10% by weight, about 0.0001 to about 1% by weight, about 0.001 to about 1% by weight, or about 0.01 to about 0.1% by weight of one or more proteases. In another embodiment, one or more cleaning compositions described herein contain from about 0.01 to about 10 mg, from about 0.01 to about 5 mg, from about 0.01 to about 2 mg, from about 0.01 to about 1 mg, from about 0.05 to about 1 mg, from about 0.5 to about 10 mg, from about 0.5 to about 5 mg, from about 0.5 to about 4 mg, from about 0.5 to about 4 mg, from about 0.5 to about 3 mg, from about 0.5 to about 2 mg, from about 0.5 to about 1 mg, from about 0.1 to about 10 mg, from about 0.1 to about 5 mg, from about 0.1 to about 4 mg, from about 0.1 to about 3 mg, from about 0.1 to about 2 mg, from about 0.1 to about 2 mg, from about 0.1 to about 1 mg, or from about 0.1 to about 0.5 mg of one or more proteases per gram of composition.

[0116] The cleaning compositions described herein are typically formulated to provide a wash water pH of about 4.0 to about 11.5, or even about 5.0 to about 11.5, or even about 5.0 to about 8.0, or even about 7.5 to about 10.5 during use in aqueous cleaning operations. Liquid product formulations are typically formulated to have a pH of about 3.0 to about 9.0 or about 3 to about 5. Granular laundry products are typically formulated to have a pH of about 9 to about 11. In some embodiments, the cleaning compositions of the present invention can be formulated to have an alkaline pH under wash conditions, for example, a pH of about 8.0 to about 12.0, or about 8.5 to about 11.0, or about 9.0 to about 11.0. In some embodiments, the cleaning compositions of the present invention can be formulated to have a neutral pH under washing conditions, for example, a pH of about 5.0 to about 8.0, or about 5.5 to about 8.0, or about 6.0 to about 8.0, or about 6.0 to about 7.5. In some embodiments, neutral pH conditions can be measured when the cleaning composition is dissolved 1:100 (weight:weight) in deionized water at 20° C. and measured using a conventional pH meter. Methods for adjusting the pH at the recommended use concentration include the use of buffers, alkalis, acids, etc., methods well known to those skilled in the art.

[0117] In some embodiments, one or more of the subtilisin variants described herein are encapsulated to protect it from other components in the composition during storage and / or to control availability of the variant during washing. In some embodiments, encapsulation increases the performance of the variant and / or additional enzymes. In some embodiments, the encapsulating material typically encapsulates at least a portion of the variants described herein. Typically, the encapsulating material is water soluble and / or water dispersible. In some embodiments, the encapsulating material has a glass transition temperature (Tg) of 0° C. or higher. Examples of 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 wax, and combinations thereof. When the encapsulating material is a carbohydrate, it is typically selected from monosaccharides, oligosaccharides, polysaccharides, and combinations thereof. In some embodiments, the encapsulant is starch (see, e.g., EP 0922499, U.S. Pat. No. 4,977,252, U.S. Pat. No. 5,354,559, and U.S. Pat. No. 5,935,826). In some embodiments, the encapsulant is a microsphere made from a plastic, such as a thermoplastic, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile, and mixtures thereof. Examples of commercially available microspheres include, but are not limited to, EXPANCEL® (Akzo Nobel Chemicals International, BV); PM6545, PM6550, PM7220, PM7228, and EXTENDOSPHERES® (Sphere One Inc.); and LUXSIL®, Q-CEL®, and SPHERICEL® (Potters Industries LLC).

[0118] There are a variety of wash conditions, including different detergent formulations, wash water volumes, wash water temperatures, and lengths of wash time to which one or more subtilisin variants described herein are exposed. A low detergent concentration system refers to a wash water containing less than about 800 ppm of detergent components. A medium detergent concentration system refers to a wash containing about 800 ppm to 2000 ppm of detergent components. A high detergent concentration system refers to a wash water containing more than about 2000 ppm of detergent components. In some embodiments, the "cold water wash" of the present invention uses a "cold water detergent" suitable for washing at temperatures between about 10°C and about 40°C, between about 20°C and about 30°C, or between about 15°C and about 25°C and between about 15°C and about 35°C or all other combined temperatures within the range of 10°C to 40°C.

[0119] Different geographies have different water hardness. Hardness is determined by the amount of calcium (Ca) in the water. 2+ ) and magnesium (Mg 2+ ) is a measure of the amount of Ca 2+ / Mg 2+ Hardness is measured in grains per gallon, a mixture of hardness minerals and hardness minerals. Most water in the United States is hard, but there are different degrees of hardness. Moderately hard water (60-120 ppm) to hard water (121-181 ppm) has hardness minerals between 60 and 181 ppm (ppm can be converted to grains per U.S. gallon by dividing ppm by 17.1).

[0120] [Table 1]

[0121] 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 adjunct ingredients. 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%, 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 adjunct ingredients.

[0122] In other embodiments, the compositions described herein comprise one or more subtilisin variants described herein and one or more additional enzymes, such as acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, DNase or nuclease, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hyaluronidase, keratinase, laccase, lactase, lignin ... The subtilisin variants are selected from the group consisting of amylase, lipase, lysozyme, mannanase, metalloprotease, oxidase, pectate lyase, pectin acetyl esterase, pectinase, pentosanase, peroxidase, phenol oxidase, phosphatase, phospholipase, phytase, polygalacturonase, additional proteases, pullulanases, reductases, rhamnogalacturonase, beta-glucanases, tannases, transglutaminase, xylan acetyl esterase, xylanase, xyloglucanases, xylosidases, and any combination or mixture thereof. Some embodiments relate to combinations of enzymes (i.e., "cocktails") that include conventional enzymes such as amylase, lipase, cutinase, and / or cellulase together with one or more subtilisin variants described herein, and / or one or more additional proteases.

[0123] 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, the additional protease is a serine protease. In another embodiment, the additional protease is an alkaline microbial protease or a trypsin-like protease. Suitable additional proteases include those of animal, vegetable or microbial origin. In some embodiments, the additional protease is a microbial protease. In other embodiments, the additional protease is a chemically or genetically modified variant. In another embodiment, the additional protease is an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases include, for example, subtilisins from Bacillus (e.g., subtilisin lentus, amyloliquefaciens, gibsonii, subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168). Examples of metalloproteases include nprE, a recombinant form of neutral metalloprotease expressed in B. subtilis (see, for example, WO 07 / 044993), and PMN, a purified neutral metalloprotease from B. amyloliquefaciens. Further examples of proteases include, but are not limited to, those described in WO 92 / 21760, WO 95 / 23221, WO 2008010925, WO 09149200, WO 09149144, WO 09149145, WO 10056640, WO 10056653, WO 20100566356, WO 2011072099, WO 201113022, WO 11140364, WO 2012151534, WO 2015038792, WO 2015 / 089441,Brochure No. 2015089447, Brochure No. 2015143360, Brochure No. 2016001449, Brochure No. 2016001450, Brochure No. 2016061438, Brochure No. 2016069544, Brochure No. 2016069548, Brochure No. 2016069552, Brochure No. 2016069557, Brochure No. 2016069563, Brochure No. 2016069569, Brochure No. 2016087617 , US Patent Application Publication No. 2016087619, US Patent Application Publication No. 2016145428, US Patent Application Publication No. 2016174234, US Patent Application Publication No. 2016183509, US Patent Application Publication No. 2016202835, US Patent Application Publication No. 2016205755, US Patent Application Publication No. 2008 / 0090747, US Patent No. 5,801,039, US Patent No. 5,340,735, US Patent No. 5,500,364, US Patent No. 5,855,625, Reissue Patent No. 34 Nos. 5,955,340, 5,700,676, 6,312,936, 6,482,628, 8,530,219, U.S. Provisional Patent Applications Nos. 62 / 331282, 62 / 351649, 62 / 437171, 62 / 437174 and 62 / 437509, and PCT Application Nos. PCT / CN2017 / 076749 and PCT / U Those described in the specification of S2017 / 029307, and also in International Publication Nos. 1999014341, 1999033960, 1999014342, 1999034003, 2007044993, 2009058303, 2009058661, 2014071410, 2014194032, and 2014194034,and metalloproteases as described in WO 2014194054 and WO 2014194117. Additional examples of proteases also include, but are not limited to, trypsin (e.g., of porcine or bovine origin) and the Fusarium proteases as described in WO 89 / 06270. Examples of commercially available proteases include, but are not limited to, MAXATASE, MAXACAL, MAXAPEM, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®, PURAFECT® OXP, PURAMAX®, EXCELLASE®, PREFERENZ™ proteases (e.g., P100, P110, P280), EFFECTENZ™ proteases (e.g., P1000, P1050, P2000), EXCELLENZ™ proteases (e.g., P1000), ULTIMASE®, and PURAFAST (Danisco US; ALCALASE®, ALCALASE® ULTRA, BLAZE®, BLAZE® EVITY®, BLAZE® EVITY® 16L, CORONASE®, SAVINASE®, SAVINASE® ULTRA, SAVINASE® EVITY®, SAVINASE®, EVERIS®, PRIMASE, DURAZYM, POLARZYME®, OVOZYME®, KANNASE®, LIQUANASE®, EVERIS®, NEUTRASE®, PROGRESS UNO®, RELASE® and ESPERASE® (Novozymes); BLAP™ and BLAP™ variants (Henkel); LAVERGY™ PRO 104L (BASF), and KAP® (B. alkalophilus subtilisin) (Kao Corporation).

[0124] Other embodiments relate to compositions comprising one or more subtilisin variants described herein and one or more lipases. In some embodiments, the compositions comprise from about 0.00001% to about 10%, 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% lipase by weight of the composition. An example of a lipase can be a variant that has been modified chemically or by genetic engineering.Examples of lipases include, but are not limited to, those of bacterial or fungal origin, such as H. lanuginosa lipase (see, e.g., EP 258068 and EP 305216), T. lanuginosus lipase (see, e.g., WO 2014 / 059360 and WO 2015 / 010009), Rhizomucor miehei lipase, and the like. miehei lipase (see, e.g., EP 238023), Candida lipases, such as C. antarctica lipase (e.g., C. antarctica lipase A or B) (see, e.g., EP 214761), Pseudomonas lipases, such as P. alcaligenes and P. pseudoalcaligenes. domonas lipase (see, e.g., EP 218272), P. cepacia lipase (see, e.g., EP 331376), P. stutzeri lipase (see, e.g., GB 1,372,034), P. fluorescens lipase, Bacillus lipase (e.g., B. subtilis lipase) (e.g., Dartois et al., Biochem. Biophys. Acta 1131:253-260 (1993)), B. stearothermophilus lipase (see, for example, JP-A-64-744992), and B. pumilus lipase (see, for example, WO 91 / 16422).Examples of 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., J. Biochem., 109:117-113 (1991)), and R. spp. lipase (Kugimiya et al., J. Biochem., 109:117-113 (1991)). al., Biosci. Biotech. Biochem. 56:716-719 (1992)), and R. oryzae lipase. Other lipolytic enzymes, such as cutinases, may also be used in one or more of the compositions described herein, including, but not limited to, cutinases derived from Pseudomonas mendocina (see WO 88 / 09367) and / or cutinases derived from Fusarium solanipisi (see WO 90 / 09446). Examples of commercially available lipases include, but are not limited to, M1 LIPASE, LUMA FAST, and LIPOMAX™ (Genencor); LIPEX®, LIPOCLEAN®, LIPOLASE®, and LIPOLASE® ULTRA (Novozymes); and LIPASE P (Amano Pharmaceutical Co. Ltd).

[0125] Yet a further embodiment relates to a composition comprising one or more subtilisin variants described herein and one or more amylases. In one embodiment, the composition comprises 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 by weight of the composition. Amylases suitable for use in alkaline solutions (e.g., alpha and / or beta) may be useful for inclusion in such compositions. An example of an amylase may be a variant that has been modified chemically or by genetic engineering. Examples of amylases include, but are not limited to, those of bacterial or fungal origin, e.g., those described in 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 99 / 14024, WO 100 / 10026 ... Publication No. 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, WO 2005018336, WO 200 No. 5019443, International Publication No. 2005066338, International Publication No. 2006002643, International Publication No. 2006012899, International Publication No. 2006012902, International Publication No. 2006031554, International Publication No. 2006063594, International Publication No. 2006066594, International Publication No. 2006066596, International Publication No. 2006136161, International Publication No. 2008000825, International Publication No. International Publication No. 2008088493, International Publication No. 2008092919, International Publication No. 2008101894, International Publication No. 2008112459, International Publication No. 2009061380, International Publication No. 2009061381, International Publication No. 2009100102, International Publication No. 2009140504, International Publication No. 2009149419, International Publication No. 2010059413, International Publication No. 2010088447, International Publication No. 2010091221, International Publication No. 2010104675, International Publication No. 2010115021, International Publication No. 2010115028, International Publication No. 2010117511, International Publication No. 2011076123, International Publication No. 2011076897, International Publication No. 2011080352, International Publication No. 2011080353, International Publication No. 2011080354, International Publication No. 2011082425,Examples of the amylase include those described in WO2011082429, WO2011087836, WO2011098531, WO2013063460, WO2013184577, WO2014099523, WO2014164777 and WO2015077126. Examples of commercially available amylases include, but are not limited to, AMPLIFY®, AMPLIFY PRIME®, BAN DURAMYL®, TERMAMYL®, TERMAMYL® ULTRA, FUNGAMYL®, STAINZYME®, STAINZYME® PLUS, STAINZYME® ULTRA, and STAINZYME® EVITY® (Novozymes); EFFECENZ™ S1000, POWERASE®, PREFERENZ™ S100, PREFERENZ™ S110, EXCELLENZ™ S2000, RAPIDASE®, and MAXAMYL® P (Danisco US).

[0126] Yet further embodiments relate to compositions comprising one or more subtilisin variants described herein and one or more cellulases. In one embodiment, the composition comprises 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% cellulase by weight of the composition. Any suitable cellulase may be used in the compositions described herein. An example of a cellulase may be a variant that has been modified chemically or by genetic engineering. Examples of cellulases include, but are not limited to, those of bacterial or fungal origin, such as those described in WO2005054475, WO2005056787, U.S. Pat. No. 7,449,318, U.S. Pat. No. 7,833,773, U.S. Pat. No. 4,435,307; EP0495257 and U.S. Provisional Patent Applications Nos. 62 / 296,678 and 62 / 435340. Examples of commercially available cellulases include, but are not limited to, CELLUCLEAN®, CELLUZYME®, CAREZYME®, CAREZYME® PREMIUM, ENDOLASE®, and RENOZYME® (Novozymes); REVITALENZ® 100, REVITALENZ® 200 / 220, and REVITALENZ® 2000 (Danisco US); and KAC-500(B) (Kao Corporation). In some embodiments, the cellulase is included as a portion or fragment of a mature wild-type or mutant cellulase, with a portion of the N-terminus removed (see, e.g., U.S. Pat. No. 5,874,276).

[0127] Yet further embodiments relate to compositions comprising one or more subtilisin variants described herein and one or more mannanases. In one embodiment, the composition comprises 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. An example of a mannanase may be a variant that has been modified chemically or by genetic engineering. Examples of mannanases include, but are not limited to, those of bacterial or fungal origin, such as those described in WO2016007929, WO2017 / 079756 and WO2017 / 079751; and U.S. Patent Nos. 6,566,114, 6,602,842 and 6,440,991. Examples of commercially available mannanases include, but are not limited to, MANNAWAY® (Novozymes), as well as EFFECTENZ™ M1000, PREFERENZ® M100, MANNASTAR® and PURABRITE™ (Danisco US).

[0128] Still further embodiments relate to compositions comprising one or more subtilisin variants described herein and one or more peroxidase and / or oxidase enzymes. In one embodiment, the composition comprises from about 0.00001% to about 10%, 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% peroxidase or oxidase by weight of the composition. Peroxidases may be used in combination with hydrogen peroxide or a source thereof (e.g., percarbonate, perborate, or persulfate) and oxidases may be used in combination with oxygen. Peroxidases and oxidases are used alone or with enhancers for "bleaching" (i.e., to prevent textile dyes from transferring from one dyed fabric to another when the fabrics are washed together in the wash liquor) (see, for example, WO 94 / 12621 and WO 95 / 01426). An example of a peroxidase and / or oxidase can be a variant that has been modified chemically or by genetic engineering. Examples of peroxidases / oxidases include, but are not limited to, those of plant, bacterial or fungal origin.

[0129] Another embodiment relates to a composition comprising one or more subtilisin variants as described herein and one or more perhydrolases, e.g., as described in WO 2005 / 056782, WO 2007 / 106293, WO 2008 / 063400, WO 2008 / 106214 and WO 2008 / 106215.

[0130] In yet another embodiment, the one or more subtilisin variants described herein and the one or more additional enzymes contained in one or more compositions described herein can each independently vary by up to about 10%, with the remainder of the cleaning composition being one or more adjunct ingredients.

[0131] In some embodiments, one or more compositions described herein are used as detergent additives, said additives being in solid or liquid form. Such additive products are intended to supplement and / or enhance 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 to about 1200 g / liter, while in other embodiments, the density ranges from about 500 to about 950 g / liter, measured at 20°C.

[0132] Some embodiments relate to laundry detergent compositions comprising one or more subtilisin variants described herein and one or more adjunct materials selected from surfactants, enzyme stabilizers, builder compounds, polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime soap dispersants, soil suspending agents, anti-redeposition agents, corrosion inhibitors, and combinations thereof. In some embodiments, the laundry composition also comprises a fabric softener.

[0133] Further embodiments relate to dishwashing compositions for hand washing comprising one or more subtilisin variants as described herein and one or more adjunct materials selected from surfactants, organic polymeric compounds, suds boosters, Group II metal ions, solvents, hydrotropes, and additional enzymes.

[0134] Other embodiments relate to one or more compositions described herein, said compositions being compact granular fabric cleaning compositions used for washing colored fabrics or providing softness through cleaning performance, or being heavy duty liquid (HDL) fabric cleaning compositions. Examples of fabric cleaning compositions and / or methods of manufacture are described in U.S. Patent Nos. 6,610,642 and 6,376,450. Other examples of cleaning compositions are described, for example, in U.S. Patent Nos. 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; 5,5 Nos. 16,448; 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.

[0135] In some embodiments, the cleaning composition comprises an acidifying particle or an amino carboxylic acid builder. Examples of amino carboxylic acid builders include amino carboxylic acids, their salts and derivatives. In some embodiments, the amino carboxylic acid builder is an amino carboxylic acid builder having the general formula MOOC-CHR-N(CH2COOM)2, where R is C 1~12where M is an alkyl group and M is an alkali metal. In some embodiments, the amino carboxylic acid builder is an amino polycarboxylic acid builder such as derivatives represented by the formula: methylglycine diacetate (MGDA), GLDA (glutamic acid-N,N-diacetate), iminodisuccinic acid (IDS), carboxymethyl inulin and its salts and derivatives, aspartic acid-N-monoacetate (ASMA), aspartic acid-N,N-diacetate (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)glutamic acid (SEGL), IDS (iminodiacetic acid) and its salts and derivatives (such as N-methyliminodiacetic acid (MIDA)), alpha-alanine-N,N-diacetic acid (alpha-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA) and its alkali metal salts and derivatives. In some embodiments, the acidified particles have a weight geometric mean particle size of about 400 μm to about 1200 μm and a bulk density of at least 550 g / L. In some embodiments, the acidified particles include at least about 5% builder.

[0136] In some embodiments, the acidified particles may contain any acid, including organic and mineral acids. The organic acid has one or two carboxyl groups, such as 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 glyoxylic acid, and may have up to 15 carbon atoms, particularly up to 10 carbon atoms. In some embodiments, the acid is citric acid. Mineral acids include hydrochloric acid and sulfuric acid. Optionally, the acidified particles are high activity particles that contain a high concentration of amino carboxylic acid builder. Sulfuric acid has also been found to contribute to the stability of the final particles.

[0137] Further embodiments relate to cleaning compositions comprising one or more subtilisin variants and one or more surfactants and / or surfactant systems, wherein the surfactants are selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof. In some embodiments, the surfactants are present at a concentration of from about 0.1% to about 60% by weight of the cleaning composition, while in other embodiments the concentration is from about 1% to about 50% and in still further embodiments the concentration is from about 5% to about 40% by weight.

[0138] In some embodiments, one or more of the compositions described herein comprises one or more detergent builders or builder systems, in one embodiment, the composition comprises from about 1% to about 0.1% to about 80%, from about 3% to about 60%, from about 5% to about 40%, or from about 10% to about 50% builder by weight of the composition. Examples of 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 acid, 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, the builders form water-soluble hardness ion complexes (e.g., sequestering builders), such as citrates and polyphosphates (e.g., sodium tripolyphosphate and sodium tripolyphosphate hexahydrate, potassium tripolyphosphate, and mixtures of sodium tripolyphosphate and potassium tripolyphosphate). Examples of builders are described, for example, in EP 2100949. In some embodiments, the builder includes a phosphate builder and a non-phosphate builder. In some embodiments, the builder is a phosphate builder. In some embodiments, the builder is a non-phosphate builder. In some embodiments, the builder includes a mixture of a phosphate builder and a non-phosphate builder. Examples of phosphate builders include, but are not limited to, monophosphates, diphosphates, tripolyphosphates, or oligomeric polyphosphates, including alkali metal salts, including sodium salts, of these compounds. In some embodiments, the builder may be sodium tripolyphosphate (STPP).In addition, the composition may contain carbonates and / or citrates.Other suitable non-phosphate builders include homopolymers and copolymers of polycarboxylic acids and their partially or completely neutralized salts, polycarboxylic acid monomers and hydroxycarboxylic acids and their salts.In some embodiments, the salts of the above compounds include ammonium and / or alkali metal salts, i.e., lithium, sodium and potassium salts, including sodium salts.Suitable polycarboxylic acids include acyclic, alicyclic, heterocyclic and aromatic carboxylic acids, which in some embodiments may contain at least two carboxyl groups, and the at least two carboxyl groups are separated from each other in each case by no more than two carbon atoms in some cases.

[0139] 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 from about 3% to about 10% of a chelating agent by weight of the composition. Examples of chelating agents include, but are not limited to, copper, iron, manganese, and mixtures thereof.

[0140] In some embodiments, one or more compositions described herein include one or more precipitation aids. Examples of precipitation aids include, but are not limited to, soil release polymers such as polyethylene glycol, polypropylene glycol, polycarboxylates, such as polyethylene terephthalic acid, clays such as kaolinite, montmorillonite, attapulgite, illite, bentonite and halloysite, and mixtures thereof.

[0141] In other embodiments, one or more compositions described herein include one or more anti-redeposition agents or non-ionic surfactants, which can prevent redeposition of soils (see, for example, EP 2100949). For example, non-ionic surfactants are used in ADW compositions to modify surfaces, particularly for sheeting purposes, to avoid film and stain formation, and to improve gloss. These non-ionic surfactants are also used to prevent redeposition of soils. In some embodiments, the non-ionic surfactants can be ethoxylated non-ionic surfactants, epoxy-capped poly(oxyalkylated) alcohols, and amine oxide surfactants.

[0142] In some embodiments, one or more compositions described herein include one or more dye transfer inhibitors. Examples of polymeric dye transfer inhibitors 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%, from about 0.01% to about 5%, or from about 0.1% to about 3% of a dye transfer inhibitor by weight of the composition.

[0143] In some embodiments, one or more compositions described herein include one or more silicates. Examples of silicates include, but are not limited to, sodium silicates, such as sodium disilicate, sodium metasilicate, and crystalline phyllosilicates. In some embodiments, the silicates are present in a concentration of about 1% to about 20% by weight, or about 5% to about 15% by weight of the composition.

[0144] In yet some further embodiments, one or more compositions described herein include one or more dispersants. Examples of water-soluble organic materials include, but are not limited to, homopolymeric or copolymeric acids or salts thereof, such as polycarboxylic acids that contain at least two carboxyl groups separated from each other by no more than two carbon atoms.

[0145] In some further embodiments, one or more compositions described herein include one or more enzyme stabilizers. In some embodiments, the enzyme stabilizer is a water-soluble source of calcium and / or magnesium ions. In some embodiments, the enzyme stabilizer includes oligosaccharides, polysaccharides, and inorganic divalent metal salts, including alkaline earth metal salts, e.g., calcium salts. In some embodiments, the enzymes used herein are stabilized by the presence in the final composition of water-soluble ion sources of zinc(II), calcium(II), and / or magnesium(II), as well as other metal ions (e.g., barium(II), scandium(II), iron(II), manganese(II), aluminum(III), tin(II), cobalt(II), copper(II), nickel(II), and oxovanadium(IV)) that provide such ions to the enzyme. Chlorides and sulfates are also used in some embodiments. Examples of oligosaccharides and polysaccharides (e.g., dextrins) are described, for example, in WO 07 / 145964. In some embodiments, reversible protease inhibitors are also used, such as 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, such as those further described in WO 2009 / 118375 and WO 2013004636.

[0146] In some embodiments, one or more compositions described herein include one or more bleaching agents, bleach activators, and / or bleach catalysts. In some embodiments, one or more compositions described herein include one or more inorganic and / or organic bleaching compounds. Examples of inorganic bleaching agents include, but are not limited to, perhydrate salts (e.g., perborates, percarbonates, perphosphates, persulfates, and persilicates). In some embodiments, the inorganic perhydrate salts are alkali metal salts. In some embodiments, the inorganic perhydrate salts are included as crystalline solids without additional protection, while in other embodiments, the salts are coated. Bleach activators are typically organic peracid precursors that enhance bleaching action during wash processes at temperatures of 60° C. or less. Examples of bleach activators include compounds that, under perhydrolysis conditions, provide aliphatic peroxycarboxylic acids having about 1 to about 10 carbon atoms or about 2 to about 4 carbon atoms, and / or optionally substituted perbenzoic acids. Examples of bleach activators are described in, for example, EP 2100949. Examples of bleach catalysts include, but are not limited to, manganese triazacyclononane and related complexes, and cobalt, copper, manganese, and iron complexes. Further examples of bleach catalysts are described in, for example, U.S. Pat. No. 4,246,612; U.S. Pat. No. 5,227,084; U.S. Pat. No. 4,810,410; WO 99 / 06521; and EP 2100949.

[0147] In some embodiments, one or more compositions described herein include one or more catalytic metal complexes. In some embodiments, a metal-containing bleach catalyst is used. In some embodiments, the metal bleach catalyst includes a catalyst system that includes a transition metal cation with a specified bleach catalytic activity (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cation), an auxiliary metal cation with little or no bleach catalytic activity (e.g., zinc or aluminum cation), and a sequestering agent with a specified stability constant for the catalyst and the auxiliary metal cation, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and water-soluble salts thereof (see, for example, U.S. Pat. No. 4,430,243). In some embodiments, one or more compositions described herein are catalyzed by manganese compounds. Such compounds and use concentrations are described, for example, in U.S. Pat. No. 5,576,282. In further embodiments, a cobalt bleach catalyst is used and included in one or more compositions described herein. Various cobalt bleach catalysts are described, for example, in US Pat. Nos. 5,597,936 and 5,595,967.

[0148] In some further embodiments, one or more compositions described herein include a transition metal complex of a macropolycyclic rigid ligand (MRL). As a practical matter, and not by way of limitation, in some embodiments, the compositions and cleaning processes described herein are tailored to provide an active MRL in the cleaning solution of at least 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, on the order of parts per billion. Examples of MRLs include, but are not limited to, crosslinked specialized ultra-rigid ligands, such as 5,12-diethyl-1,5,8,12-tetraazabicyclo(6.6.2)hexadecane. Examples of metal MRLs are described, for example, in WO 2000 / 32601 and U.S. Pat. No. 6,225,464.

[0149] In another embodiment, one or more compositions described herein comprise one or more metal care agents. In some embodiments, the composition comprises about 0.1% to about 5% by weight of the composition of the metal care agent. Examples of metal care agents include, for example, aluminum, stainless steel, and non-ferrous metals (e.g., silver and copper). Further examples of metal care agents are described, for example, in EP 2100949, WO 94 / 26860, and WO 94 / 26859. In some compositions, the metal care agent is a zinc salt.

[0150] In some embodiments, the cleaning composition is a high density liquid (HDL) composition comprising one or more subtilisin variants described herein. HDL liquid laundry detergents can include a cleaning surfactant (10%-40%), which is selected from the group of anionic cleaning surfactants (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 an optional nonionic surfactant (linear or branched or random chain substituted or unsubstituted alkyl alkoxylated alcohols, e.g., C8-C9, C10-C12, C12-C16, C16-C18, C18-C2 ... 18 Alkyl ethoxylated alcohol and / or C6-C 12 alkylphenol alkoxylates, optionally with a weight ratio of anionic detersive surfactants (having a hydrophilicity index (HIc) of 6.0 to 9) to nonionic detersive surfactants of greater than 1:1. Suitable detersive surfactants also include cationic detersive surfactants (selected from alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds and / or mixtures thereof); zwitterionic and / or amphoteric detersive surfactants (selected from alkanolamine sulfobetaines); amphoteric surfactants; semi-polar nonionic surfactants and mixtures thereof.

[0151] In another embodiment, the cleaning composition is a non-unit dose liquid or gel detergent, which may be aqueous, typically containing at least 20% to 95% water by weight, for example, up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, or up to about 35% water by weight. Other types of liquids can be included in the aqueous liquid or gel, including, but not limited to, alkanols, amines, diols, ethers, and polyols. The aqueous liquid or gel detergent may contain 0 to 30% organic solvent. The liquid or gel detergent may be non-aqueous.

[0152] The composition optionally comprises an amphiphilic alkoxylated grease cleaning polymer (alkoxylated polymers having branched hydrophilic and hydrophobic properties, e.g. selected from the group of alkoxylated polyalkyleneimines in the range of 0.05% to 10% by weight), and / or a random graft polymer (typically a hydrophilic backbone comprising monomers selected from the group consisting of unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, saturated polyalcohols such as maleic anhydride, glycerol, and mixtures thereof, and a C4-C6 carboxylic acid-based alkoxy unit, which is preferably selected from the group consisting of C1-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, glycerol, and mixtures thereof, and a C4-C6 carboxylic acid-based alkoxy unit, which is preferably selected from the group consisting of C4 ... acid-based alkoxy units, which is preferably selected from the group consisting of C4-C6 carboxylic acid-based alkoxy units, which is preferably selected from the group consisting of C4-C6 carboxylic acid-based alkoxy units, which is preferably selected from the group consisting of C4-C6 carboxylic acid-based 25 The polymer may include a surface active enhancing polymer having hydrophobic side chains selected from the group consisting of alkyl groups, polypropylene, polybutylene, vinyl esters of saturated C2-C6 monocarboxylic acids, C1-C6 alkyl esters of acrylic or methacrylic acid, and mixtures thereof.

[0153] The composition may comprise a soil release polymer (e.g., an anionic end-capped polyester, such as SRP1; a polymer comprising at least one monomer unit selected from sugars, dicarboxylic acids, polyols, and combinations thereof, in a random or block configuration; ethylene terephthalate-based polymers and copolymers thereof, in a random or block configuration, 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% to 10% by weight, for example, carboxylic acid 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 mixtures thereof; vinylpyrrolidone homopolymers and / or polyethylene glycols having a molecular weight in the range of 500 to 100,000 Da; cellulose polymers (including, for example, alkyl celluloses, alkyl alkoxyalkyl celluloses, carboxyalkyl celluloses, alkyl carboxyalkyl celluloses, (examples of which include carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixtures thereof)), and polymeric carboxylates (such as, for example, maleic acid / acrylic acid random copolymers or polyacrylate homopolymers).

[0154] The composition comprises a saturated or unsaturated fatty acid, preferably a saturated or unsaturated C 12 ~C 24It may further include fatty acids (0% to 10% by weight); deposition aids (e.g., polysaccharides, cellulose polymers, polydiallyldimethylammonium halides (DADMAC), and copolymers of DADMAC with random or block configurations of vinylpyrrolidones, acrylamides, imidazoles, imidazolinium halides, and mixtures thereof; cationic guar gum; cationic celluloses, such as cationic hydroxyethyl cellulose; cationic starches; cationic polyacrylamides; and mixtures thereof.

[0155] The composition may further comprise a dye transfer inhibitor, examples of which include manganese phthalocyanine, peroxidase, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone with N-vinylimidazole, polyvinyloxazolidone, and polyvinylimidazole, and / or mixtures thereof; chelating agents, examples of which include ethylene-diaminetetraacetic acid (EDTA); diethylenetriaminepentamethylenephosphonic acid (DTPMP); hydroxy-ethanediphosphonic acid (HEDP); ethylenediamine N,N'-disuccinic acid (EDDS); methylglycinediacetic acid (MGDA); diethylenetriaminepentaacetic acid (DTPA); propylenediaminetetraacetic acid (PDT A); 2-hydroxypyridine-N-oxide (HPNO); or methylglycine diacetate (MGDA); glutamic acid N,N-diacetate (N,N-dicarboxymethylglutamic acid tetrasodium salt (GLDA); nitrilotriacetic acid (NTA); 4,5-dihydroxy-m-benzenedisulfonic acid; citric acid and any salts thereof; N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), and derivatives thereof.

[0156] The composition may further comprise silicone or fatty acid based suds suppressors; enzyme stabilizers; color dyes, calcium and magnesium cations, visual signal components, antifoam agents (0.001% to about 4.0% by weight), and / or structurants / thickeners (0.01% to 5% by weight selected from the group consisting of diglycerides, triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulosic materials, microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof).

[0157] In some embodiments, the cleaning composition is a high density powder (HDD) composition comprising one or more subtilisin variants described herein. HDD powder laundry detergents are comprised of anionic laundry surfactants (selected from linear, branched or random chain, substituted or unsubstituted alkyl sulfates, alkyl sulfonates, alkyl alkoxylated sulfates, alkyl phosphates, alkyl phosphonates, alkyl carboxylates and / or mixtures thereof), non-ionic laundry surfactants (linear, branched or random chain, substituted or unsubstituted C8-C 18 Alkyl ethoxylates and / or C6-C 12alkylphenol alkoxylates), cationic laundry surfactants (selected from alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds and mixtures thereof); zwitterionic and / or amphoteric laundry surfactants (selected from alkanolamine sulfobetaines); amphoteric surfactants; laundry surfactants including semi-polar nonionic surfactants and mixtures thereof; builders (phosphate-free builders (e.g. zeolite builders [examples of which include Zeolite A, Zeolite X, Zeolite P and Zeolite MAP (in the range of 0% to less than 10% by weight)]); phosphate builders (e.g. sodium tripolyphosphate in the range of 0% to less than 10% by weight); citric acid, citrates and nitrilotriacetic acid or salts thereof in the range of less than 15% by weight; silicates (sodium or potassium silicate or sodium metasilicate in the range of 0% to less than 10% by weight, or layered silicates (SKS-6)); carbonates (sodium carbonate and / or or sodium bicarbonate; and bleaching agents (photobleaches, e.g., sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, xanthene dyes, and mixtures thereof); hydrophobic or hydrophilic bleach activators (e.g., dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine-TAED, and nonanoyloxybenzenesulfonate-NOBS, nitrile quaternary grades, and mixtures thereof); hydrogen peroxide; a source of hydrogen peroxide (inorganic hydrogen peroxide salts, e.g., mono- or tetrahydrates of sodium salts of perborate, percarbonate, persulfate, perphosphoric acid, or persilicic acid); hydrophobic and / or hydrophilic preformed peracids (selected from percarboxylic acids and their salts, percarbonates and their salts, perimidic acids and their salts, persulfates and their salts, and mixtures thereof) and / or bleach catalysts (e.g., imine bleach accelerators, such as iminium cations and polyions; iminium zwitterions; modified amines; modified amine oxides;N-sulfonylimines; N-phosphonylimines; N-acylimines; thiadiazole dioxides; perfluorimines; cyclic sugar ketones and mixtures thereof), metal-containing bleach catalysts (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations in combination with auxiliary metal cations such as zinc or aluminum, and sequestering agents such as ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid) and their water-soluble salts);

[0158] The composition may further include additional detergent ingredients, such as perfume microcapsules, starch encapsulated perfume accords, enzyme stabilizers, colorants, additional polymers including fabric integrity and cationic polymers, dielock components, fabric softeners, brighteners (e.g., CI optical brighteners), flocculants, chelating agents, alkoxylated polyamines, fabric precipitation aids, and / or cyclodextrins.

[0159] In some embodiments, the cleaning composition is an ADW detergent composition comprising one or more subtilisin variants described herein. The ADW detergent composition comprises two or more nonionic surfactants selected from the group of ethoxylated nonionic surfactants, alcohol alkoxylated surfactants, epoxy-capped poly(oxyalkylated) alcohol or amine oxide surfactants, present in an amount of 0-10% by weight; phosphates (monophosphates, diphosphates, tripolyphosphates, or oligomeric polyphosphates), sodium tripolyphosphate-STPP, or phosphate-free builders (amino acid based compounds, e.g., MGDA (methyl-glycine-diacetic acid), and salts and derivatives thereof, GLDA (glutamic acid-N,N-diacetic acid), and salts and derivatives thereof, IDS (imidazolidinyl esters), and / or glyceryl esters, present in an amount of 0-10% by weight. nodisuccinic 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, polycarboxylic acid monomers and hydroxycarboxylic acid monomers and their salts (in the range of 0.5% to 50% by weight) in the range of 5 to 60%; sulfonated / carboxylated polymers (to impart dimensional stability to the product) in the range of about 0.1% to about 50% by weight; and about 0.5% to about 50% by weight of sulfonated / carboxylated polymers (to impart dimensional stability to the product).drying aid polyesters, in particular anionic polyesters (optionally in combination with monomers having 3 to 6 functional groups conducive to condensation polymerization, in particular acid, alcohol or ester functional groups), selected from polycarbonate-, polyurethane- and / or polyurea-polyorganosiloxane compounds or their precursor compounds of reactive cyclic carbonate and urea type, 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 bleaching agents (for example salts of hydrogen peroxide, for example perborates, percarbonates, perphosphates, persulfates and persilicates), and and organic bleaching agents (e.g., organic peroxyacids, such as diacyl and tetraacyl peroxides, particularly diperoxydodecanedioic acid, diperoxytetradecanedioic acid and diperoxyhexadecanedioic acid); bleach activators - organic peracid precursors in the range of about 0.1% to about 10% by weight; bleach catalysts (selected from manganese triazacyclononanes and related complexes, Co, Cu, Mn and Fe bispyridylamines and related complexes, and pentaamine acetate cobalt(III) and related complexes); metal care agents (selected from benzotriazoles, metal salts and complexes, and / or silicates) in the range of about 0.1% to 5% by weight; and active enzymes per gram of detergent composition for ADW from about 0.01 to 5.Enzymes (acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, mannanase, hyaluronidase, keratin ... enzymes, oxidases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, peroxidases, phenol oxidases, phosphatases, phospholipases, phytases, polygalacturonases, proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases and mixtures thereof; and enzyme stabilizing components selected from oligosaccharides, polysaccharides, and inorganic divalent metal salts.

[0160] Further embodiments relate to compositions and methods for treating fabrics (e.g., for desizing woven fabrics) with one or more subtilisin variants described herein. Fabric treatment methods are well known in the art (see, e.g., U.S. Pat. No. 6,077,316). For example, the feel and appearance of fabrics can be improved by a method comprising contacting the fabric in a solution with a variant described herein. The fabric can be treated with the solution under pressure.

[0161] One or more subtilisin variants described herein can be applied during or after weaving of the fabric, at the desizing stage, or at one or more additional fabric processing steps. During weaving of a woven fabric, the yarns are subjected to significant mechanical strain. Prior to weaving on the loom, the warp yarns are often coated with sized starch or starch derivatives to increase their tensile strength and prevent breakage. One or more subtilisin variants described herein can be applied during or after weaving to remove the sized starch or starch derivatives. After weaving, the variants can be used to remove the sizing coating before further processing of the fabric to ensure a uniform and wash-resistant result. One or more subtilisin variants described herein can be used, for example, as detergent additives in aqueous compositions, alone or with other desizing agents and / or desizing enzymes for desizing fabrics, including cotton-containing fabrics. Amylases can also be used in compositions and methods for creating a stonewashed look on indigo dyed denim fabrics and garments. To produce garments, fabrics can be cut and sewn into garments or garments that are subsequently finished. Various enzymatic finishing methods have been developed, particularly for producing denim jeans. Denim garment finishing typically begins with enzymatic desizing, during which the garment is subjected to the action of proteolytic enzymes to impart softness to the fabric and make the cotton more amenable to subsequent enzymatic finishing steps. One or more subtilisin variants described herein can be used in denim garment finishing (e.g., "bio-stoning"), enzymatic desizing and fabric softening and / or finishing process methods.

[0162] In further embodiments, one or more of the subtilisin variants described herein can be used in industrial and industrial cleaning applications, such as, for example, industrial or industrial warewashing systems, machines, or processes; industrial or industrial commercial laundry systems, machines, or processes; and industrial or industrial laundry systems, machines, or processes.

[0163] One or more subtilisin variants described herein can be used to remove proteins from animals and their subsequent dissection or processing, such as feathers, skin, hair, and hide. In some cases, immersing an animal carcass in a solution containing one or more subtilisin variants described herein can prevent skin damage compared to traditional scalding or feather removal processes. In one embodiment, one or more subtilisin variants described herein can be sprayed onto feathers under conditions suitable for initiating feather digestion or feather decomposition. In some embodiments, the variants can be used in combination with an oxidizing agent.

[0164] In some embodiments, one or more subtilisin variants described herein can aid in the removal of oil or fat associated with raw feathers. In some embodiments, one or more subtilisin variants described herein are used in compositions for cleaning feathers and for disinfecting and partially dewatering fibers. In still other embodiments, one or more subtilisin variants described herein are used for the recovery of proteins from feathers. In some other embodiments, one or more subtilisin variants described herein are applied at about 0.5% (volume / volume) in combination with 95% ethanol or other polar organic solvents in a cleaning solution with or without surfactants. In other embodiments, one or more subtilisin variants described herein may be used alone or in combination in suitable feather treatment and protein degradation methods, such as those disclosed in International Application No. PCT / EP2013 / 065362, International Application No. PCT / EP2013 / 065363, and International Application No. PCT / EP2013 / 065364. In some embodiments, the recovered protein can then be used in animal or fish feed.

[0165] In yet another embodiment, one or more animal feed compositions, animal feed additives and / or pet foods comprise one or more subtilisin variants described herein. Other embodiments relate to methods of preparing such animal feed compositions, animal feed additive compositions and / or pet foods comprising mixing one or more subtilisin variants described herein with one or more animal feed ingredients and / or animal feed additive ingredients and / or pet food ingredients.

[0166] The term "animal" includes all non-ruminant and ruminant animals. In certain embodiments, the animal is a non-ruminant, such as horses and monogastric animals. Examples of monogastric animals include, but are not limited to, pigs, such as piglets, growing pigs, and sows; poultry, such as ostriches, ducks, chickens, broilers, and egg-laying hens; fish, such as salmon, trout, tilapia, catfish, and carp; and crustaceans, such as shrimp and prawns. In further embodiments, the animal is a ruminant, including, but not limited to, cows, calves, goats, sheep, giraffes, bison, moose, elk, yak, buffalo, deer, camels, alpacas, llamas, antelopes, pronghorns, and nilgai.

[0167] In the context of this specification, the term "pet food" is intended to be understood to mean food for domestic animals such as, but not limited to, dogs, cats, gerbils, hamsters, chinchillas, fancy rats, guinea pigs; avian pets such as canaries, parakeets and parrots; reptile pets such as turtles, lizards and snakes; and aquatic animals such as tropical fish and frogs.

[0168] The terms "animal feed composition", "feed" and "forage" are used interchangeably and include any of the following: 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 grains with solubles (DDGS) (especially corn-based distillers dried grains with solubles (cDDGS), bran, wheat middlings, wheat shorts, wheat sorghum, wheat gluten meal ... shorts), rice bran, rice hulls, oat husks, palm kernels, and citrus pulp; c) proteins obtained from sources such as soybeans, sunflowers, peanuts, lupins, peas, broad beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame, and the like; d) fats and oils obtained from vegetable and animal sources; and e) minerals and vitamins.

[0169] One or more of the subtilisin variants described herein are further used in the enzyme-assisted bleaching of paper pulps, such as chemical pulps, semi-chemical pulps, kraft pulps, mechanical pulps, pulps produced by the sulfite process, etc. Generally, the paper pulp is incubated with one or more of the subtilisin variants described herein under conditions suitable for bleaching the paper pulp.

[0170] In some embodiments, the pulp is a chlorine-free pulp bleached with oxygen, ozone, peroxide or peroxyacid. In some embodiments, one or more subtilisin variants described herein are used in the enzyme-assisted bleaching of pulps exhibiting low lignin levels produced by improved or continuous cooking processes. In some other embodiments, one or more subtilisin variants described herein are used alone or, preferably, in combination with xylanase enzymes and / or endoglucanase enzymes and / or alpha-galactosidase enzymes and / or cellobiohydrolase enzymes.

[0171] In other embodiments, one or more subtilisin variants described herein are further used in enzyme-assisted debridement of tissue, including removal of necrotic or damaged tissue, for example, from a wound to promote healing.

[0172] In yet further embodiments, one or more subtilisin variants described herein are further used in tissue culture. In particular, one or more subtilisin variants described herein can be used to suspend or resuspend cells attached to a cell culture wall, such as in a cell harvesting step. In another embodiment, one or more subtilisin variants described herein can be used to cleave protein bonds between cultured cells and the dish, suspending the cells in solution.

[0173] In yet another embodiment, one or more subtilisin variants described herein are further used as food additives, digestive aids and / or food processing aids.

[0174] In yet another embodiment, one or more of the subtilisin variants described herein are further used in leather processing by removing hair, soaking, degreasing, or demineralizing from animal hides, which is a process that involves the breakdown of nonstructural proteins during leather production. EXAMPLES

[0175] Example 1 Assay The following assays were used in the examples below. Any deviations from the assays described herein below will be addressed in the examples where such deviations occurred.

[0176] figure of merit The enzyme performance index (PI) compares the performance (measured) of a mutant to a parent enzyme selected from AprL wild type (WT) (SEQ ID NO: 2) (hereafter referred to as "AprL parent"), BliD02339WT (SEQ ID NO: 9) (hereafter referred to as "BliD parent"), or SQCBV35 mutant (SEQ ID NO: 63) (hereafter referred to as SQCBV35 parent) at the same protein concentration (theoretical or measured). The theoretical concentration of the parent enzyme can be calculated using parameters extracted from a Langmuir fit of the standard curve of the parent enzyme. A PI greater than 1 (PI>1) indicates improved performance by the mutant compared to the parent, a PI of 1 (PI=1) indicates a mutant with the same performance as the parent, and a PI less than 1 (PI<1) indicates a mutant with poorer performance than the parent.

[0177] Protein quantification assays Protein measurements of AprL or BliD mutants from culture supernatants were performed by Agilent U-HPLC or HPLC systems. For the U-HPLC method, a calibration curve (0–500 ppm) of purified parent protein was prepared and used to generate a standard curve. AprL or BliD mutants from clarified culture supernatants were diluted 10-fold with dilution buffer (25 mM Tris, pH 7.4, 5 mM CaCl2) and then mixed with acetonitrile buffer (22.5 mM Tris, pH 7.4, 4.5 mM CaCl2, 9% acetonitrile) in a 1:1 ratio. Afterwards, the samples were filtered using a 45 μm filter plate and fed through an autosampler onto a reversed-phase column (Zorbax 300 SB-C3 column, 2.1 × 100 mm and 2.1 × 50 mm, both with 1.8 μm bead size). Samples were eluted through 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 with a 4 min run and 1 min post column equilibration. Absorbance was measured at 220 nm and peaks were integrated using ChemStation software (Agilent Technologies). Protein concentrations of samples were calculated based on a standard curve of purified parent enzyme. In some cases, the Bradford method for protein determination was used with HPLC. Absorbance was measured at 595 / 450 nm on a Spectramax plate reader using Bradford reagent (Bradford Quickstart 1X dye reagent, BioRad).

[0178] Alternatively, protein determination of AprL variants from culture supernatants was performed by HPLC using an Agilent 1100 / 1200 system. A calibration curve (0–1500 ppm) with purified parent protein was made and used to generate a standard curve. AprL variants from clarified culture supernatants were analyzed by one of two methods. In some cases, culture supernatants were diluted two-fold with dilution buffer (Tris 100 mM, pH 8.6, 0.005% Tween 80) and loaded through an autosampler onto an Agilent C8 column (Agilent Poroshell 5 μm 300SB-C8 2.1 × 75 mm) pre-equilibrated at 69–70 °C. Samples were eluted from the column with a gradient of buffer A (0.1% TFA) and buffer B (100% acetonitrile and 0.1% TFA). The flow rate was 2 mL / min for a 2-min run. In other cases, clarified culture supernatants containing AprL mutants were pretreated in acid / SDS solution. 75 μL of sample was mixed with 6.4 μL of 4N HCl and mixed for 10 min on ice. 20.4 μL of 10% SDS solution was added per well, mixed, and the sample was filtered and then loaded onto an HPLC HIP-ALS Aquity 125A USEC column set at room temperature. Samples were eluted with a solution of 25 mM sodium acetate, pH 5.5, 100 mM NaCl, 0.5% SDS, at a flow rate of 0.35 mL / min for 8 min. Absorbance was measured at 220 or 230 nm, and peaks were integrated using ChemStation (Agilent Technologies) software.

[0179] For all three protein determination methods, the protein concentration of each sample was calculated based on a standard curve of the purified parent enzyme.

[0180] Protease activity The protease activity of the parent enzyme and its mutants was tested by measuring the hydrolysis of N-suc-AAPF-pNA or dimethylcasein (DMC) substrates.

[0181] The reagent solutions used for the AAPF assay were 100 mM Tris pH 8.6, 10 mM CalCl2, 0.005% Tween®-80 (Tris / Ca buffer) and 160 mM suc-AAPF-pNA / DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388). To prepare working solutions, 1 mL of suc-AAPF-pNA stock solution was added to 100 mL of Tris / Ca buffer and mixed. Enzyme samples were added to a microtiter plate (MTP) containing 1 mg / mL of suc-AAPF-pNA working solution and activity was assayed at 405 nm over 3-5 min at RT using a SpectraMax plate reader in kinetic mode. Protease activity was expressed in mOD / min.

[0182] The reagent solutions used for the DMC assay were 2.5% DMC (Sigma) in 100 mM sodium carbonate, pH 9.5, 0.075% TNBSA (2,4,6-trinitrobenzenesulfonic acid, Thermo Scientific) in Reagent A. Reagent A: 45.4 g Na2B4O7.10H2O (Merck) in 15 mL 4N NaOH, with MQ water to a final volume of 1000 mL, Diluent: 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 with slow mixing. Activity was measured at room temperature for 5 minutes at 405 nm using a SpectraMax plate reader in kinetic mode. Activity was expressed as mOD / min as described above in the AAPF assay.

[0183] Detergent Compositions Evaluated The detergent formulations used in these studies are shown in Table 1 and include heavy duty liquid laundry (HDL), heavy duty heavy duty laundry (HDD), and automatic dishwashing (ADW) formulations. Commercial detergents were heat inactivated to eliminate enzyme activity and at the doses listed in Table 1. Detergent treatments for enzyme inactivation were as follows: HDL laundry detergents containing enzymes (except Test HDL1, Test HDL2, and Persil Non-Bio HDL detergent) were inactivated by heating the raw liquid in a water bath at 95°C for 16 hours. The enzymes in HDD detergents were inactivated by preparing a solution 10 times more concentrated than the solution used in the final wash assay and heating at 95°C for 16 hours. Commercial ADW detergent formulations were inactivated by heating at 65°C for 30-45 minutes as follows: The enzyme activity was inactivated by dissolving the ADW detergent tablet in 700 mL of 21GH water and incubating this solution at 65°C for 30-45 minutes. The enzyme inactivation solution was diluted 10-fold with 21GH water prior to use in the assay. After inactivation, protease activity was assayed using AAPF substrate to confirm complete inactivation. No protease activity was detectable after heat treatment of HDD, ADW and HDL detergents. HDL detergents: Persil Non-Bio, Test HDL1 and Test HDL2 were tested for elemental boron content and had a boron content of ≦5mg / Kg and are therefore considered boron-free. GSM-B powder formulations were dissolved at 3g / L for use and the pH was not adjusted.

[0184] [Table 2]

[0185] Detergent sources: ALL, Kirkland Ultra and Kirkland Ultraclean (Sun Products), OMO Color, OMO Klein&Krachtig, 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 above detergents 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 HDL1 and Test HDL2 detergents were custom-made in-house, and their compositions are listed in Tables 1.2 and 1.3, respectively. Table 1.4 shows the composition of the GSM-B phosphate-free ADW detergent (purchased without enzymes from WFK Testgewebe GmbH, Brueggen, Germany, www.testgewebe.de). The GSM-B9 formulation was prepared by adjusting the pH of the purchased GSM-B phosphate-free detergent downward to pH 9. Table 1.5 shows the composition of the custom liquid ADW detergent formulations adjusted to test pH 7 and pH 9.

[0186] [Table 3]

[0187] [Table 4]

[0188] [Table 5]

[0189] [Table 6]

[0190] Washing performance of laundry (HDL and HDD) and ADW detergents The cleaning performance of the variants against the parent enzyme was tested on a variety of technical stains: BMI (EMPA-116, blood / milk / ink on cotton), egg (CS-38, egg yolk with heat aged dye), and POM (CFT-C10, dye / oil / milk) used in the laundry base, and egg yolk (PAS-38, egg yolk on polyacrylic fabric, aged and stained with carbon black dye) used in the dishware base. EMPA-116, CS-38, and CFT-C10 swatches were pre-rinsed for 20 min with deionized water and dried overnight at room temperature. PAS-38 fabric was pre-rinsed for 30 min at 60 °C in 10 mM CAPS buffer (pH 11) at 900 rpm in an iEMS shaker, rinsed with 2 ml of deionized water, and dried overnight. All stains were prepared by placing pre-punched swatches into MTP plates (Corning 3641) by the Center for Test materials BV, Vlaardingen, Netherlands. The microswatch-containing plates were flooded with detergent and then the enzyme was added.

[0191] General sample setup for stability assays The mutants were tested for stability under various stress conditions (buffers and detergents as shown in Table 2 below) by measuring the residual activity after incubation of the mutants at elevated temperatures. The elevated temperatures were set to give approximately 30% residual activity in the stressed samples compared to the unstressed samples. Diluted enzyme samples were mixed with the stressor and the unstressed protease activity was measured. Diluted samples in the stressor were incubated at elevated temperatures for the times shown in Table 2, followed by measurement of the activity of the stressed protease by either AAPF or DMC hydrolysis. The detergent enzymes were heat inactivated as described above before use in the stability assays.

[0192] [Table 7]

[0193] For unstressed conditions, the enzymes were immediately assayed for activity towards AAPF or DMC (see above). For stressed conditions, the PCR plates were sealed and incubated at elevated temperature for 5 min using an Eppendorf 385 MasterCycler Pro Thermocycler or a PTC-200 Peltier thermal cycler and then assayed for activity. Stressed and unstressed activities were measured by either synthetic substrate hydrolysis or the DMC method as described above. % residual activity was calculated by taking the ratio of stressed to unstressed activity and multiplying by 100. The residual activity of the mutants was divided by that of the parent enzyme to obtain the stability PI.

[0194] For the laundry assay, an aliquot of enzyme was added to the detergent-filled MTP containing the microswatches to a final volume of 200 μL with a final enzyme concentration of 0.5–5 ppm. Laundry wash assays with HDL or HDD formulations were performed at 25°C for 15–30 min, while ADW assays were performed at 40°C for 30 min. After incubation, 100–150 μL of supernatant was transferred to a fresh MTP and absorbance was read at 600 nm for EMPA-116 swatches or 405 nm for CS-10, CS-38, and PAS-38 swatches using a SpectraMax plate reader. Absorbance results were obtained by subtracting the blank control (no enzyme) value from each sample value. The absorbance of a given variant was divided by the predicted absorbance of the parent enzyme at the same concentration to obtain the wash PI for each assay condition. Values ​​for parent enzyme were determined by fitting a standard curve of purified parent enzyme samples to a Langmuir fit.

[0195] Degradation laundry wash assay (A) MTP assay: A mini stir disk was placed in each well of a 96-well MTP followed by the addition of 270 μL of 100% Persil Non-Bio detergent. 18.1 μL of enzyme supernatant diluted to approximately 500 ppm was added to the detergent-filled MTP and mixed for 5 minutes using a magnetic rotary stirrer (V&P Scientific, VP710 series). For the initial time point (T=0), 25 uL of enzyme-containing detergent was transferred to an MTP filled with 125 μL of buffer (5 mM HEPES, 250 ppm water hardness with 3:1 Ca:Mg, pH 8). A 5 μL aliquot of the enzyme diluted in detergent was then added to 185 μL buffer (5 mM HEPES, 250 ppm hardness, pH 8) that had been previously added to the MTP containing the EMPA-116BMI microwatch. At time point 0 (T=0), the wash assay was performed similarly to the method described above for fresh washes. 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 1.5, 5, or 24 hours; at the end of each time point, aliquots were removed and cleaning assays were performed similarly to the method described above for fresh washes. Residual cleaning values ​​were obtained by comparing the washes at the various remaining time points to the initial wash at T=0.

[0196] (B) Conical tube assay: Clarified enzyme culture supernatant and 100% of the test HDL1 detergent were added to 15 mL polystyrene centrifuge tubes in a final volume of 1.1 mL with a final enzyme concentration of 88 ppm in the detergent. For each mutant, a 5 μL sample was immediately removed from the tube and frozen for later assay as the initial time point (T=0). The diets were incubated at 37°C for 3 days. After incubation, a 5 μL sample was removed and frozen. To prepare samples for the washing assay, all samples were thawed and 660 μL of buffer (5 mM HEPES, 250 ppm water hardness with 3:1 Ca:Mg, pH 8) was added to the sample. A 200 μL aliquot of the diluted sample was added to the MTP containing the EMPA-116BMI microswatch. The laundry washing assay was performed for 60 min at 25°C in an iEMS incubator shaking at 1400 rpm. After incubation, 120 μL of the supernatant was transferred to a fresh MTP and the absorbance was read at 600 nm using a SpectraMax plate reader. The blank control (no enzyme) value was subtracted from each sample value to obtain the absorbance results. The residual cleanability of each variant was calculated as the ratio of the residual cleanability after 3 days to the cleanability at T=0 and reported as percent residual cleanability.

[0197] Example 2 Combinatorial design and expression of AprL-clade protease mutants The Bacillus sp. AprL subtilisin and the B. licheniformis Bra7 BliD02339 subtilisin, both members of the AprL-clade, were engineered to generate AprL-clade protease variants with improved cleaning performance and detergent stability. The AprL-clade is described in more detail in International Application No. PCT / US16 / 32514, filed May 13, 2016, and published as WO 2016183509.

[0198] The amino acid sequence of the mature AprL parent enzyme is set forth as SEQ ID NO:2. A synthetic gene (SEQ ID NO:1) encoding the AprL parent protease was synthesized by GeneArt, Life Technologies, Inc. and used to generate mutant sequences using conventional molecular biology techniques (see, e.g., Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory Press). Genes encoding AprL mutants were also synthesized. The various AprL genes were then cloned into the pHYT expression vector (from pHY300PLK (Takara-Clontech)). The expression vector contained the aprE promoter (SEQ ID NO:3), aprE signal peptide (SEQ ID NO:4), AprL propeptide (SEQ ID NO:5), and BPN' terminator sequence (SEQ ID NO:6). DNA fragments encoding the various mature protease sequences of interest (wild type and mutants) were introduced using techniques known in the art. An appropriate B. subtilis host strain was transformed with the expression plasmids containing the AprL parent and protease mutant gene sequences. Cells were grown in culture medium (MOP buffer-based enriched semi-synthetic medium with urea as the main nitrogen source, glucose as the main carbon source, supplemented with 1% soytone for robust cell growth, and antibiotic selection) in 96-well MTPs at 80% humidity, 32 °C, and 300 rpm in a shaking incubator for 2 days. After centrifugation and filtration, the clarified culture supernatant containing the proteases of interest was used for the assay. The same process was used for the construction of BliD02339 wild-type and mutant expression cassettes and subsequent transformation and cultivation of B. subtilis host strains.

[0199] In some cases, AprL mutants were generated from integration cassettes targeted to the B. subtilis aprE locus. A pool of DNA encoding genes of interest was introduced into an expression cassette, which was then integrated into the chromosome of an appropriate B. subtilis strain using methods known in the art. A DNA fragment containing the following components was part of the expression cassette: the BPN' terminator (SEQ ID NO:6), a chloramphenicol resistance marker from Staphylococcus aureus (SEQ ID NO:165), the B. subtilis aprE promoter (SEQ ID NO:3), the B. subtilis aprE signal peptide sequence (SEQ ID NO:4), and the AprL propeptide (SEQ ID NO:5). The transformed cells were cultured as above, and the proteins of interest were similarly isolated.

[0200] Example 3 Performance evaluation of AprL mutants in laundry applications Mutants of the AprL parent protease (SEQ ID NO: 2) (hereinafter referred to as "AprL mutants") prepared as described in Example 2 and shown in Table 3 were evaluated for cleaning performance and stability as described in Examples 3-7.

[0201] The AprL variants listed in Table 3 were tested in the laundry cleaning performance assay described in Example 1 using the EMPA-116BMI stain and the following laundry detergents: Kirkland Ultra HDD, OMO Color HDD, Surf Excel, Kirkland Ultraclean HDL, OMO Klein&Krachtig HDL and Blue Moon HDL. The AprL variants from Table 3 that have a PI>1.1 relative to the AprL parent enzyme are listed in Table 4.

[0202] [Table 8]

[0203] [Table 9]

[0204] Example 4 Performance evaluation of AprL mutants in dishwashing applications The AprL variants listed in Table 3 were tested in the ADW cleaning performance assay described in Example 1 using PAS-38 egg yolk stain and the following ADW detergents: GSM-B pH 10, GSM-B pH 9, SUN® All in One, Finish® Quantum, and Finish® All in One. AprL variants (shown in Table 3) with a PI>1.01 in at least one of these ADW detergents against the AprL parent enzyme are shown in Table 5. AprL variants from Table 3 with a PI>1.01 in all of these ADW detergents against the AprL parent enzyme are shown in Table 6.

[0205] [Table 10]

[0206] [Table 11]

[0207] Example 5 AprL mutants with improved stability The AprL variants listed 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% Kirkland Ultra HDL pH 8.2 at 56° C., OMO Klein&Krachtig HDL pH 8.2 at 51° C. AprL variants from Table 3 that (i) have a stability PI>1.1 relative to the AprL parent enzyme in at least one of the stability assays described in Example 1, and (ii) have a wash PI>1.01 in both the wash and ADW wash performance assays described in Example 1 are listed in Table 7. AprL variants from Table 3 that have a PI>1.1 relative to the AprL parent enzyme in at least one of the wash (HDL or HDD), ADW, or stability assays described in Example 1 are listed in Table 8.

[0208] [Table 12]

[0209] [Table 13]

[0210] Example 6 Performance evaluation of additional AprL mutants The AprL variants listed in Table 3 were tested in the protease activity assay (AAPF or DMC assay) as described in Example 1, the laundry cleaning performance assay as described in Example 1 with EMPA-116 BMI stain and All HDL detergent and OMO Klein&Krachtig HDL detergent, the ADW cleaning performance assay as described in Example 1 with PAS-38 egg yolk stain and GSM-B pH 10 and Finish® Quantum dish detergent, and the stability assay as described in Example 1 in the following buffers: LAS-EDTA buffer at 53.5° C. and Tris-EDTA buffer at 51° C. The AprL variants from Table 3 that have a laundry cleaning PI>1.01 relative to the AprL parent enzyme are listed in Table 9. The AprL variants from Table 3 that have an ADW cleaning PI>1.01 relative to the AprL parent enzyme are listed in Table 10. The AprL variants from Table 3 that have a stability PI>1.01 relative to the AprL parent enzyme are listed in Table 11. The AprL variants from Table 3 that have laundry cleaning, ADW cleaning and stability PIs > 1.01 relative to the AprL parent enzyme in at least one detergent are shown in Table 12.

[0211] [Table 14]

[0212] [Table 15]

[0213] [Table 16]

[0214] [Table 17]

[0215] Example 7 Cleaning performance of AprL mutants in boron-free detergents The cleaning performance of purified AprL mutants, M123I-Q136R, M123I-Q136R-G165Q, V045R-Q136R-G165Q-S258E, V045R-M123I-Q136R-G165Q-S258E and V045R-M123I-Q136R, was tested in the following detergents, All 3X, Purex® Dirt Lift and Persil Non Bio, which do not contain detectable amounts of boron or boron-containing compounds. These AprL mutants were tested in the laundry cleaning performance assay described in Example 1. The results are shown in Table 13 as calculated PI relative to the AprL parent enzyme. The proteases were tested at a dosage of 2.5 ppm. The amount of boron present in the boron-free detergents of this example was quantified by elemental analysis, which in all cases detected only trace amounts (less than 5 mg) of boron per kg of detergent.

[0216] [Table 18]

[0217] Example 8 Cleaning performance and detergent stability of AprL and BliD02339 protease mutants The AprL mutants shown in Table 14 were generated as described in Example 2. The cleaning performance and stability of the mutants in Table 14 were tested in one or more of the cleaning performance and stability assays described in Example 1.

[0218] Laundry cleaning performance of the AprL variants shown in Table 14 was tested on EMPA-116 microswatches in test HDL1 and Persil Non-Bio detergents (Table 1.1, assay conditions 8 and 10). Enzyme stability was tested at 52.5°C and / or 54°C in 10% test HDL1 detergent (Table 2, assay condition 5). Results are shown in Table 15 as PI values ​​compared to the AprL parent enzyme, where ND means not determined.

[0219] Table 17 shows the performance of AprL variants tested in laundry cleaning performance and stability assays described in Example 1: (i) cleaning performance on EMPA-116, CFT CS-38 and CFT-C10 microswatches in Persil Non-Bio, Test HDL1 and Test HDL2 detergents, and (ii) stability in 10% PNB at 63° C. for 15-20 minutes and / or in 10% Test HDL1 at 60° C. for 15-20 minutes. Cleaning performance results are reported as PI values ​​for the SQCBV35 variant (SEQ ID NO: 63) and stability results are reported as % residual activity of the sample after stress relative to non-stress conditions. ND means that the value was not determined.

[0220] [Table 19]

[0221] [Table 20]

[0222] [Table 21]

[0223] [Table 22]

[0224] [Table 23]

[0225] [Table 24]

[0226] [Table 25]

[0227] [Table 26]

[0228] [Table 27]

[0229] [Table 28]

[0230] Mutants of the BliD02339 parent protease (SEQ ID NO: 9) listed in Table 18 (hereinafter "BliD mutants") were generated as described in Example 2 and tested for cleaning performance and stability as described in Example 1.

[0231] Table 18 shows BliD variants that showed improvement over the BliD parent enzyme in cleaning performance against three technical stains microwatch, namely EMPA-116, CFT CS-38, and CFT-C10, when tested in Persil Non-Bio and Test HDL2 laundry detergent, and also showed improved stability when tested in 10% Persil Non-Bio (PNB) laundry detergent (stressed at 63° C. for 15-20 minutes). The cleaning performance and stability results are shown in Table 19. Cleaning performance results are reported as PI values ​​relative to the BliD parent enzyme (SEQ ID NO: 9) and stability results are reported as % residual activity of samples after stress relative to non-stress conditions. ND means that the value was not determined.

[0232] [Table 29]

[0233] [Table 30]

[0234] Example 9 Comparison of AprL protease to related molecules Identification of homologous proteases Proteins related to AprL protease (SEQ ID NO:2) were identified by a BLAST search (Altschul et al., Nucleic Acids Res, 25:3389-402, 1997) against the non-redundant NCBI protein database, and a subset is shown in Table 20. A similar search was performed against the Genome Quest Patent database with search parameters set to default values, and a 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) for both search sets is defined by dividing the number of identical residues by the number of aligned residues in the pairwise alignment. The column entitled "Sequence Length" corresponds to the length (in amino acids) of the protein identified by the accession number in the column entitled "Accession Number", and "Alignment Length" refers to the length (in amino acids) of the sequence used for the alignment and PID calculation.

[0235] [Table 31]

[0236] [Table 32]

[0237] [Table 33]

[0238] Alignment of subtilisin sequences with AprL Using the CLUSTALW software (Thompson et al., Nucleic Acids Research, 22:4673-4680, 1994) with default parameters, the amino acid sequences of the mature AprL (SEQ ID NO:2) and BliD02339 (SEQ ID NO:9) proteins were aligned with the mature sequences of several subtilisins in Tables 20 and 21. The alignment is shown in FIG. 1 with boxes drawn around one or more amino acid motifs selected from SEQ ID NOs:166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178 and 179.

[0239] Example 10 Phylogenetic tree of subtilisins including AprL and BliD02339 Phylogenetic trees for the mature sequences of AprL protein (SEQ ID NO:2), BliD02339 protein (SEQ ID NO:9), AprL protease variants: namely SQM-30 (SEQ ID NO:36), SQCBV20 (SEQ ID NO:57), SQCBV35 (SEQ ID NO:63), SQCBV37 (SEQ ID NO:65), SQCBV188 (SEQ ID NO:105), SQCBV269 (SEQ ID NO:140), SQBLV134 (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:384) were constructed using a subset of the subtilisin sequences shown in Tables 20 and 21. The sequences were entered into Vector NTI Advance suite and a dendrogram was generated using the Neighbor-Joining (NJ) method (Saitou, N.; and Nei, M. (1987) Mol Biol Evol 4, 406-425). The NJ method works with a distance matrix between all the sequence pairs analyzed. These distances relate to the degree of dissimilarity between the sequences. The dendrogram was calculated after sequence alignment. The tree construction was calculated using the following parameters: Kimura's sequence distance correction and sites containing gaps were ignored. The MEGA 6 program was used to display the phylogenetic tree shown in Figure 2. Based on the phylogenetic distribution, certain subtilisins were grouped to form the AprL-clade as shown in Figure 2.

[0240] Example 11 Additional AprL mutants The AprL mutants shown in Table 22 were generated as described in Example 2.

[0241] [Table 34]

[0242] [Table 35]

[0243] [Table 36]

[0244] [Table 37]

[0245] [Table 38]

[0246] [Table 39]

[0247] [Table 40]

[0248] Example 12 Laundry cleaning performance and detergent stability of additional AprL mutants The AprL variants shown in Table 22 and SQCBV35 were tested for laundry washing performance and stability as described in Example 1. BMI (EMPA-116) microswatches were used in Persil Non-Bio, Blue Moon and Liby detergents (Table 2, assay conditions 6, 10, and 16). Enzyme stability was tested at 62-65°C in 10% Persil Non-Bio, Blue Moon and Liby detergents (Table 2).

[0249] Table 23 shows the results of cleaning performance in Persil Non-Bio as PI values ​​compared to the SQCBV35 variant (SEQ ID NO: 63). Table 23 also shows the results for stability in 10% PNB at 62°C for 15 minutes, 10% Blue Moon at 64°C for 15 minutes, and 10% Liby at 65°C for 15 minutes, reported as % residual activity of the sample after stress relative to non-stress conditions. Table 24 shows the results of cleaning performance in Persil Non-Bio (PNB), Blue Moon and Liby detergents as performance index (PI) values ​​compared to the SQCBV35 variant (SEQ ID NO: 63). Table 24 also shows the results for stability in 10% PNB detergent at 62°C for 15 minutes, 10% Blue Moon detergent at 64°C for 15 minutes, and 10% Liby detergent at 65°C for 15 minutes, reported as % residual activity of the sample after stress relative to non-stress conditions. Table 25 shows the results of cleaning performance in Persil Non-Bio detergent using EMPA-116, CS-38 and CFT-10 stains as PI values ​​compared to the SQCBV35 variant (SEQ ID NO: 63). Table 25 also shows the results of stability in 10% PNB at 66°C for 20 minutes, 10% Blue Moon at 68°C for 20 minutes and 10% Liby at 69°C for 20 minutes, reported as % residual activity of the sample after stress relative to non-stress conditions. Table 26 shows the results of laundry cleaning performance and stability of the various AprL variants in Liby, Blue Moon and PNB detergents. The stability conditions are listed in Table 2 of Example 1. Table 27 shows the performance of the various AprL variants in the degraded laundry cleaning assay (performed as described in Example 1: Degraded laundry cleaning assay (A)). Degraded cleaning performance is reported as % cleaning activity remaining after incubating samples in 100% Blue Moon detergent at 37°C for 3 weeks.

[0250] [Table 41]

[0251] [Table 42]

[0252] [Table 43]

[0253] [Table 44]

[0254] [Table 45]

[0255] [Table 46]

[0256] Example 13 ADW cleaning performance and detergent stability of additional AprL mutants The cleaning performance of AprL protease variants, AprL wt and SQCBV35, shown in Table 22, was measured in dish-based use (ADW) using various detergent formulations shown in Table 1.1 and pre-punched egg yolk microswatches (PAS-38) that were pre-rinsed or not pre-rinsed before the assay. To prepare pre-rinsed PAS-38 swatches, 180 μl of 10 mM CAPS buffer (pH 11) was added to the MTP containing the microswatches, the plate was sealed and incubated in an iEMS incubator at 60° C. and 1100 rpm for 30 minutes. The buffer was then removed and the swatches were rinsed with deionized water and air-dried. To measure enzyme cleaning in ADW formulations, 175 μL of detergent solution (3 g / L detergent prepared with 374 ppm water hardness) was added to each well of the MTP, followed by 5 μL of enzyme. The MTP was sealed and incubated in an iEMS incubator at 40°C with a shaking speed of 1100 rpm for 30 minutes. After incubation, 100 μL of wash solution from each well was transferred to a new MTP and the absorbance of the supernatant was measured at 405 nm on a SpectraMax plate reader. The blank control (no enzyme) value was subtracted from each sample in the GSM-B pH 10.5 formulation to obtain the net absorbance. When using the custom liquid ADW detergent formulation (at pH 7 and 9), all the supernatant was removed from the MTP wells after the enzyme incubation and transferred to another MTP to determine the absorbance at 405 nm. The swatches in the MTP were rinsed with deionized water, then 180 μL of 10 mM CAPS buffer (pH 11) was added per well and the plate was incubated for an additional 10 minutes at 40°C with 1100 rpm in an iEMS incubator. After this final incubation, 100 μL of the resulting wash solution was transferred to a new MTP and the absorbance was measured at 405 nm. To determine the overall cleaning performance in the custom liquid ADW detergent formulations (at pH 7 and 9), the two resulting absorbance measurements were added and this sum was used to calculate the performance index (PI) for each enzyme.

[0257] In all cases, the PI for each condition and each protease variant was calculated by dividing the blank-subtracted total absorbance by that of the parent protease (tested at the same concentration). The parent protease values ​​were determined from the standard curve of the parent protease included in the study and fitted to a Langmuir fit. The ADW performance results of AprL variants (using either rinsed or unrinsed swatches) tested in either GSM-B pH 10.5 or custom liquid ADW detergent formulations (adjusted to pH 7 and pH 9) are shown in Table 28. Results are reported as PI values ​​compared to AprL wild-type protease ("AprL wt").

[0258] [Table 47]

Claims

1. 2. A subtilisin variant derived from a parent comprising the amino acid sequence of SEQ ID NO: 2, comprising the following at the position corresponding to SEQ ID NO: 2: 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-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;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-A202 V-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-S250 G-S258P-Q274A; P9E-T77N-N96S-G165Q-G203E-S250G; P9E-T77N-N96S-G165E-G203E-T210P-S250G; A1Q-T3V-S86H-G130S-S155G-G165 Q-N184Q-A202V-G203N-T210P-Q274A; A1Q-T3V-S86H-G165Q-N184Q-N239S-S250G-S258P; A1Q-T77N-S86H-V147L-A151G-N184Q-N217S- 1. A subtilisin variant comprising a set of amino acid substitutions selected from: S86H-T115F-S155G-G165Q-N184Q-A202V-Q274A; and A68S-S86H-T115F-N184Q-A202V-G203N-N239S-S250G-S258P-Q274A, and having improved thermostability as an improved property compared to a reference subtilisin comprising the amino acid sequence of SEQ ID NO:2, said variant having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

2.

2. 2. A subtilisin variant derived from a parent comprising the amino acid sequence of SEQ ID NO: 2, comprising the following at the position corresponding to SEQ ID NO: 2: A068S-T077N-A128P-G165Q-N217S; A068S-T077N-V087S-A128P-S155G-G165Q-N217S; A068S-T077 N-A128P-G165Q; A068S-T077N-V087S-A128P-S155G-G165Q; I035A-A068S-T077N-V087S-A128P-V1 47L-G165Q-N217S; A068S-T077N-V087S-A128P-S155G-G165Q-N217S-A242G; A068S-T077N-V087S- A128P-V147L-S155E-G165Q; A068S-T077N-V087S-A128P-S155E-G165Q-N217S; A068S-T077N-S086 N-V087S-A128P-S155G-G165Q-N217S-A242G; I035A-A068S-T077N-A128P-V147L-G165Q-N217S; I0 35A-A068S-T077N-A128P-S155G-G165Q-A242G; I035A-A068S-T077N-A128P-V147L-G165Q-A242G; and A068S-T077N-V087S-A128P-S155G-G165Q-A242G, and having improved thermostability as an improved property compared to a reference subtilisin comprising the amino acid sequence of SEQ ID NO:9, said variant having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

2.

3. 2. A subtilisin variant derived from a parent comprising the amino acid sequence of SEQ ID NO: 2, comprising the following at the position corresponding to SEQ ID NO: 2: 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;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;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-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-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-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-G127; 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T3V-A68S-T77N-M123I-G127S-A12 8P-A143Q-G165Q-N184Q-A202V-N217S-S258P; T3V-A68S-T77N-G117S-M123I-G127S-A128P-G165Q-N184Q-A202V-N21 7S-S258P; T3V-A48V-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A48T-A68S-T77N-M12 3I-G127S-A128P-G165Q-N184Q-A202V-N217S-S258P; T3V-A29S-A68S-T77N-M123I-G127S-A128P-G165Q-N184Q-A202 V-N217S-S258P; T3V-A68S-T77N-G127S-A128P-G165Q-N184Q-A202V-N217S; T3V-A68S-T77N-G127S-A128P-G165Q-N 184Q-N217S-S258P; T3V-A68S-T77N-A128P-G165Q-N184Q-A202V-N217S-S258P; A68S-T77N-M123I-G127S-A128P-G16 and A68S-T77N-M123I-A128P-G165Q-N184Q-A202V-N217S-S258P, and having improved thermostability as an improved property compared to a reference subtilisin comprising the amino acid sequence of SEQ ID NO:63, said variant having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

2.

4. The subtilisin variant of any one of claims 1 to 3, wherein the variant has protease activity and is isolated.

5. 5. The subtilisin variant of any one of claims 1 to 4, wherein said improved thermostability said variant has a stability PI > 1 or a % residual activity 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 higher of > 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more.

6. 6. The subtilisin variant of any one of claims 1 to 5, wherein the variant further has one or more improved properties compared to a reference subtilisin; the improved properties being selected from improved protease activity, improved cleaning performance in detergents, and improved degraded laundry cleanability in detergents.

7. The one or more improved properties include: (i) improved protease activity, wherein the variant has a PI>1 with N-suc-AAPF-pNA or dimethylcasein substrate; (ii) improved cleaning performance in detergents, wherein the variant has a BMI, POM, egg and / or egg yolk cleaning PI>1; (iii) improved degraded laundry detergency in detergents, wherein the variants have: (a) a degraded laundry detergency PI of >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%, 98%, 99%, 100% or more; and / or (c) a residual cleaning activity after 3 days or 3 weeks at 37°C of >10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more. The subtilisin mutant of claim 6.

8. 8. The subtilisin variant of claim 6 or 7, wherein the detergent is a boron-free composition.

9. A composition comprising one or more subtilisin variants according to any one of claims 1 to 8.

10. The composition of claim 9, wherein the composition is a detergent composition.

11. 11. The composition of claim 10, wherein the detergent composition is selected from laundry detergents, fabric softeners, dishwashing detergents and hard surface cleaning detergents.

12. The compositions may contain 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 adjunct materials, and / or one or more enzymes selected from the group consisting of acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, DNase or nuclease, endo-beta-1,4-glucanase, endo-beta-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hyaluronidase, keratinase, lacquer 12. The composition of any one of claims 9 to 11, further comprising one or more additional enzymes or enzyme derivatives selected from the group consisting of: beta-glucanase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, oxidase, pectate lyase, pectin acetyl esterase, pectinase, pentosanase, peroxidase, phenoloxidase, phosphatase, phospholipase, phytase, polygalacturonase, protease, pullulanase, reductase, rhamnogalacturonase, beta-glucanase, tannase, transglutaminase, xylan acetyl-esterase, xylanase, xyloglucanase, xylosidase, metalloprotease, additional serine protease, and combinations thereof.

13. 13. The composition according to any one of claims 9 to 12, wherein the composition contains or does not contain phosphate and / or contains or does not contain boron.

14. 14. The composition of any one of claims 9 to 13, wherein the composition is in the form of a granule, powder, solid, bar, liquid, tablet, gel, paste, or unit dose composition.

15. A method of cleaning comprising contacting a surface or item in need of cleaning with a subtilisin variant according to any one of claims 1 to 8 or a composition according to any one of claims 9 to 14.

16. 16. The method of claim 15, further comprising rinsing the surface or article after contacting the surface or article with the variant or composition.

17. 17. The cleaning method of claim 15 or 16, wherein the item is a dish or a fabric.

18. A polynucleotide comprising a nucleic acid sequence encoding the subtilisin variant of any one of claims 1 to 8.

19. 20. The polynucleotide of claim 18, which is an isolated polynucleotide.

20. 20. An expression vector or expression cassette comprising the polynucleotide of claim 18 or 19.

21. 21. The expression vector or expression cassette of claim 20, wherein the nucleic acid sequence is operably linked to a promoter.

22. 22. A recombinant host cell comprising an expression vector or expression cassette according to claim 20 or 21, or a polynucleotide according to claim 18 or 19.

23. 9. A composition comprising the subtilisin variant of any one of claims 1 to 8, wherein the composition is for disinfection, industrial or institutional cleaning, medical equipment cleaning, animal feed, contact lens cleaning, wound debridement, or textile, leather, or feather processing.

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