Subtilisin variants and methods of use

Abstract

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

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 382,942, filed on November 9, 2022, which is hereby incorporated by reference in its entirety.

[0003] Disclosed herein are one or more subtilisin variants, nucleic acids encoding the same, and compositions and methods related to their production and use, wherein the one or more subtilisin variants include one or more subtilisin variants having improved stability and / or soil removal as compared to one or more reference subtilisins.

[0004] Reference to a sequence listing submitted electronically

[0005] An official copy of the sequence listing was submitted electronically via the Patent Center as a sequence listing in XML format, with the file name 20231018_NB41985PCT_SeqLst , created on October 18, 2023 , and having a size of 11,443 bytes, and was submitted contemporaneously with this specification. The sequence listing contained in the XML - formatted file is part of this specification and is hereby incorporated by reference in its entirety. Background of the Invention

[0006] Proteases (also known as proteolytic enzymes) are enzymes that have the ability to break down other proteins. Proteases have the ability to initiate proteolysis for protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the peptide or polypeptide chains that form proteins. This activity of proteases as protein - digesting enzymes is called proteolytic activity. There are many well - known procedures for measuring proteolytic activity (Kalisz, “Microbial Proteinases,” in: Fiechter (ed.), Advances in Biochemical Engineering / Biotechnology [Advances in Biochemical Engineering / Biotechnology], (1988)). For example, proteolytic activity can be determined by comparative assays that analyze the ability of individual proteases to hydrolyze commercial substrates. Exemplary substrates that can be used to assay proteases or proteolytic activity include, but are not limited to, dimethyl casein (Sigma C - 9801), bovine collagen (Sigma C - 9879), bovine elastin (Sigma E - 1625), and Keratin Azure (Sigma - Aldrich K8500). Colorimetric assays using these substrates are well known in the art (see, for example, WO 99 / 34011 and U.S. Patent No. 6,376,450, both of which are incorporated by reference herein).

[0007] Serine proteases are enzymes (EC number 3.4.21) that have an active site serine that initiates the hydrolysis of protein peptide bonds. Serine proteases encompass a wide variety of enzymes with broad specificities and biological functions, and based on the structures of these enzymes, they are further classified into chymotrypsin-like (trypsin-like) and subtilisin-like. The prototype subtilisin (EC number 3.4.21.62) was originally obtained from Bacillus subtilis. Subtilisin and its homologues are members of the S8 peptidase family of the MEROPS classification scheme (Rawlings, N.D. et al. (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors [Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors]. Nucleic Acids Res [Nucleic Acid Research] 44, D343-D350). Members of the S8 family have a catalytic triad in their amino acid sequence in the order Asp, His, and Ser. Although many variant proteases have been developed for cleaning applications, there is still a need for improved protease variants. Summary of the Invention

[0008] One embodiment relates to a subtilisin variant that comprises two or more mutations selected from the group consisting of: X009E, X074D, X085D, X099E, X157D, X176E, X188E, X189E, X211L, X242D, and X256E, wherein the positions are numbered by correspondence to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to a subtilisin having the amino acid sequence of SEQ ID NO:1 or 7.

[0009] Another embodiment relates to a subtilisin variant that comprises three mutations selected from the group consisting of: X009E, X074D, X085D, X099E, X157D, X176E, X188E, X189E, X242D, and X256E, wherein the positions are numbered by correspondence to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to a subtilisin having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0010] Still other embodiments relate to methods for producing the variants described herein, the method comprising stably transforming a host cell with an expression vector comprising a polynucleotide encoding one or more subtilisin variants described herein. Still further embodiments relate to polynucleotides comprising nucleic acid sequences encoding one or more subtilisin variants described herein. Detailed Description

[0011] In one embodiment, the present disclosure provides one or more subtilisin variants comprising one, two, three or more amino acid substitutions at positions selected from the group consisting of: 9, 74, 85, 99, 157, 176, 188, 189, 211, 242 and 256, where these positions are numbered by correspondence to the amino acid sequence of SEQ ID NO:1. In some embodiments, when compared to subtilisin having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7, the variants provided herein exhibit one or more improved properties, such as improved cleaning performance, or improved stability, or both improved cleaning performance and improved stability. In some embodiments, the variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin (e.g., SEQ ID NO:1 or SEQ ID NO:7). The subtilisin variants provided herein can be used to prepare cleaning compositions (e.g., automatic dishwashing compositions or laundry detergent compositions). In addition, the subtilisin variants provided herein can also be used in cleaning methods (e.g., dishwashing methods or laundry washing methods) using such variants or compositions comprising such subtilisin variants.

[0012] In some embodiments, the variant subtilisin provided herein has a greater robustness improvement factor (RIF) than the parental subtilisin. The parameter referred to herein as the "robustness improvement factor, RIF" is determined for a given subtilisin as follows: Multiply the percentage of residual activity value by the cleaning performance index calculated for the C-05 stain and the cleaning performance index calculated for the C-S-39 stain, and divide that number by the product of the corresponding parameters obtained for the parental subtilisin (e.g., SEQ ID NO:1 or SEQ ID NO:7) tested under the same conditions. As shown in Tables 2 and 3, the RIF values obtained for the examples of variant enzymes provided herein reflect the overall ability of the variant enzymes to deliver performance benefits in liquid laundry detergents.

[0013] Unless otherwise indicated herein, one or more subtilisin variants described herein can be prepared and used by a variety of techniques used in the fields of molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, recombinant DNA, and the use and development of industrial enzymes. Undefined terms and abbreviations shall conform to their conventional meanings as used in the art. Unless otherwise defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any definitions provided herein will be interpreted in the context of the specification as a whole. As used herein, unless the context clearly indicates otherwise, the singular forms "a / an" and "the" include the plural. Unless otherwise indicated, nucleic acid sequences are written from left to right in the 5' to 3' direction; and amino acid sequences are written from left to right in the amino to carboxyl direction. Each numerical range used herein includes every narrower numerical range falling within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0014] As used herein in connection with a numerical value, the term "about" means a range of + / - 0.5 of the numerical value, unless the term is otherwise specifically defined in the context. For example, the phrase "a pH value of about 6" means a pH value of 5.5 to 6.5, unless the pH value is otherwise specifically defined.

[0015] The nomenclature for amino acid substitutions of one or more subtilisin variants described herein uses one or more of the following: position; position: one or more amino acid substitutions; or one or more starting amino acids: position: one or more substituted amino acids. References to "position" (e.g., 5, 8, 17, 22, etc.) encompass any starting amino acid that may be present at such position, and any substitution that may be present at such position. References to "position: one or more amino acid substitutions" (e.g., 1S / T / G, 3G, 17T, etc.) encompass any starting amino acid that may be present at such position and one or more amino acids that may substitute for such starting amino acid. References to position can be listed in several forms, e.g., position 003 can also be referred to as position 03 or 3. References to starting or substituted amino acids can further be represented by several starting or substituted amino acids separated by a foreslash (" / "). For example, D275S / K means that position 275 is substituted by serine (S) or lysine (K), and P / S197K means that the starting amino acid proline (P) or serine (S) at position 197 is substituted by lysine (K). References to X as an amino acid at a position refer to any amino acid at the recited position.

[0016] The positions of the amino acid residues in a given amino acid sequence are numbered by correspondence to the amino acid sequence of SEQ ID NO:1. That is, the amino acid sequence of SEQ ID NO:1 is used as a reference sequence for numbering the positions of the amino acid residues. For example, the amino acid sequences of one or more subtilisin variants described herein are aligned with the amino acid sequence of SEQ ID NO:1 using an alignment algorithm as described herein, and each amino acid residue in the given amino acid sequence that aligns (preferably, optimally) with an amino acid residue in SEQ ID NO:1 is conveniently numbered by reference to the numerical position of the corresponding amino acid residue. When compared to a query sequence (sometimes also referred to as the "reference sequence"), for example, a sequence alignment algorithm as described herein will identify one or more positions in the subject sequence where insertions or deletions have occurred. For example, as provided in Figure 1 of PCT Publication No. WO 2019108599, amino acid alignments can be used to determine sequence alignments with other subtilisin amino acid sequences.

[0017] The terms "protease" and "proteinase" refer to enzymes that have the ability to break down proteins and peptides. Proteases have the ability to perform "proteolysis" by hydrolysis of the peptide bonds that link amino acids together in a peptide or polypeptide chain that forms a protein. This activity of proteases as protein-digesting enzymes is referred to as "proteolytic activity". There are many well-known procedures for measuring proteolytic activity. For example, proteolytic activity can be determined by comparative assays that analyze the ability of the respective protease to hydrolyze a suitable substrate. Exemplary substrates that can be used to analyze protease or proteolytic activity include, but are not limited to, dimethyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625), and Keratin Azure (Sigma-Aldrich K8500). Colorimetric assays using these substrates are well known in the art (see, for example, WO 99 / 34011 and US 6,376,450). The pNA peptidyl assay (see, for example, DelMar et al., Anal Biochem [Analytical Biochemistry], 99:316-320, 1979) can also be used to determine the concentration of active enzyme. This assay measures the rate of release of p-nitroaniline when the enzyme hydrolyzes a soluble synthetic substrate such as succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (suc-AAPF-pNA). The rate of formation of the yellow color from the hydrolysis reaction is measured on a spectrophotometer at 405 or 410 nm and this rate is proportional to the concentration of active enzyme. Additionally, absorbance measurements at 280 nanometers (nm) can be used to determine the total protein concentration in a purified protein sample. The activity of the substrate divided by the protein concentration gives the enzyme specific activity.

[0018] As used herein, "Bacillus" includes all species within the genus "Bacillus" known to those skilled in the art, including but not limited to: Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus gibsonii, Bacillus pumilus, and Bacillus thuringiensis. It should be recognized that the genus Bacillus is constantly undergoing taxonomic reorganization. Thus, the genus is intended to include species that have been reclassified, including but not limited to organisms such as Bacillus stearothermophilus (now named "Geobacillus stearothermophilus") or Bacillus polymyxa (now "Paenibacillus polymyxa"). The production of resistant endospores under stress environmental conditions is considered a defining characteristic of the genus Bacillus, although this feature also applies to the recently named genera Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

[0019] "Bacillus lentus subtilisin" includes any subtilisin obtained from or derived from a Bacillus lentus source, including P29600. In one embodiment, the present invention provides a "GG36 variant" (or "P29600 variant" or "GG36 subtilisin variant"), wherein these mutations are present in the mature GG36 amino acid sequence shown in SEQ ID NO:1. In other embodiments, Bacillus lentus subtilisin and its variants include those polypeptides having an amino acid sequence that has at least 60% sequence identity with SEQ ID NO:1.

[0020] "Bacillus gibsonii subtilisin" includes any subtilisin obtained from or derived from a Bacillus gibsonii source. In one embodiment, the subtilisin variants provided herein can be derived from Bacillus gibsonii clade subtilisins (such as those described in WO 2015 / 089447, and those described in WO 2016 / 205755). Other Bacillus gibsonii subtilisins include those described in US Patent Application Publication No. 20090275493 and their variants, those described in International Patent Application Publication No. WO 2016 / 087403 and their variants, and those described in US Patent No. 7,449,187 and their variants. In other embodiments, Bacillus gibsonii subtilisin includes those polypeptides having an amino acid sequence that has at least 60% sequence identity with SEQ ID NO:7.

[0021] The term "vector" refers to a nucleic acid construct used to introduce or transfer one or more nucleic acids into a target cell or target tissue. Typically, a vector is used to introduce foreign DNA into a cell or tissue. Vectors include plasmids, cloning vectors, phages, viruses (e.g., viral vectors), cosmids, expression vectors, shuttle vectors, etc. Typically, a vector includes an origin of replication, a multiple cloning site, and a selectable marker. Typically, the process of inserting a vector into a target cell is called transformation. In some embodiments, the present 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 presequence (such as a secretion, signal peptide sequence, etc.), the vector being capable of achieving expression of the DNA sequence in a suitable host, and folding and translocation of the recombinant polypeptide chain.

[0022] As used herein, in the context of introducing a nucleic acid sequence into a cell, the term "introducing" refers to any method suitable for transferring a nucleic acid sequence into a cell. Such introducing methods include, but are not limited to: protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. Transformation refers to the genetic alteration of a cell caused by the uptake, optional genomic incorporation, and expression of genetic material (such as DNA).

[0023] The term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid molecule of the present disclosure. Expression may also refer to the translation of mRNA into a polypeptide. Thus, the term "expression" includes any steps involved in the "production of a polypeptide", including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0024] The phrase "expression cassette" or "expression vector" refers to a nucleic acid construct or vector that is recombinantly or synthetically produced for expressing a nucleic acid of interest (such as a foreign nucleic acid or transgene) in a target cell. Typically, the nucleic acid of interest expresses a target protein. Typically, an expression vector or expression cassette contains a promoter nucleotide sequence that drives or facilitates the expression of the foreign nucleic acid. Typically, an expression vector or expression cassette also includes other designated nucleic acid elements that permit the transcription of a specific nucleic acid in a target cell. A recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Some expression vectors have the ability to incorporate and express a heterologous DNA fragment in a host cell or the host cell genome. Many prokaryotic and eukaryotic expression vectors are commercially available. Selecting an appropriate expression vector for expressing a protein from the nucleic acid sequences incorporated into the expression vector is within the knowledge of those skilled in the art.

[0025] As used herein, a nucleic acid is "operably linked" to another nucleic acid sequence when 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 affects the transcription of the coding sequence. A ribosome binding site can be operably linked to a coding sequence if it is positioned to facilitate the translation of the coding sequence. Typically, "operably linked" DNA sequences are contiguous. However, an enhancer need not be contiguous. Linking is achieved by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide linkers or adaptors can be used according to conventional practice.

[0026] The term "gene" refers to a polynucleotide (such as a DNA segment) that encodes a polypeptide and includes the regions before and after the coding region. In some cases, a gene includes intervening sequences (introns) between individual coding segments (exons).

[0027] When used with reference to cells, the term "recombinant" typically indicates that the cell has been modified by the introduction of an exogenous nucleic acid sequence, or that the cell is derived from a cell so modified. For example, a recombinant cell can contain a gene that is not present in the same form in a cell in its natural (non-recombinant) form, or a recombinant cell can contain a native gene (found in the native form of the cell) that has been modified and reintroduced into the cell. A recombinant cell can contain a nucleic acid that is endogenous to the cell that has been modified but not removed from the cell; such modifications include those obtained by gene replacement, site-specific mutagenesis, and related techniques known to those of ordinary skill in the art. Recombinant DNA technology includes techniques for producing recombinant DNA in vitro and transferring the recombinant DNA into a cell in which it can be expressed or propagated to produce a recombinant polypeptide. "Recombination" and "recombining" of polynucleotides or nucleic acids generally refers to the assembly or combination of two or more nucleic acid or polynucleotide strands or fragments to produce a new polynucleotide or nucleic acid.

[0028] If, in its native state or when manipulated by methods known to those of skill in the art, a nucleic acid or polynucleotide can be transcribed and / or translated to produce a polypeptide or a fragment thereof, then the nucleic acid or polynucleotide can be said to "encode" the polypeptide. The antisense strand coding sequence of such a nucleic acid can also be said to encode.

[0029] The terms "host strain" and "host cell" refer to a host that is suitable for an expression vector containing a DNA sequence of interest.

[0030] "Protein" or "polypeptide" encompasses a polymeric sequence of amino acid residues. The terms "protein" and "polypeptide" are used interchangeably herein. Throughout this disclosure, the single-letter and three-letter codes for amino acids as defined by the Joint Commission on Biochemical Nomenclature (JCBN) of the International Union of Pure and Applied Chemistry (IUPAC) and the International Union of Biochemistry and Molecular Biology (IUB) are used. The single letter X refers to any of the twenty amino acids. It should also be understood that, due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

[0031] The term "prosequence" or "propeptide sequence" refers to the amino acid sequence between the signal peptide sequence and the mature protease sequence that is necessary for the correct folding and secretion of the protease; they are sometimes referred to as intramolecular chaperones. Cleavage of the prosequence or propeptide sequence gives rise to the mature active protease. Bacterial serine proteases are usually expressed as proenzymes. For example, examples of modified propeptides are provided in WO 2016 / 205710.

[0032] The terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that can participate in the secretion or directed transport of a protein in its mature or precursor form. Typically, the signal sequence is located at the N-terminus of the precursor or mature protein sequence. The signal sequence can be endogenous or exogenous. The signal sequence is generally not present in the mature protein. Typically, after protein transport, the signal sequence is cleaved from the protein by signal peptidase.

[0033] The term "mature" form of a protein, polypeptide or peptide refers to the functional form of the protein, polypeptide or peptide that does not have a signal peptide sequence and a propeptide sequence.

[0034] The term "precursor" form of a protein or peptide refers to the mature form of the protein that has a presequence operably linked to the amino or carbonyl terminus of the protein. The precursor can also have a "signal" sequence operably linked to the amino terminus of the presequence. The precursor can also have additional polypeptides involved in post-translational activities (e.g., polypeptides that are cleaved from it to leave the mature form of the protein or peptide).

[0035] With respect to a polypeptide, the term "wild-type" refers to a naturally occurring polypeptide that does not include artificial substitutions, insertions or deletions at one or more amino acid positions. Similarly, with respect to a polynucleotide, the term "wild-type" refers to a naturally occurring polynucleotide that does not include artificial substitutions, insertions or deletions at one or more nucleotides. However, a polynucleotide encoding a wild-type polypeptide is not limited to a naturally occurring polynucleotide and encompasses any polynucleotide encoding a wild-type or parental polypeptide.

[0036] With respect to polypeptides, the term "parent" includes reference to a naturally occurring or wild-type polypeptide, or a naturally occurring polypeptide in which one or more amino acid positions have been artificially substituted, inserted, or deleted, which is used as a basis for introducing substitutions or additional substitutions to generate variant enzymes provided herein. With respect to polypeptides, the term "parent" also includes any polypeptide having protease activity that serves as a starting polypeptide for alteration (such as substitution, addition, and / or deletion) to produce a variant having one or more alterations compared to the starting polypeptide. That is, the parent or reference polypeptide is not limited to a naturally occurring wild-type polypeptide and encompasses any wild-type, parent, or reference polypeptide. Similarly, with respect to polynucleotides, the term "parent" can refer to a naturally occurring polynucleotide or a polynucleotide that indeed includes artificial substitutions, insertions, or deletions at one or more nucleotides. With respect to polynucleotides, the term "parent" also includes any polynucleotide encoding a polypeptide having protease activity that serves as a starting polynucleotide for alteration to produce a variant protease having modifications such as substitutions, additions, and / or deletions compared to the starting polynucleotide. That is, the polynucleotide encoding a wild-type, parent, or reference polypeptide is not limited to a naturally occurring polynucleotide and encompasses any polynucleotide encoding a wild-type, parent, or reference polypeptide. In some embodiments, the parent polypeptides herein include polypeptides having the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:7.

[0037] The term "naturally occurring" refers to, for example, sequences found in nature and the residues contained therein (e.g., polypeptide sequences and the amino acids contained therein or nucleotide sequences and the nucleotides contained therein). In contrast, the term "non-naturally occurring" refers to, for example, sequences not found in nature and the residues contained therein (e.g., polypeptide sequences and the amino acids contained therein or nucleotide sequences and the nucleic acids contained therein).

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

[0039] The terms "derived from" and "obtained from" refer not only to a protein produced or producible by a strain of the organism in question, but also to a protein encoded by a DNA sequence isolated from such a strain and produced in a host organism containing such a DNA sequence. Additionally, the term refers to a protein encoded by a synthetic and / or cDNA-derived DNA sequence and having the identifying characteristics of the protein in question. For example, a "protease derived from Bacillus" refers to those enzymes having proteolytic activity that are naturally produced by Bacillus, as well as serine proteases such as those produced from a Bacillus source but produced by other host cells transformed with a nucleic acid encoding a serine protease using genetic engineering techniques.

[0040] In the context of two polynucleotide or polypeptide sequences, the term "identity" means that the nucleotides or amino acids in the two sequences are the same when aligned in a maximum correspondence, as measured using the sequence comparison or analysis algorithms described below and known in the art.

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

[0042] As used herein, "homologous protein" or "homologous protease" refers to proteins having varying degrees of similarity in primary, secondary, and / or tertiary structure. When comparing proteins, protein homology can refer to the similarity of linear amino acid sequences. Homology can be determined, for example, by amino acid sequence alignment using programs such as BLAST, MUSCLE, or CLUSTAL. Homology searches of protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST with a threshold (E-value cutoff) of 0.001. (Altschul et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res, vol. 25, no. 17, pp. 3389-402 (1997)). The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most relevant sequences according to biological similarity, but is not recommended for query sequences less than 20 residues (Altschul et al., Nucleic Acids Res, 25: 3389-3402, 1997 and Schaffer et al., Nucleic Acids Res, 29: 2994-3005, 2001). Exemplary default BLAST parameters for nucleic acid sequence searches include: word length threshold for adjacent words = 11; E-value cutoff = 10; scoring matrix = NUC.3.1 (match = 1, mismatch = -3); gap open = 5; and gap extension = 2. Exemplary default BLAST parameters for amino acid sequence searches include: word length = 3; E-value cutoff = 10; scoring matrix = BLOSUM62; gap open = 11; and gap extension = 1. Using this information, protein sequences can be grouped and / or a phylogenetic tree can be constructed therefrom. The amino acid sequences can be entered, for example, into a program such as the Vector NTI Advance suite, and a bootstrap tree can be created using the Neighbor Joining (NJ) method (Saitou and Nei, Mol Biol Evol, 4: 406-425, 1987). The tree structure can be calculated using the Kimura correction for sequence distance and ignoring positions with gaps. Programs such as AlignX can display the calculated distance values in parentheses after the molecular names shown on the phylogenetic tree.

[0043] Understanding the homology between molecules can reveal the evolutionary history of the molecules as well as their functional information; if a newly sequenced protein is homologous to a protein that has already been characterized, there is a strong indication of the biochemical function of the new protein. Two molecules are said to be homologous if they are derived from a common ancestor. Homologous molecules or homologs can be divided into two categories: paralogs and orthologs. Paralogs are homologs that exist within a single species. Paralogs often differ in their detailed biochemical functions. Orthologs are homologs that exist in different species and have very similar or identical functions. A protein superfamily is the largest grouping (clade) of proteins from which a common ancestor can be inferred. Typically this common ancestor is based on sequence alignment and mechanistic similarity. Typically, a superfamily contains several protein families that show sequence similarity within the family. Based on the MEROPS protease classification system, the term "protein clan" is commonly used for protease superfamilies. As used herein, the term "subtilisin" includes any member of the S8 serine protease family as described in the MEROPS - peptidase database (Rawlings, N.D et al. (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors [Nucleic Acids Res 44, D343 - D350]).

[0044] The CLUSTAL W algorithm is another example of a sequence alignment algorithm (see Thompson et al., Nucleic Acids Res 22:4673 - 4680, 1994). The default parameters of the CLUSTAL W algorithm include: 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 transition weight = 0.50; list hydrophilic residues = GPSNDQEKR; use negative matrix = off; switch special residue penalty = on; switch hydrophilic penalty = on; and switch end - gap separation penalty = off. In the CLUSTAL algorithm, deletions occurring at either end are included. For example, a variant having a five - amino - acid deletion at either end (or within the polypeptide) of a 500 - amino - acid polypeptide will have a percent sequence identity of 99% (495 / 500 identical residues × 100) relative to the "reference" polypeptide. Such a variant would be encompassed by a variant having "at least 99% sequence identity" to the polypeptide.

[0045] A nucleic acid or polynucleotide is "isolated" when it is at least partially or completely separated from other components, including but not limited to, for example, other proteins, nucleic acids, cells, etc. Similarly, a polypeptide, protein, or peptide is "isolated" when it is at least partially or completely separated from other components, including but not limited to, for example, other proteins, nucleic acids, cells, etc. The isolated species is more abundant than other species on a molar basis. For example, the isolated species can represent 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. Preferably, the species of interest is purified to substantial homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods). Many techniques well known in the art, such as agarose or polyacrylamide gel electrophoresis of nucleic acid or protein samples, followed by visualization after staining, can be used to determine purity and homogeneity. If desired, high-resolution techniques such as high-performance liquid chromatography (HPLC) or similar methods can be used to purify the substance.

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

[0047] The term "cleaning activity" refers to the cleaning performance achieved by a serine protease polypeptide, variant, or reference subtilisin under the prevailing conditions during proteolytic, hydrolytic, cleaning, or other processes of the present disclosure. 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 enzyme-sensitive stains on an article or surface (e.g., stains caused by food, grass, blood, ink, milk, oil, and / or egg protein). The cleaning performance of one or more of the subtilisin variants or reference subtilisins described herein can be determined by subjecting the stains on an article or surface to one or more standard washing conditions and evaluating the degree of stain removal by 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 US 6,605,458, as well as those cleaning assays and methods included in the examples provided below.

[0048] The term "effective amount" of one or more of the subtilisin variants or reference subtilisins described herein refers to the amount of protease that achieves a desired level of enzyme activity in a particular cleaning composition. Such an effective amount can be readily determined by one of ordinary skill in the art and is based on many factors, such as the particular protease 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, etc.

[0049] The term "adjuvant material" refers to any liquid, solid, or gaseous substance, or recombinant polypeptide or active fragment thereof, contained in a cleaning composition in addition to one or more of the subtilisin variants described herein. In some embodiments, the cleaning compositions of the present disclosure include one or more cleaning adjuvant materials. Typically, each cleaning adjuvant material is 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). Preferably, each cleaning adjuvant material is compatible with the protease used in the composition.

[0050] Cleaning compositions and cleaning preparations include any composition suitable for cleaning, bleaching, disinfecting, and / or sterilizing any object, article, and / or surface. Such compositions and preparations include, but are not limited to, for example, liquid and / or solid compositions, including cleaning compositions or detergent compositions (such as liquid, tablet, gel, bar, granular, and / or solid fabric cleaning or detergent compositions) and fine fabric detergent compositions; hard surface cleaning compositions and preparations, such as for glass, wood, ceramic, and metal countertops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile, clothing enhancing cleaning or detergent compositions, clothing additive cleaning compositions, and clothing pre-spotter cleaning compositions; dishwashing compositions, including hand wash or manual dishwashing compositions (such as "hand wash" or "manual" dish detergents) and automatic dishwashing compositions (such as "automatic dish detergents"). The present invention can also be in the form of single dose units, including but not limited to pills, tablets, gelcaps, or other single dose units such as pre-measured powders or liquids.

[0051] Unless otherwise indicated, cleaning compositions or cleaning preparations as used herein include general or heavy-duty detergents in particulate or powder form, especially cleaning detergents; general detergents in liquid, particulate, gel, solid, tablet, paste, or unit dosage form, especially so-called heavy-duty liquid (HDL) detergents or heavy-duty dry cleaning (HDD) detergent types; liquid fine fabric detergents; hand wash or manual dish detergents, including those of the high-foaming type; hand wash or manual dish detergents, automatic dish detergents, or dish or tableware detergents, including various tablet, powder, solid, particulate, liquid, gel, and rinse aid types for household and institutional use; liquid cleaning and disinfectants, including antibacterial hand wash types, cleaning bars, mouthwashes, denture cleaners, car shampoos, carpet shampoos, bathroom cleaners; hair shampoos and / or hair bleaches for humans and other animals; body washes and bubble baths and metal cleaners; and cleaning aids such as bleach additives and "stain sticks" or pretreatment types. In some embodiments, the particulate composition is in a "compact" form; in some embodiments, the liquid composition is in a "concentrate" form.

[0052] The terms "detergent composition" or "detergent formulation" are used with respect to compositions intended for use in a washing medium for cleaning soiled or dirty objects, including specific fabric and / or non-fabric objects or articles. In some embodiments, the detergents of the present disclosure comprise one or more of the subtilisin variants described herein, and additionally comprise one or more surfactants, one or more transferases, hydrolases, oxidoreductases, builders (e.g., builder salts), bleaches, bleach activators, blueing agents, fluorescent dyes, anti-caking agents, masking agents, enzyme stabilizers, calcium, enzyme activators, antioxidants, and / or solubilizers. In some cases, the builder salts are a mixture of silicate and phosphate, preferably having more silicate (e.g., sodium metasilicate) than phosphate (e.g., sodium tripolyphosphate). Some embodiments relate to cleaning compositions or detergent compositions that do not contain any phosphate (e.g., phosphate or phosphate builder).

[0053] The phrases "one or more substantially boron-free compositions" or "one or more substantially boron-free detergents" respectively refer to one or more compositions or one or more detergents containing trace amounts of boron (e.g., less than about 1000 ppm (1 mg / kg or 1 mg / L 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), which boron may be from other composition or detergent ingredients.

[0054] The term "bleaching" refers to treating a material (e.g., fabric, clothing, pulp, etc.) or surface for a sufficient length of time and / or under appropriate pH and / or temperature conditions to effect whitening (i.e., becoming white) and / or cleaning of the material. Examples of chemicals suitable for bleaching include, but are not limited to, for example, ClO2, H2O2, peracids, NO2, etc. Bleaching agents also include enzymatic bleaching agents such as perhydrolases and arylesterases. Another embodiment relates to a composition comprising one or more of the subtilisin variants described herein and one or more perhydrolases, such as the perhydrolases described in WO 2005 / 056782, WO 2007 / 106293, WO 2008 / 063400, WO 2008 / 106214, and WO 2008 / 106215.

[0055] The "washing performance" of the term protease (e.g., one or more subtilisin variants as described herein, or recombinant polypeptides or active fragments thereof) refers to the cleaning contribution that one or more subtilisin variants as described herein provide additional cleaning performance for washing compared to a detergent that does not add one or more subtilisin variants as described herein to the composition. The washing performance is compared under relevant washing conditions. In some test systems, other relevant factors, such as detergent composition, sud concentration, water hardness, washing mechanics, time, pH, and / or temperature can be controlled in such a way that one or more conditions typical for household applications in certain market segments (e.g., hand dishwashing or manual dishwashing, automatic dishwashing, tableware cleaning, countertop appliance cleaning, fabric cleaning, etc.) are mimicked.

[0056] In this document, the phrase "relevant washing conditions" is used to indicate the conditions actually used in the home in the hand dishwashing, automatic dishwashing, or laundry detergent market segments, in particular the washing temperature, time, washing mechanics, sud concentration, detergent type, and water hardness.

[0057] The term "dishwashing" refers to both household dishwashing and industrial dishwashing and encompasses both automatic dishwashing (e.g., washing with a dishwashing machine) and manual dishwashing (e.g., washing by hand).

[0058] The "compact" form of the cleaning composition in this document is best reflected by density and, in terms of the composition, by the amount of inorganic filler salts. Inorganic filler salts are a conventional component of the detergent composition in powder form. In a conventional detergent composition, the filler salts are present in a substantial amount, typically about 17% to about 35% by weight of the total composition. In contrast, in a compact composition, the filler salts are present in an amount less than about 15% of the total composition. In some embodiments, the filler salts are present in an amount not exceeding about 10%, or more preferably about 5%, by weight of the composition. In some embodiments, the inorganic filler salts are selected from the alkali and alkaline earth metal salts of sulfates and chlorides. In some embodiments, the filler salt is sodium sulfate.

[0059] One or more subtilisin variants that can be used in cleaning applications, cleaning methods, and various industrial applications are disclosed herein. One or more isolated, recombinant, substantially pure, or non-naturally occurring subtilisin variants are also disclosed herein. In some embodiments, one or more subtilisin variants as described herein can be used in cleaning applications and can be incorporated into a cleaning composition that can be used in a method for cleaning an item or surface in need of cleaning (e.g., a clothing item or textile).

[0060] In one embodiment, a subtilisin variant is provided, wherein the variant comprises two, three, four, or more amino acid substitutions at positions selected from the group consisting of: 9, 74, 85, 99, 157, 176, 188, 189, 211, 242, and 256, wherein these positions are numbered according to SEQ ID NO:1, and wherein the variant has at least 60% identity to the amino acid sequence of SEQ ID NO:1 or 7.

[0061] In some embodiments, the variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least a 2-fold, 3-fold, 4-fold, or greater improvement in RIF relative to the parental subtilisin, and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0062] In one embodiment, a subtilisin variant is provided, wherein the variant comprises two, three, four, five, or more amino acid substitutions at positions selected from the group consisting of: X009E, X074D, X085D, X099E, X157D, X176E, X188E, X189E, X211L, X242D, and X256E, wherein these positions are numbered according to SEQ ID NO:1, and wherein the variant has at least 60% identity to the amino acid sequence of SEQ ID NO:1 or 7.

[0063] In some embodiments, the subtilisin variant does not comprise a combination of substitutions selected from: a) the mutation X074D in combination with X009E, X157D, X176E, X188E, and X256E; b) the combination X099E-X256E; c) the combination X189E-X256E; and d) a combination of two or more substitutions selected from X009E, X157D, X176E, and X256E; wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to the amino acid sequence of SEQ ID NO:1 or 7.

[0064] In some embodiments, the subtilisin variants comprise a combination of substitutions selected from the group consisting of: X009E-X085D, X009E-X099E, X009E-X188E, X009E-X189E, X009E-X242D, X074D-X085D, X074D-X099E, X074D-X189E, X074D-X242D, X085D-X099E, X085D-X157D, X085D-X176E, X085D-X188E, X085D-X189E, X085D-X242D, X085D-X256E, X099E-X157D, X099E-X176E, X099E-X188E, X099E-X189E, X099E-X242D, X157D-X188E, X157D-X189E, X157D-X242D, X176E-X188E, X176E-X189E, X176E-X242D, X176E-X256E, X188E-X189E, X188E-X242D, X188E-X256E, X189E-X242D, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to the amino acid sequence of SEQ ID NO:1 or 7.

[0065] In some embodiments, the substituted combinations are selected from the group consisting of: S009E-S085D, S009E-S099E, S009E-A188E, S009E-G189E, S009E-N242D, N074D-S085D, N074D-S099E, N074D-G189E, N074D-N242D, S085D-S099E, S085D-G157D, S085D-Q176E, S085D-A188E, S085D-G189E, S085D-N242D, S085D-L256E, S099E-G157D, S099E-Q176E, S099E-A188E, S099E-G189E, S099E-N242D, G157D-A188E, G157D-G189E, G157D-N242D, Q176E-A188E, Q176E-G189E, Q176E-N242D, Q176E-L256E, A188E-G189E, A188E-N242D, A188E-L256E, G189E-N242D, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, such variant subtilisins have a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisins provided herein have at least a 2-fold, 3-fold, 4-fold or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1) and have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1.

[0066] In some embodiments, the substituted combinations are selected from the group consisting of: T009E-N085D, T009E-S099E, T009E-T188E, T009E-G189E, T009E-N242D, N074D-N085D, N074D-S099E, N074D-G189E, N074D-N242D, N085D-S099E, N085D-G157D, N085D-Q176E, N085D-T188E, N085D-G189E, N085D-N242D, N085D-Q256E, S099E-G157D, S099E-Q176E, S099E-T188E, S099E-G189E, S099E-N242D, G157D-T188E, G157D-G189E, G157D-N242D, Q176E-T188E, Q176E-G189E, Q176E-N242D, Q176E-Q256E, T188E-G189E, T188E-N242D, T188E-Q256E, G189E-N242D, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, such variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least a 2-fold, 3-fold, 4-fold or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:7) and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:7.

[0067] In one embodiment, a subtilisin variant is provided, wherein the variant has an improved robustness factor (RIF) compared to the parental subtilisin, and the variant comprises two or more substitutions selected from the group consisting of: X009E, X074D, X085D, X099E, X157D, X176E, X188E, X189E, X211L, X242D, and X256E, wherein the positions are numbered relative to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to subtilisin having the amino acid sequence of SEQ ID NO:1.

[0068] In one embodiment, a subtilisin variant is provided, wherein the variant has an improved robustness factor (RIF) compared to the parental subtilisin, and the variant comprises two or more substitutions selected from the group consisting of: X009E, X074D, X085D, X099E, X157D, X176E, X188E, X189E, X211L, X242D, and X256E, wherein the positions are numbered relative to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to subtilisin having the amino acid sequence of SEQ ID NO:7.

[0069] In another embodiment, a subtilisin variant is provided, wherein the variant comprises three, four, five or more amino acid substitutions at positions selected from the group consisting of: X009E, X074D, X085D, X099E, X157D, X176E, X188E, X189E, X242D, and X256E, wherein the positions are numbered according to SEQ ID NO:1, and wherein the variant has at least 60% identity to the amino acid sequence of SEQ ID NO:1 or 7.

[0070] In some embodiments, the subtilisin variant does not comprise a combination of substitutions selected from: a) the mutation X074D in combination with two or more substitutions selected from X009E, X157D, X176E, X188E, and X256E; b) a combination of three substitutions selected from X009E, X074D, X085D, X176E, and X242D; and c) a combination of three substitutions selected from X009E, X157D, X176E, and X256E; wherein the positions are numbered relative to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to the amino acid sequence of SEQ ID NO:1 or 7.

[0071] In some embodiments, the subtilisin variants comprise a combination of substitutions selected from the group consisting of: X009E-X085D-X099E, X009E-X085D-X188E, X009E-X085D-X189E, X009E-X085D-X242D, X009E-X099E-X188E, X009E-X099E-X189E, X009E-X099E-X242D, X009E-X188E-X189E, X009E-X188E-X242D, X009E-X189E-X242D, X074D-X085D-X099E, X074D-X085D-X189E, X074D-X085D-X242D, X074D-X099E-X189E, X074D-X099E-X242D, X074D-X189E-X242D, X085D-X099E-X157D, X085D-X099E-X176E, X085D-X099E-X188E, X085D-X099E-X189E, X085D-X099E-X242D, X085D-X157D-X188E, X085D-X157D-X189E, X085D-X157D-X242D, X085D-X176E-X188E, X085D-X176E-X189E, X085D-X176E-X242D, X085D-X188E-X189E, X085D-X188E-X242D, X085D-X188E-X256E, X085D-X189E-X242D, X085D-X242D-X256E, X099E-X157D-X188E, X099E-X157D-X189E, X099E-X157D-X242D, X099E-X176E-X188E, X099E-X176E-X189E, X099E-X176E-X242D, X099E-X188E-X189E, X099E-X188E-X242D, X099E-X189E-X242D, X157D-X188E-X189E, X157D-X188E-X242D, X157D-X189E-X242D, X176E-X188E-X189E, X176E-X188E-X242D, X176E-X189E-X242D, X188E-X189E-X242D, and X188E-X242D-X256E, where the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and where the variant has at least 60% identity with the amino acid sequence of SEQ ID NO:1 or 7.

[0072] In some embodiments, the substituted combinations are selected from the group consisting of: S009E-S085D-S099E, S009E-S085D-A188E, S009E-S085D-G189E, S009E-S085D-N242D, S009E-S099E-A188E, S009E-S099E-G189E, S009E-S099E-N242D, S009E-A188E-G189E, S009E-A188E-N242D, S009E-G189E-N242D, N074D-S085D-S099E, N074D-S085D-G189E, N074D-S085D-N242D, N074D-S099E-G189E, N074D-S099E-N242D, N074D-G189E-N242D, S085D-S099E-G157D, S085D-S099E-Q176E, S085D-S099E-A188E, S085D-S099E-G189E, S085D-S099E-N242D, S085D-G157D-A188E, S085D-G157D-G189E, S085D-G157D-N242D, S085D-Q176E-A188E, S085D-Q176E-G189E, S085D-Q176E-N242D, S085D-A188E-G189E, S085D-A188E-N242D, S085D-A188E-L256E, S085D-G189E-N242D, S085D-N242D-L256E, S099E-G157D-A188E, S099E-G157D-G189E, S099E-G157D-N242D, S099E-Q176E-A188E, S099E-Q176E-G189E, S099E-Q176E-N242D, S099E-A188E-G189E, S099E-A188E-N242D, S099E-G189E-N242D, G157D-A188E-G189E, G157D-A188E-N242D, G157D-A188E-L256E, G157D-G189E-N242D, Q176E-A188E-G189E, Q176E-A188E-N242D, Q176E-G189E-N242D, A188E-G189E-N242D, and A188E-N242D-L256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1,And the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, such variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least a 2-fold, 3-fold, 4-fold or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1), and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1.,

[0073] In some embodiments, the substituted combinations are selected from the group consisting of: T009E-N085D-S099E, T009E-N085D-T188E, T009E-N085D-G189E, T009E-N085D-N242D, T009E-S099E-T188E, T009E-S099E-G189E, T009E-S099E-N242D, T009E-T188E-G189E, T009E-T188E-N242D, T009E-G189E-N242D, N074D-N085D-S099E, N074D-N085D-G189E, N074D-N085D-N242D, N074D-S099E-G189E, N074D-S099E-N242D, N074D-G189E-N242D, N085D-S099E-G157D, N085D-S099E-Q176E, N085D-S099E-T188E, N085D-S099E-G189E, N085D-S099E-N242D, N085D-G157D-T188E, N085D-G157D-G189E, N085D-G157D-N242D, N085D-Q176E-T188E, N085D-Q176E-G189E, N085D-Q176E-N242D, N085D-T188E-G189E, N085D-T188E-N242D, N085D-T188E-Q256E, N085D-G189E-N242D, N085D-N242D-Q256E, S099E-G157D-T188E, S099E-G157D-G189E, S099E-G157D-N242D, S099E-Q176E-T188E, S099E-Q176E-G189E, S099E-Q176E-N242D, S099E-T188E-G189E, S099E-T188E-N242D, S099E-G189E-N242D, G157D-T188E-G189E, G157D-T188E-N242D, G157D-T188E-Q256E, G157D-G189E-N242D, Q176E-T188E-G189E, Q176E-T188E-N242D, Q176E-G189E-N242D, T188E-G189E-N242D, and T188E-N242D-Q256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1,And the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, such variant subtilisins have a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisins provided herein have at least a 2-fold, 3-fold, 4-fold or greater improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:7), and have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:7.,

[0074] In some embodiments, the substituted combinations are selected from the group consisting of: X009E-X085D, X009E-X099E, X009E-X107D, X009E-X182D, X009E-X189E, X074D-X099E, X074D-X107D, X074D-X189E, X085D-X099E, X085D-X157D, X085D-X176E, X085D-X188E, X085D-X189E, X085D-X256E, X099E-X176E, X099E-X107D, X099E-X182D, X099E-X188E, X099E-X189E, X157D-X107D, X157D-X188E, X157D-X189E, X176E-X107D, X176E-X182D, X176E-X188E, X176E-X189E, X176E-X242D, X107D-X188E, X107D-X189E, X107D-X256E, X182D-X188E, X182D-X189E, X182D-X256E, X188E-X189E, X188E-X256E and X189E-X242D, where the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7. In some embodiments, such variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least a 2-fold, 3-fold, 4-fold or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1 or SEQ ID NO:7), and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0075] In some embodiments, the substituted combinations are selected from the group consisting of: S009E-S085D, S009E-S099E, S009E-Q107D, S009E-S182D, S009E-G189E, N074D-S099E, N074D-Q107D, N074D-G189E, S085D-S099E, S085D-G157D, S085D-Q176E, S085D-A188E, S085D-G189E, S085D-L256E, S099E-Q176E, S099E-Q107D, S099E-S182D, S099E-A188E, S099E-G189E, G157D-Q107D, G157D-A188E, G157D-G189E, Q176E-Q107D, Q176E-S182D, Q176E-A188E, Q176E-G189E, Q176E-N242D, Q107D-A188E, Q107D-G189E, Q107D-L256E, S182D-A188E, S182D-G189E, S182D-L256E, A188E-G189E, A188E-L256E, and G189E-N242D, where positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7. In some embodiments, such variant subtilisins have a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisins provided herein have at least a 2-fold, 3-fold, 4-fold, or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1 or SEQ ID NO:7), and have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0076] In some embodiments, the combination of substitutions is selected from the group consisting of: T009E-N085D, T009E-S099E, T009E-Q107D, T009E-N182D, T009E-G189E, N074D-S099E, N074D-Q107D, N074D-G189E, N085D-S099E, N085D-G157D, N085D-Q176E, N085D-T188E, N085D-G189E, N085D-Q256E, S099E-Q176E, S099E-Q107D, S099E-N182D, S099E-T188E, S099E-G189E, G157D-Q107D, G157D-T188E, G157D-G189E, Q176E-Q107D, Q176E-N182D, Q176E-T188E, Q176E-G189E, Q176E-N242D, Q107D-T188E, Q107D-G189E, Q107D-Q256E, N182D-T188E, N182D-G189E, N182D-Q256E, T188E-G189E, T188E-Q256E, and G189E-N242D, where positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7. In some embodiments, such variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least a 2-fold, 3-fold, 4-fold, or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1 or SEQ ID NO:7), and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7. In some embodiments, the combination of substitutions is selected from the group consisting of: X009E-X085D-X099E, X009E-X085D-X107D, X009E-X085D-X182D, X009E-X085D-X188E, X009E-X085D-X189E, X009E-X085D-X242D, X009E-X099E-X107D,X009E-X099E-X182D, X009E-X099E-X188E, X009E-X099E-X189E, X009E-X099E-X242D, X009E-X107D-X182D, X009E-X107D-X188E, X009E-X107D-X189E, X009E-X107D-X242D, X009E-X182D-X188E, X009E-X182D-X189E, X009E-X182D-X242D, X009E-X188E-X189E, X009E-X189E-X242D, X074D-X085D-X099E, X074D-X085D-X107D, X074D-X085D-X189E, X074D-X099E-X107D, X074D-X099E-X189E, X074D-X099E-X242D, X074D-X107D-X189E, X074D-X107D-X242D, X074D-X189E-X242D, X085D-X099E-X157D, X085D-X099E-X176E, X085D-X099E-X107D, X085D-X099E-X182D, X085D-X099E-X188E, X085D-X099E-X189E, X085D-X099E-X242D, X085D-X157D-X107D, X085D-X157D-X182D, X085D-X157D-X188E, X085D-X157D-X189E, X085D-X157D-X242D, X085D-X176E-X107D, X085D-X176E-X182D, X085D-X176E-X188E, X085D-X176E-X189E, X085D-X176E-X242D, X085D-X107D-X188E, X085D-X107D-X189E, X085D-X107D-X256E, X085D-X182D-X188E, X085D-X182D-X189E, X085D-X182D-X256E, X085D-X188E-X189E, X085D-X188E-X242D, X085D-X188E-X256E, X085D-X189E-X242D, X085D-X242D-X256E, X099E-X157D-X107D, X099E-X157D-X182D, X099E-X157D-X188E, X099E-X157D-X189E, X099E-X176E-X107DX099E-X176E-X182D, X099E-X176E-X188E, X099E-X176E-X189E, X099E-X176E-X242D, X099E-X107D-X182D, X099E-X107D-X188E, X099E-X107D-X189E, X099E-X107D-X242D, X099E-X182D-X188E, X099E-X182D-X189E, X099E-X182D-X242D, X099E-X188E-X189E, X099E-X188E-X242D, X099E-X189E-X242D, X157D-X107D-X182D, X157D-X107D-X188E, X157D-X107D-X189E, X157D-X107D-X242D, X157D-X182D-X188E, X157D-X182D-X189E, X157D-X188E-X189E, X157D-X188E-X242D, X157D-X189E-X242D, X176E-X107D-X182D, X176E-X107D-X188E, X176E-X107D-X189E, X176E-X107D-X242D, X176E-X182D-X188E, X176E-X182D-X189E, X176E-X182D-X242D, X176E-X188E-X189E, X176E-X188E-X242D, X176E-X189E-X242D, X107D-X182D-X188E, X107D-X182D-X189E, X107D-X182D-X256E, X107D-X188E-X189E, X107D-X188E-X242D, X107D-X188E-X256E, X107D-X189E-X242D, X107D-X242D-X256E, X182D-X188E-X189E, X182D-X188E-X242D, X182D-X188E-X256E, X182D-X189E-X242D, X182D-X242D-X256E, X188E-X189E-X242D and X188E-X242D-X256E, wherein the positions are numbered by correspondence to the amino acid sequence of SEQ ID NO:1, and the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,An amino acid sequence having 97% or 98% amino acid sequence identity. In some embodiments, such variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least a 2-fold, 3-fold, 4-fold or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1 or SEQ ID NO:7), and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0077] In some embodiments, the substituted combinations are selected from the group consisting of: S009E-S085D-S099E, S009E-S085D-Q107D, S009E-S085D-S182D, S009E-S085D-A188E, S009E-S085D-G189E, S009E-S085D-N242D, S009E-S099E-Q107D, S009E-S099E-S182D, S009E-S099E-A188E, S009E-S099E-G189E, S009E-S099E-N242D, S009E-Q107D-S182D, S009E-Q107D-A188E, S009E-Q107D-G189E, S009E-Q107D-N242D, S009E-S182D-A188E, S009E-S182D-G189E, S009E-S182D-N242D, S009E-A188E-G189E, S009E-G189E-N242D, N074D-S085D-S099E, N074D-S085D-Q107D, N074D-S085D-G189E, N074D-S099E-Q107D, N074D-S099E-G189E, N074D-S099E-N242D, N074D-Q107D-G189E, N074D-Q107D-N242D, N074D-G189E-N242D, S085D-S099E-G157D, S085D-S099E-Q176E, S085D-S099E-Q107D, S085D-S099E-S182D, S085D-S099E-A188E, S085D-S099E-G189E, S085D-S099E-N242D, S085D-G157D-Q107D, S085D-G157D-S182D, S085D-G157D-A188E, S085D-G157D-G189E, S085D-G157D-N242D, S085D-Q176E-Q107D, S085D-Q176E-S182D, S085D-Q176E-A188E, S085D-Q176E-G189E, S085D-Q176E-N242D, S085D-Q107D-A188E, S085D-Q107D-G189E, S085D-Q107D-L256E, S085D-S182D-A188E, S085D-S182D-G189E, S085D-S182D-L256E, S085D-A188E-G189E, S085D-A188E-N242D,S085D - A188E - L256E, S085D - G189E - N242D, S085D - N242D - L256E, S099E - G157D - Q107D, S099E - G157D - S182D, S099E - G157D - A188E, S099E - G157D - G189E, S099E - Q176E - Q107D, S099E - Q176E - S182D, S099E - Q176E - A188E, S099E - Q176E - G189E, S099E - Q176E - N242D, S099E - Q107D - S182D, S099E - Q107D - A188E, S099E - Q107D - G189E, S099E - Q107D - N242D, S099E - S182D - A188E, S099E - S182D - G189E, S099E - S182D - N242D, S099E - A188E - G189E, S099E - A188E - N242D, S099E - G189E - N242D, G157D - Q107D - S182D, G157D - Q107D - A188E, G157D - Q107D - G189E, G157D - Q107D - N242D, G157D - S182D - A188E, G157D - S182D - G189E, G157D - A188E - G189E, G157D - A188E - N242D, G157D - G189E - N242D, Q176E - Q107D - S182D, Q176E - Q107D - A188E, Q176E - Q107D - G189E, Q176E - Q107D - N242D, Q176E - S182D - A188E, Q176E - S182D - G189E, Q176E - S182D - N242D, Q176E - A188E - G189E, Q176E - A188E - N242D, Q176E - G189E - N242D, Q107D - S182D - A188E, Q107D - S182D - G189E, Q107D - S182D - L256E, Q107D - A188E - G189E, Q107D - A188E - N242D, Q107D - A188E - L256E, Q107D - G189E - N242D, Q107D - N242D - L256E, S182D - A188E - G189E, S182D - A188E - N242D, S182D - A188E - L256E, S182D - G189E - N242D, S182D - N242D - L256EA188E-G189E-N242D and A188E-N242D-L256E, wherein positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7. In some embodiments, such variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least a 2-fold, 3-fold, 4-fold or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1 or SEQ ID NO:7), and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0078] In some embodiments, the substituted combinations are selected from the group consisting of: T009E-N085D-S099E, T009E-N085D-Q107D, T009E-N085D-N182D, T009E-N085D-T188E, T009E-N085D-G189E, T009E-N085D-N242D, T009E-S099E-Q107D, T009E-S099E-N182D, T009E-S099E-T188E, T009E-S099E-G189E, T009E-S099E-N242D, T009E-Q107D-N182D, T009E-Q107D-T188E, T009E-Q107D-G189E, T009E-Q107D-N242D, T009E-N182D-T188E, T009E-N182D-G189E, T009E-N182D-N242D, T009E-T188E-G189E, T009E-G189E-N242D, N074D-N085D-S099E, N074D-N085D-Q107D, N074D-N085D-G189E, N074D-S099E-Q107D, N074D-S099E-G189E, N074D-S099E-N242D, N074D-Q107D-G189E, N074D-Q107D-N242D, N074D-G189E-N242D, N085D-S099E-G157D, N085D-S099E-Q176E, N085D-S099E-Q107D, N085D-S099E-N182D, N085D-S099E-T188E, N085D-S099E-G189E, N085D-S099E-N242D, N085D-G157D-Q107D, N085D-G157D-N182D, N085D-G157D-T188E, N085D-G157D-G189E, N085D-G157D-N242D, N085D-Q176E-Q107D, N085D-Q176E-N182D, N085D-Q176E-T188E, N085D-Q176E-G189E, N085D-Q176E-N242D, N085D-Q107D-T188E, N085D-Q107D-G189E, N085D-Q107D-Q256E, N085D-N182D-T188E, N085D-N182D-G189E, N085D-N182D-Q256E, N085D-T188E-G189E, N085D-T188E-N242D,N085D-T188E-Q256E, N085D-G189E-N242D, N085D-N242D-Q256E, S099E-G157D-Q107D, S099E-G157D-N182D, S099E-G157D-T188E, S099E-G157D-G189E, S099E-Q176E-Q107D, S099E-Q176E-N182D, S099E-Q176E-T188E, S099E-Q176E-G189E, S099E-Q176E-N242D, S099E-Q107D-N182D, S099E-Q107D-T188E, S099E-Q107D-G189E, S099E-Q107D-N242D, S099E-N182D-T188E, S099E-N182D-G189E, S099E-N182D-N242D, S099E-T188E-G189E, S099E-T188E-N242D, S099E-G189E-N242D, G157D-Q107D-N182D, G157D-Q107D-T188E, G157D-Q107D-G189E, G157D-Q107D-N242D, G157D-N182D-T188E, G157D-N182D-G189E, G157D-T188E-G189E, G157D-T188E-N242D, G157D-G189E-N242D, Q176E-Q107D-N182D, Q176E-Q107D-T188E, Q176E-Q107D-G189E, Q176E-Q107D-N242D, Q176E-N182D-T188E, Q176E-N182D-G189E, Q176E-N182D-N242D, Q176E-T188E-G189E, Q176E-T188E-N242D, Q176E-G189E-N242D, Q107D-N182D-T188E, Q107D-N182D-G189E, Q107D-N182D-Q256E, Q107D-T188E-G189E, Q107D-T188E-N242D, Q107D-T188E-Q256E, Q107D-G189E-N242D, Q107D-N242D-Q256E, N182D-T188E-G189E, N182D-T188E-N242D, N182D-T188E-Q256E, N182D-G189E-N242D, N182D-N242D-Q256ET188E-G189E-N242D and T188E-N242D-Q256E, wherein positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7. In some embodiments, such variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least a 2-fold, 3-fold, 4-fold or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1 or SEQ ID NO:7), and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0079] In another embodiment, there is provided a subtilisin variant, wherein the variant comprises the substitution X085D and at least two additional substitutions selected from the group consisting of: X009E, X074D, X099E, X157D, X176E, X188E, X189E, X242D and X256E, wherein positions are numbered according to SEQ ID NO:1, and wherein the variant has at least 60% identity to the amino acid sequence of SEQ ID NO:1 or 7. In some embodiments, such variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least a 2-fold, 3-fold, 4-fold or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1 or 7), and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0080] In some embodiments, a subtilisin variant comprising substitution X085D does not comprise additional combinations of substitutions selected from: a) two mutations selected from X009E, X074D, X176E, and X242D; b) mutation X074D further combined with one of X009E, X157D, X176E, X188E, and X256E; c) combination X099E-X256E; and d) combination X189E-X256E; wherein positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1.

[0081] In some embodiments, the variant comprises substitution X085D in a combination of substitutions selected from the group consisting of: X009E-X099E, X009E-X157D, X009E-X176E, X009E-X188E, X009E-X189E, X009E-X242D, X009E-X256E, X074D-X099E, X074D-X189E, X074D-X242D, X099E-X157D, X099E-X176E, X099E-X188E, X099E-X189E, X099E-X242D, X157D-X176E, X157D-X188E, X157D-X189E, X157D-X242D, X157D-X256E, X176E-X188E, X176E-X189E, X176E-X242D, X176E-X256E, X188E-X189E, X188E-X242D, X188E-X256E, X189E-X242D, and X242D-X256E, wherein positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or 7. In some embodiments, such variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least a 2-fold, 3-fold, 4-fold, or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1 or 7) and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or 7.

[0082] In another embodiment, a subtilisin variant is provided, wherein the variant comprises the substitution X188E and at least two additional substitutions selected from the group consisting of: X009E, X085D, X099E, X157D, X176E, X189E, X242D, and X256E, wherein the positions are numbered according to SEQ ID NO:1, and wherein the variant has at least 60% identity to the amino acid sequence of SEQ ID NO:1 or 7. In some embodiments, such a variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least a 2-fold, 3-fold, 4-fold, or greater improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1 or 7), and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0083] In some embodiments, a subtilisin variant comprising the substitution X188E does not comprise additional combinations of substitutions selected from the combinations X099E-X256E and X189E-X256E; wherein the positions are numbered by correspondence to the amino acid sequence of SEQ ID NO:1.

[0084] In some embodiments, the variant comprises substitution X188E in a combination of substitutions selected from the group consisting of: X009E-S085D, X009E-X099E, X009E-X157D, X009E-X176E, X009E-X189E, X009E-X242D, X009E-X256E, S085D-X099E, S085D-X157D, S085D-X176E, S085D-X189E, S085D-X242D, S085D-X256E, X099E-X157D, X099E-X176E, X099E-X189E, X099E-X242D, X157D-X176E, X157D-X189E, X157D-X242D, X157D-X256E, X176E-X189E, X176E-X242D, X176E-X256E, and X189E-X242D, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or 7. In some embodiments, such variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least 2-fold, 3-fold, 4-fold, or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1 or 7), and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or 7.

[0085] In another embodiment, a subtilisin variant is provided, wherein the variant comprises the substitution X099E and at least two additional substitutions selected from the group consisting of: X009E, X085D, X157D, X176E, X188E, X189E, X242D, and X256E, wherein the positions are numbered according to SEQ ID NO:1, and wherein the variant has at least 60% identity to the amino acid sequence of SEQ ID NO:1 or 7. In some embodiments, such variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least a 2-fold, 3-fold, 4-fold or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1 or 7), and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0086] In some embodiments, a subtilisin variant comprising the substitution X099E does not comprise additional combinations of substitutions selected from the following combinations: a) the mutation X074D further combined with one of X009E, X157D, X176E, X188E, and X256E; b) the combination X099E-X256E; and c) the combination X189E-X256E; wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1.

[0087] In some embodiments, the variant comprises a substitution X099E in a combination of substitutions selected from the group consisting of: X009E-X085D, X009E-X157D, X009E-X176E, X009E-X188E, X009E-X189E, X009E-X242D, X074D-X085D, X074D-X189E, X074D-X242D, X085D-X157D, X085D-X176E, X085D-X188E, X085D-X189E, X085D-X242D, X157D-X176E, X157D-X188E, X157D-X189E, X157D-X242D, X176E-X188E, X176E-X189E, X176E-X242D, X188E-X189E, X188E-X242D, and X189E-X242D, where positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or 7. In some embodiments, such variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisin provided herein has at least a 2-fold, 3-fold, 4-fold, or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1 or 7), and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or 7.

[0088] In another embodiment, the specification provides variant subtilisins that comprise a combination of mutations selected from the group consisting of: G157D-A188E-N242D, G157D-Q176E-A188E, G157D-Q176E-N242D, N074D-G189E-N242D, N074D-S085D-G189E, N074D-S085D-N242D, N074D-S085D-S099E, N074D-S099E-G189E, N074D-S099E-N242D, Q176E-A188E-N242D, Q176E-G189E-N242D, S009E-S085D-L256E, S085D-A188E-N242D, S085D-G157D-A188E, S085D-G157D-G189E, S085D-G157D-N242D, S085D-G189E-N242D, S085D-Q176E-G189E, S085D-Q176E-N242D, S085D-S099E-A188E, S085D-S099E-G157D, S085D-S099E-G189E, S085D-S099E-N242D, S085D-S099E-Q176E, S099E-A188E-N242D, S099E-G157D-A188E, S099E-G157D-G189E, S099E-G157D-N242D, S099E-G157D-Q176E, S099E-G189E-N242D, S099E-Q176E-A188E, and S099E-Q176E-N242D, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, such variant subtilisins have a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisins provided herein have at least a 2-fold, 3-fold, 4-fold, or greater improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:1) and have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1.

[0089] In another embodiment, the specification provides variant subtilisins that comprise combinations of mutations selected from the group consisting of: N085D-G157D-Q176E-M211L, N085D-Q176E-T188E-M211L, T009E-N085D-M211L-Q256E, T009E-T188E-M211L-Q256E, G157D-Q176E-T188E-M211L, N085D-G157D-T188E-M211L, N074D-N085D-S099E-M211L, N085D-S099E-Q176E-M211L, S099E-G157D-Q176E-M211L, T009E-S099E-M211L-Q256E, S099E-Q176E-T188E-M211L, N074D-S099E-G189E-M211L, N074D-N085D-S099E-M211Q, T009E-N085D-M211Q-Q256E, N085D-Q176E-G189E-M211L, N074D-N085D-G189E-M211L, G157D-Q176E-M211L-N242D, N085D-Q176E-M211L-N242D, N085D-S099E-T188E-M211L, N085D-S099E-G157D-M211L, N074D-N085D-M211L-N242D, Q176E-T188E-M211L-N242D, N074D-G189E-M211L-N242D, N074D-S099E-M211L-N242D, N085D-G157D-M211L-N242D, N085D-G157D-G189E-M211L, G157D-Q176E-G189E-M211L, T009E-M211L-N242D-Q256E, N085D-T188E-M211L-N242D, S039E-N085D-Q176E-M211L, S039E-S099E-M211L-Q256E, T009E-S039E-M211L-Q256E, T009E-S039E-N085D-M211L, S039E-Q176E-M211L-N242D, S039E-S099E-T188E-M211L, S039E-S099E-G157D-M211L, S039E-N074D-G189E-M211L, and S039E-N074D-N085D-M211L, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:7,And the variant subtilisin comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, such a variant subtilisin has a greater robustness improvement factor (RIF) than the parental subtilisin. In some embodiments, the variant subtilisins provided herein have at least a 2-fold, 3-fold, 4-fold or higher improvement in RIF relative to the parental subtilisin (e.g., SEQ ID NO:7), and have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:7.,

[0090] Another embodiment relates to one or more subtilisin variants described herein, provided that one or more substitutions are non-naturally occurring. Yet even still additional embodiments relate to one or more subtilisin variants described herein, wherein the variant (i) is derived from Bacillus lentus or Bacillus gibsonii subtilisin; (ii) is isolated; (iii) has proteolytic activity; or (iv) comprises a combination of (i) to (iii). Still yet another embodiment relates to one or more subtilisin variants described herein, wherein the variant is derived from a parental or reference polypeptide that (i) has 60%, 65%, 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 the amino acid sequence of SEQ ID NO:1 or 7; or (ii) has 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or 7. In still another embodiment, the parent comprises the amino acid sequence of SEQ ID NO:1 or 7. Even additional embodiments relate to one or more subtilisin variants described herein, wherein the variant comprises an amino acid sequence that (i) has 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less than 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or 7.

[0091] The present disclosure includes subtilisin variants that have one or more modifications at surface-exposed amino acids. The surface modifications of the enzyme variants can be used in detergent compositions by having a minimum performance index for wash performance, stability of the enzyme in the detergent composition, and thermal stability of the enzyme, while having at least one of these characteristics improved relative to the parent subtilisin. In some embodiments, the surface modification alters the hydrophobicity and / or charge of the amino acid at that position. Hydrophobicity can be determined using techniques known in the art, such as those described by White and Wimley (White, S.H. and Wimley, W.C., (1999) Annu. Rev. Biophys. Biomol. Struct 28:319-65).

[0092] As used herein, "surface properties" can be used to refer to electrostatic charge, and properties such as hydrophobicity and hydrophilicity exhibited by the surface of a protein. In even still further embodiments, one or more of the subtilisin variants described herein have one or more improved properties when compared to a reference subtilisin or a parent subtilisin; wherein the improved property is selected from improved detergent cleaning performance, improved stability, and combinations thereof.

[0093] In another embodiment, the parent subtilisin comprises the amino acid sequence of SEQ ID NO: 1 or 7. In another embodiment, the parent subtilisin is a polypeptide having the amino acid sequence of SEQ ID NO: 1 or 7. In yet another embodiment, the improved property is (i) improved detergent cleaning performance, wherein the variant has improved cleaning performance on blood / milk / ink stains or egg stains on woven cotton compared to the parent subtilisin; and / or (ii) improved stability, wherein the variant has higher residual activity compared to the parent or reference subtilisin. In still yet another embodiment, the detergent cleaning performance is measured according to the cleaning performance assay of Example 2; and / or the stability is measured according to the stability assay of Example 2.

[0094] In the context of oxidation, chelating agent, denaturing agent, surfactant, heat and / or pH stable proteases, the term "enhanced stability" or "improved stability" refers to a subtilisin variant that has a higher retained proteolytic activity over time compared to a reference or parent subtilisin (e.g., a wild-type protease or a parent protease, such as SEQ ID NO: 1 or 7). Autolysis has been identified as a mode of loss of subtilisin activity in liquid detergents. (Stoner et al., 2004 Protease autolysis in heavy-duty liquid detergent formulations: effects of thermodynamic stabilizers and protease inhibitors, Enzyme and Microbial Technology 34: 114-125).

[0095] Regarding protease variants, the terms "thermally stable" and "thermostable" and "thermostability" refer to proteases that retain a greater amount of residual activity after exposure to varying temperatures over a given period of time under conditions (or "stress conditions") commonly found in proteolysis, hydrolysis, cleaning, or other processes, as compared to the parental or reference protease. Residual activity is the amount of activity remaining after testing compared to the initial activity of the sample and can be reported as a percentage, e.g., residual activity %. "Altered temperature" encompasses both temperature increases and decreases. In some embodiments, the variant proteases provided herein retain at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98%, or about 99% proteolytic activity after exposure to a temperature in the range of 40°C to 80°C for a given period of time (e.g., at least about 5 minutes, at least about 20 minutes, at least about 60 minutes, about 90 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 minutes, about 360 minutes, about 420 minutes, about 480 minutes, about 540 minutes, about 600 minutes, about 660 minutes, about 720 minutes, about 780 minutes, about 840 minutes, about 900 minutes, about 960 minutes, about 1020 minutes, about 1080 minutes, about 1140 minutes, or about 1200 minutes). In some embodiments, using the method shown in Example 2, the variant subtilisin proteases provided herein have a higher residual activity than the parental or reference protease. In some embodiments, when measured after 20 minutes at 37 - 42 degrees Celsius in a liquid detergent, the variant subtilisin proteases provided herein have at least 5% improved residual activity compared to the parental subtilisin protease. In some embodiments, when measured after 20 minutes at 37 - 42 degrees Celsius in a liquid detergent, the variant subtilisin proteases provided herein have at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% improved residual activity compared to the parental subtilisin protease.

[0096] The subtilisin protease variants provided herein can be used to produce various compositions, such as enzyme compositions and cleaning compositions or detergent compositions. The enzyme compositions comprise a subtilisin protease variant as provided herein. The enzyme compositions can be in any form, such as granules, liquid formulations, or enzyme slurries.

[0097] Enzyme granules can be made in the following ways: for example, rotary atomization, wet granulation, dry granulation, spray drying, disk granulation, extrusion, pan coating, spheronization, drum granulation, fluidized bed agglomeration, high shear granulation, fluidized bed spray coating, crystallization, precipitation, emulsion gelation, rotary disk atomization and other casting methods as well as spheroidization processes. The core of the granule can be the granule itself or the inner core of a layered granule.

[0098] The core can contain one or more water-soluble agents or one or more water-dispersible agents, including but not limited to sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate and ammonium sulfate, citric acid, sugars (e.g., sucrose, lactose, glucose, granular sucrose, maltodextrin and fructose), plasticizers (e.g., polyols, urea, dibutyl phthalate and dimethyl phthalate), fibrous materials (e.g., cellulose and cellulose derivatives such as hydroxypropyl methylcellulose, carboxymethylcellulose and hydroxyethylcellulose), phosphates, calcium, protease inhibitors and combinations thereof. Suitable dispersing agents include but are not limited to clay, sugar pills (a combination of sugar and starch; e.g., starch-sucrose sugar pill - ASNP), talc, silicates, carboxymethylcellulose, starch and combinations thereof.

[0099] In some embodiments, the core mainly contains sodium sulfate. In some embodiments, the core consists essentially of sodium sulfate. In certain embodiments, the core consists only of sodium sulfate.

[0100] In some embodiments, the core contains a subtilisin variant as provided herein. In other embodiments, the core contains one or more enzymes in addition to the protease. In other embodiments, the core is inert and does not contain enzymes.

[0101] In some embodiments, the core is an enzyme powder, including UFC containing the enzyme. The enzyme powder can be spray dried and can optionally be admixed with any of the water-soluble agents or water-dispersible agents listed herein. The enzyme can be or can include the protease to be stabilized, in which case the enzyme powder should further include a stabilizer.

[0102] In some embodiments, the core is coated with at least one coating. In certain embodiments, the core is coated with at least two coatings. In another certain embodiment, the core is coated with at least three coatings. The materials for one or more coatings can be suitable for use in cleaning compositions and / or detergent compositions (see, for example, US20100124586, WO 9932595 and US5324649).

[0103] In some embodiments, the coating comprises one or more of the following materials: inorganic salts (e.g., sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate), citric acid, sugars (e.g., sucrose, lactose, glucose, and fructose), plasticizers (e.g., polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous materials (e.g., cellulose and cellulose derivatives such as hydroxypropyl methylcellulose, carboxymethylcellulose, and hydroxyethylcellulose), clay, sugar pills (a combination of sugar and starch), silicates, carboxymethylcellulose, phosphates, starch (e.g., corn starch), fats, oils (e.g., rapeseed oil and paraffin oil), lipids, vinyl polymers, vinyl copolymers, polyvinyl alcohol (PVA), plasticizers (e.g., polyols, urea, dibutyl phthalate, dimethyl phthalate, and water), anti-caking agents (e.g., talc, clay, amorphous silica, and titanium dioxide), defoaming agents (such as FOAMBLAST and EROL ), and talc. Suitable components for the coating are detailed in US20100124586, WO9932595, and US 5324649.

[0104] In some embodiments, the coating comprises sugars (e.g., sucrose, lactose, glucose, granulated sucrose, maltodextrin, and fructose). In some embodiments, the coating comprises a polymer such as polyvinyl alcohol (PVA). Suitable PVAs for incorporation into one or more coatings of the multi-layer particles include partially hydrolyzed, fully hydrolyzed, and moderately hydrolyzed PVAs having low to high viscosities. In some embodiments, the coating comprises an inorganic salt such as sodium sulfate.

[0105] In some embodiments, at least one coating is an enzyme coating. In some embodiments, the core is coated with at least two enzyme layers. In another embodiment, the core is coated with at least three or more enzyme layers.

[0106] In some embodiments, the enzyme granules comprise a subtilisin variant in combination with one or more additional enzymes as provided herein, the one or more additional enzymes being selected from the group consisting of: acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersin, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloprotease, nuclease (e.g., DNase and / or RNase), oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenoloxidase, phosphatase, phosphodiesterase, phospholipase, phytase, polygalacturonase, polyesterase, additional protease, pullulanase, reductase, rhamnogalacturonase, β-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof. Typically, at least one enzyme coating comprises at least one subtilisin variant as provided herein.

[0107] The above enzyme list is merely illustrative and is not meant to be exclusive. Any enzyme can be used in the particles described herein, including wild-type enzymes, recombinant enzymes, and variant enzymes from bacterial, fungal, yeast sources, as well as acidic, neutral, or basic enzymes.

[0108] Another embodiment relates to a method of cleaning a surface, the method comprising contacting the surface or article in need of cleaning with an effective amount of one or more subtilisin variants as provided herein or a composition comprising one or more subtilisin variants as provided herein. In some embodiments, the surface or article in need of cleaning comprises a protein stain on the surface. In some embodiments, the surface or article in need of cleaning comprises a protein stain. The term "stain" encompasses any type of dirt on the surface of an article (e.g., a hard surface article such as a tableware or a textile). In some embodiments, the stain is a protein stain. As used herein, a "protein stain" is a stain or dirt containing protein.

[0109] Additional embodiments relate to a method of cleaning a protein stain, the method comprising contacting the surface or article in need of cleaning with an effective amount of one or more subtilisin variants as provided herein or a composition comprising one or more subtilisin variants as provided herein.

[0110] Another embodiment relates to a method for cleaning egg stains, the method comprising contacting a surface or article to be cleaned with an effective amount of one or more subtilisin variants as provided herein or a composition comprising one or more such subtilisin variants.

[0111] Another embodiment relates to a method for cleaning BMI stains, the method comprising contacting a surface or article to be cleaned with an effective amount of one or more subtilisin variants as provided herein or a composition comprising one or more such subtilisin variants.

[0112] One or more of the subtilisin variants described herein can be subject to various changes, such as one or more amino acid insertions, deletions, and / or substitutions (conservative or non-conservative), including cases where such changes do not substantially alter the enzyme activity of the variant. Similarly, the nucleic acids of the present invention can also be subject to various changes, such as one or more substitutions of one or more nucleotides in one or more codons such that a particular codon encodes the same or a different amino acid, resulting in silent changes (e.g., when the encoded amino acid is not altered by the nucleotide mutation) or non-silent changes; one or more deletions of one or more nucleotides (or codons) in the sequence; one or more additions or insertions of one or more nucleotides (or codons) in the sequence; and / or cleavage or one or more truncations of one or more nucleotides (or codons) in the sequence. Many such changes in the nucleic acid sequence do not substantially alter the enzyme 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 one or more of the same amino acids.

[0113] One or more isolated, non-naturally occurring, or recombinant polynucleotides are described herein, the polynucleotide comprising a nucleic acid sequence encoding one or more of the subtilisin variants described herein, or a recombinant polypeptide or an active fragment thereof. One or more of the nucleic acid sequences described herein can be used in the recombinant production (e.g., expression) of one or more of the subtilisin variants described herein, typically by expressing a plasmid expression vector comprising a sequence encoding one or more of the subtilisin variants described herein or a fragment thereof. One embodiment provides a nucleic acid encoding one or more of the subtilisin variants described herein, wherein the variant is a mature form having proteolytic activity. In some embodiments, one or more of the subtilisin variants described herein are recombinantly expressed with a homologous propeptide sequence. In other embodiments, one or more of the subtilisin variants described herein are recombinantly expressed with a heterologous propeptide sequence (e.g., the propeptide sequence from Bacillus lentus (SEQ ID NO: 4) or a variant thereof).

[0114] One or more of the nucleic acid sequences described herein can be produced by using any suitable synthetic, manipulative, and / or isolation techniques or combinations thereof. For example, one or more of the polynucleotides described herein can be produced using standard nucleic acid synthesis techniques well known to those skilled in the art, such as solid-phase synthesis techniques. In such techniques, typically fragments of up to 50 or more nucleotide bases are synthesized and then ligated (e.g., by enzymatic or chemical ligation methods) to substantially form any desired continuous nucleic acid sequence. The synthesis of one or more of the polynucleotides described herein can also be facilitated by any suitable method known in the art, including but not limited to chemical synthesis using the following methods: the classical phosphoramidite method (see, e.g., Beaucage et al., Tetrahedron Letters 22:1859-69 (1981)), or the method described in Matthes et al., EMBO J. 3:801-805 (1984), as typically practiced in automated synthesis methods. One or more of the polynucleotides described herein can also be produced using an automated DNA synthesizer. Custom nucleic acids can be ordered from a variety of commercial sources (e.g., ATUM (DNA 2.0), Newark, CA, USA; Life Tech (GeneArt), Carlsbad, CA, USA; GenScript, Ontario, Canada; Base Clear B.V., Leiden, Netherlands; Integrated DNA Technologies, Skokie, IL, USA; Ginkgo Bioworks (Gen9), Boston, MA, USA; and Twist Bioscience, San Francisco, CA, USA). Other techniques and related principles for synthesizing nucleic acids are described by, for example, Itakura et al., Ann. Rev. Biochem. 53:323 (1984) and Itakura et al., Science 198:1056 (1984).

[0115] Recombinant DNA techniques for modifying nucleic acids are well-known in the art, such as restriction endonuclease digestion, ligation, reverse transcription and cDNA production, and polymerase chain reaction (e.g., PCR). One or more of the polynucleotides described herein can also be obtained by screening a cDNA library using one or more oligonucleotide probes that can hybridize to or be PCR amplified from polynucleotides encoding one or more of the subtilisin 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 references known to those skilled in the art. One or more of the polynucleotides described herein can be obtained by altering the naturally occurring polynucleotide backbone (e.g., the polynucleotide backbone encoding one or more of the subtilisin variants or a reference subtilisin described herein) by, for example, known mutagenesis procedures (e.g., site-directed mutagenesis, site saturation mutagenesis, and in vitro recombination). A variety of methods suitable for generating modified polynucleotides described herein encoding one or more of the subtilisin variants described herein are known in the art, including but not limited to, for example, site saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed evolution, and various other recombination methods.

[0116] Additional embodiments relate to one or more vectors that contain one or more of the subtilisin variants described herein (e.g., polynucleotides encoding one or more of the subtilisin variants described herein); expression vectors or expression cassettes that contain one or more of the nucleic acids or polynucleotide sequences described herein; isolated, substantially pure, or recombinant DNA constructs that contain one or more of the nucleic acids or polynucleotide sequences described herein; isolated or recombinant cells that contain one or more of the polynucleotide sequences described herein; and compositions that contain one or more such vectors, nucleic acids, expression vectors, expression cassettes, DNA constructs, cells, cell cultures, or any combination or mixture thereof.

[0117] Some embodiments relate to one or more recombinant cells that contain one or more of the vectors described herein (e.g., expression vectors or DNA constructs) that contain one or more of the nucleic acids or polynucleotide sequences described herein. Some such recombinant cells are transformed or transfected with at least one such vector, although other methods are available and known in the art. Such cells are typically referred to as host cells. Some such cells include bacterial cells, including but not limited to cells of the genus Bacillus, such as Bacillus subtilis cells. Other embodiments relate to recombinant cells (e.g., recombinant host cells) that contain one or more of the subtilisins described herein.

[0118] In some embodiments, one or more of the vectors described herein are expression vectors or expression cassettes that comprise one or more of the polynucleotide sequences described herein operably linked to one or more additional nucleic acid segments required for effective gene expression (e.g., a promoter of one or more of the polynucleotide sequences described herein). The vector may include a transcription terminator and / or a selectable gene (e.g., an antibiotic resistance gene) that enables continuous culture maintenance of host cells plasmid-infected by growth in a medium containing an antimicrobial agent.

[0119] 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 [editors], Chapter 3, Molecular Biological Methods for Bacillus, John Wiley & Sons (1990)); replication plasmids suitable for Bacillus subtilis include those listed on page 92). (See also, Perego, “Integrational Vectors for Genetic Manipulations in Bacillus subtilis”; Sonenshein et al., [editors]; “Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular Genetics”, American Society for Microbiology, Washington, D.C. (1993), pp. 615-624; and p2JM103BBI).

[0120] For the 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 comprising one or more copies (and in some cases multiple copies) of a polynucleotide encoding one or more subtilisin variants described herein are transformed into a cell under conditions suitable for the expression of the variant. In some embodiments, the polynucleotide sequence encoding one or more subtilisin variants described herein (and other sequences contained in the vector) is integrated into the genome of the host cell; however, in other embodiments, the plasmid vector containing the polynucleotide sequence encoding one or more subtilisin variants described herein remains as an autonomous extrachromosomal element within the cell. Some embodiments provide extrachromosomal nucleic acid elements as well as input nucleotide sequences integrated into the genome of the host cell. The vectors described herein can be used to produce 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 is capable of integrating the polynucleotide encoding the variant into the host chromosome and optionally amplifying it in the host chromosome. Examples of integration sites are well known to those of skill in the art. In some embodiments, transcription of the polynucleotide encoding one or more subtilisin variants described herein is effected by a promoter that is the wild-type promoter of the parental subtilisin. In some other embodiments, the promoter is heterologous to one or more subtilisin variants described herein but is functional in the host cell. Exemplary promoters for bacterial host cells include but are not limited to amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpaII promoters; the promoter of the Bacillus stearothermophilus maltogenic amylase gene; the Bacillus amyloliquefaciens (BAN) amylase gene; the Bacillus subtilis alkaline protease gene; the Bacillus clausii alkaline protease gene; the Bacillus pumilis xylosidase gene; Bacillus thuringiensis cryIIIA; and the Bacillus licheniformis α-amylase gene. Additional promoters include but are not limited to the A4 promoter, and the bacteriophage λPR or PL promoters and the Escherichia coli lac, trp or tac promoters.

[0121] One or more subtilisin variants described herein can be produced in host cells of any suitable microorganism, including bacteria and fungi. In some embodiments, one or more subtilisin variants described herein can be produced in Gram-positive bacteria. In some embodiments, the host cell is a Bacillus species, Streptomyces species, Escherichia species, Aspergillus species, Trichoderma species, Pseudomonas species, Corynebacterium species, Saccharomyces species or Pichia species. In some embodiments, one or more subtilisin variants described herein are produced by a Bacillus species host cell. Examples of Bacillus species host cells useful for the production of one or more subtilisin variants described herein include, but are not limited to: Bacillus licheniformis, Bacillus gibsonii, Bacillus lentus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus stearothermophilus, Bacillus alcalophilus, Bacillus coagulans, Bacillus circulans, Bacillus pumilus, Bacillus thuringiensis, Bacillus clausii and Bacillus megaterium, and other organisms within the genus Bacillus. In some embodiments, a Bacillus subtilis host cell is used for the production of the variants described herein. USPN 5,264,366 and 4,760,025 (RE34,606) describe various Bacillus host strains that can be used for the production of one or more subtilisin variants described herein, but other suitable strains can be used.

[0122] Several 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 species strains, as well as variants of naturally occurring strains and / or recombinant strains. In some embodiments, the host strain is a recombinant strain in which 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 Bacillus subtilis host strain, particularly a recombinant Bacillus subtilis host strain. Many Bacillus subtilis strains are known, including but not limited to, for example, 1A6 (ATCC 39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753 to PB1758, PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP 211 strains (see, for example, Hoch et al., Genetics 73:215-228 (1973); see also, US 4,450,235; US 4,302,544; and EP 0134048). The use of Bacillus subtilis as an expression host cell is well known in the art (see, for example, 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)).

[0123] In some embodiments, the Bacillus species host cell is a Bacillus species comprising a mutation or deletion in at least one of the following genes: degU, degS, degR, and degQ. In some embodiments, the mutation is in the degU gene, and in some embodiments, the mutation is degU(Hy)32 (see, e.g., Msadek et al., J. Bacteriol. 172:824 - 834 (1990); and Olmos et al., Mol. Gen. Genet. 253:562 - 567 (1997)). In some embodiments, the Bacillus host comprises a mutation or deletion in scoC4 (see, e.g., Caldwell et al., J. Bacteriol. 183:7329 - 7340 (2001)); spoIIE (see, e.g., Arigoni et al., Mol. Microbiol. 31:1407 - 1415 (1999)); and / or oppA or other genes of the opp operon (see, e.g., Perego et al., Mol. Microbiol. 5:173 - 185 (1991)). Indeed, any mutation in the opp operon that results in the same phenotype as a mutation in the oppA gene is expected to be useful in some embodiments of the altered Bacillus strains described herein. In some embodiments, these mutations occur singly, while in other embodiments, combinations of mutations are present. In some embodiments, the altered Bacillus host cell strain that can be used to produce one or more of the subtilisin variants described herein is a Bacillus host strain that already comprises a mutation in one or more of the above genes. Additionally, a Bacillus species host cell comprising one or more mutations and / or one or more deletions in an endogenous protease gene can be used. In some embodiments, the Bacillus host cell comprises deletions of the aprE and nprE genes. In other embodiments, the Bacillus species host cell comprises deletions of 5 protease genes, while in other embodiments, the Bacillus host cell comprises deletions of 9 protease genes (see, e.g., US2005 / 0202535).

[0124] Transform a host cell 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 acid (e.g., DNA) into Bacillus cells or E. coli cells using plasmid DNA constructs or vectors and transforming such plasmid DNA constructs or vectors into such cells are well known. In some embodiments, the plasmid is subsequently isolated from the E. coli cells and transformed into Bacillus cells. However, the use of an intervening microorganism such as E. coli is not necessary, and in some embodiments, the DNA construct or vector is introduced directly into the Bacillus host.

[0125] Exemplary methods for introducing one or more of the nucleic acid sequences described herein into Bacillus cells are described, for example, in Ferrari et al., “Genetics,” in Hardwood et al. [Eds.], Bacillus, Plenum Publishing Corp. (1989), pp. 57-72; Saunders et al., J. Bacteriol., 157:718-726 (1984); Hoch et al., J. Bacteriol., 93:1925-1937 (1967); Mann et al., Current Microbiol., 13:131-135 (1986); Holubova, Folia Microbiol., 30:97 (1985); Chang et al., Mol. Gen. Genet. 168:11-115 (1979); Vorobjeva et al., FEMS Microbiol. Lett. 7:261-263 (1980); Smith et al., Appl. Env. Microbiol 51:634 (1986); Fisher et al., Arch. Microbiol., 139:213-217 (1981); and McDonald, J. Gen. Microbiol 130:203 (1984)). Indeed, methods such as transformation (including protoplast transformation and transfection, transduction, and protoplast fusion) are well known and suitable for use herein. Methods for transforming Bacillus cells known in the art include, for example, methods such as plasmid marker rescue transformation, which involves the uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (see, Contente et al., Plasmid 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, the incoming donor plasmid recombines with a homologous region of the resident “helper” plasmid during a process that mimics chromosomal transformation.

[0126] In addition to the methods commonly used, in some embodiments, host cells are directly transformed with a DNA construct or vector comprising a nucleic acid encoding one or more of the 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 the host cell). Introducing the DNA construct or vector described herein into a host cell includes those physical and chemical methods known in the art for introducing a nucleic acid sequence (e.g., a DNA sequence) into a host cell without insertion into the host genome. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, and liposomes. In additional embodiments, the DNA construct or vector is co-transformed with a plasmid without insertion into the plasmid. In further embodiments, selectable markers are deleted from the modified Bacillus strain by methods known in the art (see, Stahl et al., J. Bacteriol. 158:411 - 418 (1984); and Palmeros et al., Gene 247:255 - 264 (2000)).

[0127] In some embodiments, the transformed cells are cultured in a conventional nutrient medium. Appropriate specific culture conditions, such as temperature, pH, etc., are known to those skilled in the art and are described in detail in the scientific literature. Some embodiments provide a culture (e.g., a cell culture) that contains one or more of the subtilisin variants or nucleic acid sequences described herein.

[0128] In some embodiments, host cells transformed with one or more polynucleotide sequences encoding one or more of the subtilisin variants described herein are cultured in a suitable nutrient medium under conditions that permit expression of the variant, and the resulting variant is then recovered from the culture. In some embodiments, the variant produced by the cells is recovered from the medium by conventional procedures, which include, but are not limited to, separating the host cells from the medium by, for example, centrifugation or filtration, precipitating the protein components of the supernatant or filtrate with a salt (e.g., ammonium sulfate), and purifying by chromatography (e.g., ion exchange, gel filtration, affinity, etc.).

[0129] In some embodiments, one or more subtilisin variants produced by a recombinant host cell are secreted into the culture medium. A nucleic acid sequence encoding a purification facilitating domain can be used to facilitate purification of the variant. 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 that facilitates purification of the variant (see, for example, Kroll et al., DNA Cell Biol. 12:441-53 (1993)). Such purification facilitating domains include, but are not limited to, for example, metal chelating peptides such as the histidine-tryptophan module that permits purification on immobilized metal (see Porath, Protein Expr. Purif. 3:263-281

[1992] ), the protein A domain that permits purification on immobilized immunoglobulin, and the domain employed in the FLAGS extension / affinity purification system. It has also been found that inclusion of a cleavable linker sequence such as factor XA or enterokinase (e.g., the sequence available from Invitrogen, San Diego, Calif.) between the purification domain and the heterologous protein can be used to facilitate purification.

[0130] The variant proteins of the invention can be produced in host cells using methods well known in the art, such as by secretion or intracellular expression. Fermentation, isolation, and concentration techniques are well known in the art, and conventional methods can be used to prepare concentrated, enzyme-containing solutions. The host cells can be further processed, for example, by heating or by changing the pH or salt content or by enzymatic treatment with enzymes including egg white lysozyme, T4 lysozyme, or the enzymes described in WO 2022047149, for example, to release the enzyme or to improve cell separation. For production-scale recovery, the variant polypeptide can be enriched or partially purified via removal of cells with polymer flocculation as generally described above. Alternatively, the enzyme can be enriched or purified by microfiltration and then concentrated by ultrafiltration using available membranes and equipment. However, for some applications, the enzyme does not need to be enriched or purified, and the whole culture broth can be lysed and used without further treatment. The enzyme can then be processed, for example, into granules.

[0131] A variety of methods can be used to determine the production level of one or more mature subtilisin variants described herein in a host cell. Such methods include, but are not limited to, for example, methods utilizing polyclonal or monoclonal antibodies specific for the protease. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), and fluorescence-activated cell sorting (FACS). These and other assays are well known in the art (see, for example, Maddox et al., J. Exp. Med. 158:1211 (1983)).

[0132] Some other embodiments provide methods for preparing or producing one or more of the mature subtilisin variants described herein. The mature subtilisin variants do not include signal peptide or propeptide sequences. Some methods include preparing or producing one or more of the subtilisin variants described herein in a recombinant bacterial host cell (such as a Bacillus species cell (e.g., Bacillus subtilis cell)). Other embodiments provide methods for producing one or more of the subtilisin variants described herein, which methods include culturing a recombinant host cell containing a recombinant expression vector under conditions favorable for the production of the variant, the recombinant expression vector containing a nucleic acid sequence encoding one or more of the subtilisin variants described herein. Some such methods further include recovering the variant from the culture.

[0133] Additional embodiments provide methods for producing one or more of the subtilisin variants described herein, which methods include: (a) introducing a recombinant expression vector containing a nucleic acid encoding the variant into a population of cells (such as bacterial cells, e.g., Bacillus subtilis cells); and (b) culturing the cells in a medium under conditions favorable for the production of the variant encoded by the expression vector. Some such methods further include: (c) isolating the variant from the cells or from the medium.

[0134] Additional embodiments relate to methods of improving the cleaning performance or stability of subtilisin, which methods include modifying subtilisin to include one or more substitutions, or combinations of substitutions, as provided herein.

[0135] Unless otherwise indicated, all component or composition levels provided herein are given with reference to the active level of the component or composition and do not include impurities that may be present in commercially available sources, such as residual solvents or by-products. The enzyme component weight is based on the total active protein. Unless otherwise indicated, all percentages and ratios are by weight. Unless otherwise indicated, all percentages and ratios are based on the total composition. The compositions described herein include cleaning compositions, such as detergent compositions. In the exemplary detergent compositions, the enzyme level is expressed as the pure enzyme by weight of the total composition and, unless otherwise specified, the detergent ingredients are expressed by weight of the total composition.

[0136] In one embodiment, one or more of the subtilisin variants described herein can be used in cleaning applications, such as but not limited to cleaning dishware items or tabletop appliance items, fabrics, medical devices, and items having a hard surface (e.g., the hard surfaces of tables, tabletops, walls, furniture items, floors, ceilings). In other embodiments, one or more of the subtilisin variants described herein can be used in disinfection applications, such as but not limited to disinfecting automatic dishwashers or washing machines.

[0137] Another embodiment relates to a composition 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 the group consisting of laundry detergent compositions, automatic dishwashing (ADW) compositions, hand wash (manual) dish detergent compositions, hard surface cleaning compositions, eyewear cleaning compositions, medical device cleaning compositions, disinfectant (e.g., malodor or microbial) compositions, and personal care cleaning compositions. In yet other embodiments, the composition is a laundry detergent composition, an ADW composition, or a hand wash (manual) dish detergent composition. Even still further embodiments relate to fabric cleaning compositions, while other embodiments relate to non-fabric cleaning compositions. In some embodiments, the cleaning composition is boron-free. In other embodiments, the cleaning composition is phosphate-free. In still other embodiments, the composition comprises one or more subtilisin variants described herein and one or more excipients, adjuvants, and / or additional enzymes.

[0138] In another embodiment, the present disclosure provides detergent compositions (e.g., ADW compositions) that comprise a surfactant and at least one subtilisin variant as provided herein. Such compositions may further comprise one or more of excipients, adjuvants, and / or additional enzymes.

[0139] In still yet another embodiment, the compositions described herein contain phosphate, are phosphate-free, contain boron, are boron-free, or a combination thereof. In other embodiments, the composition is a boron-free composition. In some embodiments, the boron-free composition is a composition to which no borate stabilizer has been added. In another embodiment, the boron-free composition is a composition containing less than 5.5% boron. In still another embodiment, the boron-free composition is a composition containing less than 4.5% boron. In yet still another embodiment, the boron-free composition is a composition containing less than 3.5% boron. In still yet another embodiment, the boron-free composition is a composition containing less than 2.5% boron. In even another embodiment, the boron-free composition is a composition containing less than 1.5% boron. In another embodiment, the boron-free composition is a composition containing less than 1.0% boron. In still another embodiment, the boron-free composition is a composition containing less than 0.5% boron. In other embodiments, the composition is a composition that is free or substantially free of enzyme stabilizers or peptide inhibitors.

[0140] In another embodiment, one or more of the compositions described herein are in a form selected from gels, tablets, powders, granules, solids, liquids, unit doses, and combinations thereof. In yet another embodiment, one or more of the compositions described herein are in a form selected from low-water compact formulations, low-water HDLs or unit doses (UDs), or high-water formulations or HDLs. In some embodiments, the cleaning compositions described herein are in unit dosage forms. In other embodiments, the unit dose form is selected from pills, tablets, capsules, caplets, sachets, pouches, multi-compartment pouches, and pre-measured powders or liquids. In some embodiments, the unit dosage form is designed to provide controlled release of the ingredients within a multi-compartment pouch (or other unit dosage form). Suitable unit doses and controlled release forms are described, for example, in EP 2100949, WO 02 / 102955, US 4,765,916, US 4,972,017, and WO 04 / 111178. In some embodiments, the unit dosage form is a tablet or powder contained within a water-soluble film or pouch.

[0141] Exemplary laundry detergent compositions include, but are not limited to, for example, liquid and powder laundry detergent compositions. Exemplary hard surface cleaning compositions include, but are not limited to, for example, compositions for cleaning the hard surfaces of non-tableware items, non-tabletop appliance items, tables, tabletops, furniture items, walls, floors, and ceilings. Exemplary hard surface cleaning compositions are described, for example, in USPN 6,610,642, 6,376,450, and 6,376,450. Exemplary personal care compositions include, but are not limited to, compositions for cleaning dentures, teeth, hair, contact lenses, and skin. Exemplary components of such oral care compositions are those described, for example, in US 6,376,450.

[0142] In some embodiments, one or more of the subtilisin variants described herein clean at low temperatures. In other embodiments, one or more of the compositions described herein clean at low temperatures. In other embodiments, one or more of the compositions described herein contain an effective amount of one or more of the subtilisin variants described herein, which are useful or effective for cleaning surfaces where protein stains need to be removed.

[0143] In some embodiments, adjunct materials are incorporated, e.g., to assist or enhance cleaning performance; to treat substrates to be cleaned; or to alter the aesthetics of the cleaning composition, such as in the case of fragrances, colorants, dyes, etc. One embodiment relates to a composition comprising one or more adjunct materials and one or more subtilisin variants described herein. Another embodiment relates to a composition comprising one or more adjunct materials and one or more subtilisin variants described herein, wherein the adjunct material is selected from: bleach catalysts, additional enzymes, enzyme stabilizers (including, e.g., enzyme stabilizing systems), chelants, optical brighteners, soil release polymers, dye transfer agents, dispersants, foam inhibitors, dyes, fragrances, colorants, fillers, photoactivators, fluorescent agents, fabric conditioners, hydrolysable surfactants, preservatives, antioxidants, anti-shrink agents, anti-wrinkle agents, bactericides, fungicides, color accents, silver care agents, anti-tarnish agents, anti-corrosion agents, alkaline sources, solubilizers, carriers, processing aids, pigments, pH control agents, surfactants, builders, chelating agents, dye transfer inhibitors, deposition aids, catalytic materials, bleach activators, bleach boosters, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, polymeric dispersants, clay soil removal / anti-redeposition agents, structural elasticizers, fabric softeners, carriers, hydrotropes, processing aids, pigments, and combinations thereof. Exemplary adjunct materials and levels of use can be found in USPN 5,576,282, 6,306,812, 6,326,348, 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014, and 5,646,101. In embodiments where one or more cleaning adjunct materials are incompatible with one or more subtilisin variants described herein, methods are used to keep the adjunct material and one or more variants separate (i.e., not in contact with each other) until the combination of the two components is appropriate. Such separation methods include any suitable methods known in the art (e.g., capsule tablets, encapsulation, tablets, physical separation, etc.).

[0144] Some embodiments relate to cleaning additive products comprising one or more subtilisin variants described herein. In some embodiments, the additive is encapsulated in a dosage form for addition to a cleaning process. In some embodiments, the additive is encapsulated in a dosage form for addition to a cleaning process in which a peroxide source is used and an enhanced bleaching effect is desired.

[0145] Exemplary fillers or carriers for particulate compositions include, but are not limited to, various salts such as sulfates, carbonates, and silicates; talc; and clays. Exemplary fillers or carriers for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols (including polyols and diols such as methanol, ethanol, propanol, and isopropanol). In some embodiments, the composition contains from about 5% to about 90% of such fillers or carriers. Acidic fillers may be included in such compositions to lower the pH of the resulting solution in the cleaning method or application.

[0146] In one embodiment, one or more of the cleaning compositions described herein comprise an effective amount of one or more of the subtilisin variants described herein, which are either alone or in combination with one or more additional enzymes. Typically, the cleaning composition comprises at least about 0.0001 wt% to about 20 wt%, from about 0.0001 wt% to about 10 wt%, from about 0.0001 wt% to about 1 wt%, from about 0.001 wt% to about 1 wt%, or from about 0.01 wt% to about 0.2 wt% of one or more of the subtilisin variants described herein. In another embodiment, one or more of the cleaning compositions described herein comprise from about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.01 to about 2 mg, about 0.01 to about 1 mg, about 0.05 to about 1 mg, about 0.5 to about 10 mg, about 0.5 to about 5 mg, about 0.5 to about 4 mg, about 0.5 to about 3 mg, about 0.5 to about 2 mg, about 0.5 to about 1 mg, about 0.1 to about 10 mg, about 0.1 to about 5 mg, about 0.1 to about 4 mg, about 0.1 to about 3 mg, about 0.1 to about 2 mg, about 0.1 to about 2 mg, about 0.1 to about 1 mg, or about 0.1 to about 0.5 mg of one or more of the subtilisin variants described herein per gram of composition.

[0147] Typically, the cleaning compositions described herein are formulated such that during use in aqueous cleaning operations, the wash water will have a pH from about 4.0 to about 11.5, or even from about 5.0 to about 11.5, or even from about 5.0 to about 8.0, or even from about 7.5 to about 10.5. Liquid product formulations are typically formulated to have a pH from about 3.0 to about 9.0 or even from about 3 to about 5. Granular laundry products are typically formulated to have a pH from about 8 to about 11. In some embodiments, the cleaning compositions of the present invention can be formulated to have an alkaline pH under washing conditions, such as a pH from about 8.0 to about 12.0, or from about 8.5 to about 11.0, or from about 9.0 to about 11.0. In some embodiments, the cleaning compositions of the present invention can be formulated to have a neutral pH under washing conditions, such as a pH from about 5.0 to about 8.0, or from about 5.5 to about 8.0, or from about 6.0 to about 8.0, or from about 6.0 to about 7.5. In some embodiments, neutral pH conditions can be measured when the cleaning composition is dissolved in deionized water at 20 °C at a 1:100 (wt:wt) ratio, using a conventional pH meter. Techniques for controlling the pH at the recommended use levels include using buffers, alkalis, acids, etc., and are well known to those skilled in the art.

[0148] In some embodiments, one or more of the subtilisin variants described herein are encapsulated to protect them from the effects of other components in the composition during storage and / or to control the availability of the variant during cleaning. In some embodiments, encapsulation enhances the performance of the variant and / or additional enzymes. In some embodiments, the encapsulating material typically encapsulates at least a portion of the subtilisin variant 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. Exemplary encapsulating materials include, but are not limited to: carbohydrates, natural or synthetic gums, chitin, chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin wax, and combinations thereof. When the encapsulating material is a carbohydrate, it is typically selected from monosaccharides, oligosaccharides, and combinations thereof. In some embodiments, the encapsulating material is starch (see, for example, EP0922499, US 4,977,252, US 5,354,559, and US 5,935,826). In some embodiments, the encapsulating material is microspheres made of plastics (such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile, and mixtures thereof). Exemplary commercial microspheres include, but are not limited to (Stockviksverken, Sweden); and PM6545, PM 6550, PM 7220, PM 7228, and (PQ Corp., Valley Forge, PA)

[0149] There are various washing conditions, including different detergent formulations, washing water volumes, washing water temperatures, and lengths of washing time to which one or more of the subtilisin variants described herein may be exposed. Low detergent concentration systems involve wash water containing less than about 800 ppm of detergent components. Medium detergent concentration systems involve wash water containing from about 800 ppm to about 2000 ppm of detergent components. High detergent concentration systems involve wash water containing greater than about 2000 ppm of detergent components. In some embodiments, the "cold water wash" of the present invention utilizes a "cold water detergent" suitable for washing at temperatures in the range of from about 10°C to about 40°C, from about 20°C to about 30°C, or from about 15°C to about 25°C, and all other combinations within the range of about 15°C to about 35°C or 10°C to 40°C.

[0150] Different geographical locations have different water hardnesses. Hardness is a measure of the amount of calcium (Ca 2+ ) and magnesium (Mg 2+ ) in water. Water hardness is typically described as a mixture of Ca 2+ / Mg 2+ in grains per gallon (gpg). In the United States, most water is hard water, but the hardness varies. Moderately hard (60 - 120 ppm) to hard (121 - 181 ppm) water has hardness minerals of 60 to 181 ppm (ppm can be converted to grains per US gallon by dividing ppm by 17.1).

[0151] Water Grains per gallon Parts per million Soft Less than 1.0 Less than 17 Slightly hard 1.0 to 3.5 17 to 60 Moderately hard 3.5 to 7.0 60 to 120 Hard 7.0 to 10.5 120 to 180 Very hard Greater than 10.5 Greater than 180

[0152] 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 of the subtilisin variants described herein, and from about 99.999% to about 90.0% by weight of the composition of one or more auxiliary materials. In another embodiment, the cleaning composition comprises from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, or from about 0.005% to about 0.5% by weight of the composition of one or more subtilisin variants, and from about 99.9999% to about 90.0%, from about 99.999% to about 98%, from about 99.995% to about 99.5% by weight of the composition of one or more auxiliary materials.

[0153] In other embodiments, the compositions described herein comprise one or more subtilisin variants described herein and one or more additional enzymes. The one or more additional enzymes are selected from acyltransferases, α-amylases, β-amylases, α-galactosidases, arabinosidases, arylesterases, β-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, dispase, endo-β-1,4-glucanases, endo-β-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hexosaminidases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, lysozymes, mannanases, metalloproteinases, nucleases (e.g., DNases and / or RNases), oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phosphodiesterases, phospholipases, phytases, polygalacturonases, polyesterases, additional proteases, pullulanases, reductases, rhamnogalacturonases, β-glucanases, tannases, transglutaminases, xanthan lyases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, and any combination or mixture thereof. Some embodiments relate to combinations (i.e., "mixtures") of enzymes comprising conventional enzymes such as amylases, lipases, cutinases, mannanases, and / or cellulases, which combinations are combined with one or more subtilisin variants described herein and / or one or more additional proteases.

[0154] In another embodiment, one or more of the compositions described herein comprise one or more of the 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 a metalloprotease, a fungal subtilisin, or an alkaline microbial protease or a trypsin-like protease. Suitable additional proteases include those of animal, plant, 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 mutant. In another embodiment, the additional protease is an alkaline microbial protease or a trypsin-like protease. In other embodiments, the additional protease does not contain epitopes that cross-react with the subtilisin variant, as measured by antibody binding or other assays available in the art. Exemplary alkaline proteases include subtilisins derived from, for example, Bacillus (e.g., BPN’, Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168), or of fungal origin (e.g., those described in U.S. Patent No. 8,362,222).Exemplary additional proteases include, but are not limited to, those described in WO 92 / 21760, WO 95 / 23221, WO 2008 / 010925, WO 09 / 149200, WO 09 / 149144, WO 09 / 149145, WO 10 / 056640, WO 10 / 056653, WO 2010 / 0566356, WO11 / 072099, WO 2011 / 13022, WO 11 / 140364, WO 12 / 151534, WO 2015 / 038792, WO 2015 / 089447, WO 2015 / 089441, WO 2017 / 215925 / US Publication No. 2008 / 0090747, US 5,801,039, US5,340,735, US 5,500,364, US 5,855,625, RE 34,606, US 5,955,340, US 5,700,676, US 6,312,936, US 6,482,628, US 8,530,219, US Provisional Application Nos. 62 / 180673 and 62 / 161077, and PCT Application Nos. PCT / US2015 / 021813, PCT / US2015 / 055900, PCT / US2015 / 057497, PCT / US2015 / 057492, PCT / US2015 / 057512, PCT / US2015 / 057526, PCT / US2015 / 057520, PCT / US2015 / 057502, PCT / US2016 / 022282 and PCT / US16 / 32514, and the metalloproteases described in WO 1999014341, WO1999033960, WO 1999014342, WO 1999034003, WO 2007044993, WO 2009058303, WO2009058661, WO 2014071410, WO 2014194032, WO 2014194034, WO 2014194054, WO 2014 / 194117, EP 3380599, WO 2017215925 and WO 2016203064. Exemplary additional proteases include, but are not limited to, trypsin (e.g., of porcine or bovine origin) and the Fusarium protease described in WO 89 / 06270. Exemplary commercial proteases include, but are not limited to. MAXACAL TM 、MAXAPEM TM 、

[0155]

[0156] OXP, PURAMAX TM , EXCELLASE TM , PREFERENZ TM Proteases (e.g., P100, P110, P280, P300), EFFECTENZ TM Proteases (e.g., P1000, P1050, P2000), EXCELLENZ TM Protease (e.g., P1000), and PURAFAST TM (DuPont / Danisco / Genencor); ULTRA, variants, 16L, ULTRA, DURAZYM TM , LIQUANASE PROGRESS and (Novozymes); BLAP TM and BLAP TM variants (Henkel); LAVERGY TM PRO 104L (BASF), KAP (Bacillus alkalophilus subtilisin (Kao)) and (AB Enzymes).

[0157] Another embodiment relates to a composition comprising one or more subtilisin variants described herein and one or more lipases. In some embodiments, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% lipase, by weight of the composition. Exemplary lipases can be chemically or genetically modified mutants. Exemplary lipases include, but are not limited to, those from bacterial or fungal sources, such as Humicola lanuginosa lipase (see, e.g., EP258068 and EP 305216), Thermomyces lanuginosa lipase (see, e.g., WO 2014 / 059360 and WO 2015 / 010009), Rhizomucor miehei lipase (see, e.g., EP238023), Candida lipases such as Candida antarctica lipase (e.g., Candida antarctica lipase A or B) (see, e.g., EP 214761), Pseudomonas lipases such as Pseudomonas alcaligenes and Pseudomonas pseudoalcaligenes lipase (see, e.g., EP218272), Pseudomonas cepacia lipase (see, e.g., EP 331376), Pseudomonas stutzeri lipase (see, e.g., GB 1,372,034), Pseudomonas fluorescens lipase, Bacillus lipases (e.g., Bacillus subtilis lipase (Dartois et al., Biochem. Biophys. Acta [Biochimica et Biophysica Acta] 1131:253-260 (1993)), Bacillus stearothermophilus lipase (see, e.g., JP 64 / 744992), and Bacillus pumilus lipase (see, e.g., WO 91 / 16422)).Exemplary lipases that can be cloned include, but are not limited to, Penicillium camembertii lipase (see Yamaguchi et al., Gene 103:61-67 (1991)); Geotrichum candidum lipase (see Schimada et al., J. Biochem., 106:383-388 (1989)); and various Rhizopus lipases such as Rhizopus delemar lipase (see Hass et al., Gene 109:117-113 (1991)), Rhizopus niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 (1992)) and Rhizopus oryzae lipase. Other lipolytic enzymes (e.g., cutinase) can also be used in one or more of the compositions described herein, including but not limited to, for example, cutinases derived from Pseudomonas mendocina (see WO 88 / 09367) and / or Fusarium solanipisi (see WO90 / 09446). Exemplary commercial lipases include, but are not limited to, M1 LIPASE. TM , LUMAFAST TM , LIPOMAX TM and PREFERENZ TM L100 (DuPont); and ULTRA (Novozymes); and LIPASE P TM (Amano Pharmaceutical Co., Ltd).

[0158] Still other embodiments relate to compositions comprising one or more subtilisin variants described herein and one or more amylases. In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% amylase, by weight of the composition. Any amylase suitable for use in an alkaline solution (e.g., α-amylase and / or β-amylase) can be used in such compositions. Exemplary amylases can be chemically or genetically modified mutants. Exemplary amylases include, but are not limited to, those of bacterial or fungal origin, such as those described in GB 1,296,839, WO 9100353, WO9402597, WO 94183314, WO 9510603, WO 9526397, WO 9535382, WO 9605295, WO 9623873, WO9623874, WO 9630481, WO 9710342, WO 9741213, WO 9743424, WO 9813481, WO 9826078, WO9902702, WO 9909183, WO 9919467, WO 9923211, WO 9929876, WO 9942567, WO 9943793, WO9943794, WO 9946399, WO 0029560, WO 0060058, WO 0060059, WO 0060060, WO 0114532, WO0134784, WO 0164852, WO 0166712, WO 0188107, WO 0196537, WO 02092797, WO 0210355, WO0231124, WO 2004055178, WO 2004113551, WO 2005001064, WO 2005003311, WO2005018336, WO 2005019443, WO 2005066338, WO 2006002643, WO 2006012899, WO2006012902, WO 2006031554, WO 2006063594, WO 2006066594, WO 2006066596, WO2006136161, WO 2008000825, WO 2008088493, WO 2008092919, WO 2008101894, WO2008 / 112459, WO 2009061380, WO 2009061381, WO 2009100102, WO 2009140504, WO 2009149419, WOThe amylases in 2010 / 059413, WO 2010088447, WO 2010091221, WO 2010104675, WO 2010115021, WO10115028, WO 2010117511, WO 2011076123, WO 2011076897, WO 2011080352, WO2011080353, WO 2011080354, WO 2011082425, WO 2011082429, WO 2011087836, WO2011098531, WO 2013063460, WO 2013184577, WO 2014099523, WO 2014164777, WO2015077126, and WO 2018184004. Exemplary commercial amylases include, but are not limited to STAINZYME STAINZYME STAINZYME and BAN TM (Novozymes A / S); EFFECTENZ TM S1000, POWERASE TM 、PREFERENZ TM S100, PREFERENZ TM S110, PREFERENZ TM S210, EXCELLENZ TM S2000, and P (DuPont). In some embodiments, the subtilisin variants provided herein can be combined with one or more amylases and their variants, and combinations of the one or more amylases and their variants, the one or more amylases selected from the group consisting of: AA707, AA560, AAI10, BspAmy24, SP722, and CspAmy1.

[0159] Yet still another embodiment relates to a composition comprising one or more of the subtilisin variants described herein and one or more cellulases. In one embodiment, the composition comprises from about 0.00001% to about 10%, 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% cellulase, by weight of the composition. Any suitable cellulase can be used in the compositions described herein. Exemplary cellulases can be chemically or genetically modified mutants. Exemplary cellulases include, but are not limited to, those from bacterial or fungal sources, such as those described in: WO 2005054475, WO 2005056787, US 7,449,318, US 7,833,773, US4,435,307; EP 0495257; and U.S. Provisional Application No. 62 / 296,678. Exemplary commercial cellulases include, but are not limited to and PREMIUM (Novozymes); REVITALENZ TM 100, REVITALENZ TM 200 / 220, and 2000 (DuPont); and KAC-500(B) TM (Kao Corporation). In some embodiments, the cellulase is incorporated as part or fragment of a mature wild-type or variant cellulase (where a portion of the N-terminus is deleted) (see, e.g., US 5,874,276).

[0160] Yet still another embodiment relates to a composition comprising one or more of the subtilisin variants described herein and one or more mannanases. In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% mannanase, by weight of the composition. Exemplary mannanases can be chemically or genetically modified mutants. Exemplary mannanases include, but are not limited to, those from bacterial or fungal sources, such as those described in: WO 99 / 64619, WO 2016 / 007929; USPN 6,566,114, 6,602,842, and 6,440,991; and U.S. Provisional Application No. 62 / 251516, 62 / 278383, and 62 / 278387. Exemplary commercial mannanases include, but are not limited to (Novozymes) and EFFECTENZ TM M 1000, EFFECTENZ TM M 2000, M 100, and PURABRITE TM (DuPont).

[0161] Still other embodiments relate to compositions comprising one or more subtilisin variants described herein and one or more nucleases (e.g., DNase or RNase). In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% nuclease, by weight of the composition. Exemplary nucleases include, but are not limited to, those described in WO 2015181287, WO2015155350, WO 2016162556, WO 2017162836, WO 2017060475 (e.g., SEQ ID NO:21), WO2018184816, WO 2018177936, WO 2018177938, WO2018 / 185269, WO 2018185285, WO2018177203, WO 2018184817, WO 2019084349, WO 2019084350, WO 2019081721, WO2018076800, WO 2018185267, WO 2018185280, WO 2018206553, and WO 2020099490. Other nucleases that can be used in combination with the subtilisin variants provided herein in the compositions and methods provided herein include those described in: Nijland R, Hall MJ, Burgess JG (2010) Dispersal ofBiofilms by Secreted,Matrix Degrading,Bacterial DNase [Dispersal of Biofilms by Secreted, Matrix Degrading, Bacterial DNase]. PLoS ONE [Public Library of Science: General] 5(12) and Whitchurch, C.B., Tolker-Nielsen, T., Ragas, P.C., Mattick, J.S. (2002) Extracellular DNA required for bacterial biofilm formation [Extracellular DNA required for bacterial biofilm formation]. Science [Science] 295:1487.

[0162] Yet even still additional embodiments relate to compositions comprising one or more of the subtilisin variants described herein and one or more peroxidases and / or oxidases. In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% peroxidase or oxidase by weight of the composition. The peroxidase can be used in combination with hydrogen peroxide or a source thereof (such as percarbonate, perborate, or persulfate), and the oxidase can be used in combination with oxygen. The peroxidase and oxidase, alone or in combination with a synergist, are used for "solution bleaching" (i.e., to prevent the transfer of textile dyes from one dyed fabric to another when the fabrics are washed together in a wash liquor) (see, e.g., WO 94 / 12621 and WO95 / 01426). Exemplary peroxidases and / or oxidases can be chemically or genetically modified mutants. Exemplary peroxidase / oxidases include, but are not limited to, those of plant, bacterial, or fungal origin.

[0163] Another embodiment relates to compositions comprising one or more of the subtilisin variants described herein and one or more perhydrolases, such as the perhydrolases described in WO 2005 / 056782, WO 2007 / 106293, WO 2008 / 063400, WO 2008 / 106214, and WO 2008 / 106215.

[0164] In yet another embodiment, one or more of the subtilisin variants described herein and one or more additional enzymes contained in one or more of the compositions described herein can each independently vary up to about 10% by weight of the composition, with the balance of the cleaning composition being one or more auxiliary materials.

[0165] In some embodiments, one or more of the compositions described herein can be used as a detergent additive, wherein the additive is 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, it ranges from about 500 to about 950 g / liter as measured at 20°C.

[0166] Some embodiments relate to laundry detergent compositions that comprise one or more of the Bacillus subtilis protease variants described herein and one or more adjunct materials selected from the group consisting of: surfactants, enzyme stabilizers, builder compounds, polymeric compounds, bleaches, additional enzymes, foam inhibitors, dispersants, calcium soap dispersants, soil suspending agents, anti-redeposition agents, corrosion inhibitors, and combinations thereof. In some embodiments, the laundry composition further contains a fabric softener.

[0167] Additional embodiments relate to manual dishwashing compositions that comprise one or more of the Bacillus subtilis protease variants described herein and one or more adjunct materials selected from the group consisting of: surfactants, organic polymeric compounds, foam boosters, Group II metal ions, solvents, hydrotropes, and additional enzymes.

[0168] Other embodiments relate to one or more of the compositions described herein, wherein the composition is a compact granular fabric cleaning composition for colored fabric washing or providing softening through washing capacity, or a heavy-duty liquid (HDL) fabric cleaning composition. Exemplary fabric cleaning compositions and / or methods of preparation are described in USPN 6,610,642 and 6,376,450. Other exemplary cleaning compositions are described, for example, in USPN 6,605,458; 6,294,514; 5,929,022; 5,879,584; 5,691,297; 5,565,145; 5,574,005; 5,569,645; 5,565,422; 5,516,448; 5,489,392; and 5,486,303; 4,968,451; 4,597,898; 4,561,998; 4,550,862; 4,537,706; 4,515,707; and 4,515,705.

[0169] In some embodiments, the cleaning composition comprises acidifying particles or aminocarboxylic acid builders. Examples of aminocarboxylic acid builders include aminocarboxylic acids, their salts, and derivatives. In some embodiments, the aminocarboxylic acid builder is an aminopolycarboxylic acid builder such as glycine-N,N-diacetic acid or having the general formula MOOC-CHR-N(CH2COOM)2 (wherein R is C 1-12Derivatives of (wherein R is alkyl and M is an alkali metal). In some embodiments, the aminocarboxylic acid builder can be methylglycine diacetic acid (MGDA), GLDA (glutamic acid - N,N - diacetic acid), iminodisuccinic acid (IDS), carboxymethyl inulin and its salts and derivatives, aspartic acid - N - monoacetic acid (ASMA), aspartic acid - N,N - diacetic acid (ASDA), aspartic acid - N - monopropionic acid (ASMP), iminodiacetic acid (IDA), N-(2 - sulfomethyl)aspartic acid (SMAS), N-(2 - sulfonylethyl)aspartic acid (SEAS), N-(2 - sulfomethyl)glutamic acid (SMGL), N-(2 - sulfonylethyl)glutamic acid (SEGL), IDA (iminodiacetic acid) and its salts and derivatives such as N - methyliminodiacetic acid (MIDA), α - alanine - N,N - diacetic acid (α - 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), sulfamic acid - N,N - diacetic acid (SLDA), taurine - N,N - diacetic acid (TUDA) and sulfomethyl - N,N - diacetic acid (SMDA), and their alkali metal salts and derivatives. In some embodiments, the weight geometric mean particle size of the acidified particles is from about 400 μ to about 1200 μ, and the bulk density is at least 550 g / L. In some embodiments, the acidified particles contain at least about 5% of the builder.

[0170] In some embodiments, the acidified particles can contain any acid, including organic acids and mineral acids. The organic acids can have one or two carboxyl groups and in some cases can have up to 15 carbons, particularly up to 10 carbons, 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 hydrate. In some embodiments, the acid is citric acid. Mineral acids include hydrochloric acid and sulfuric acid. In some cases, the acidified particles are highly active particles containing a high level of aminocarboxylic acid builder. It has also been found that sulfuric acid further contributes to the stability of the final particles.

[0171] Further embodiments relate to cleaning compositions comprising one or more variants of subtilisin and one or more surfactants and / or surfactant systems, wherein the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, zwitterionic surfactants, semi - polar nonionic surfactants and mixtures thereof. In some embodiments, the surfactant is present at a level of from about 0.1% to about 60% by weight of the cleaning composition, while in alternative embodiments, the level is from about 1% to about 50%, and in yet further embodiments, the level is from about 5% to about 40%.

[0172] In some embodiments, one or more of the compositions described herein comprise one or more detergent builders or builder systems. In one embodiment, the composition comprises from at least about 0.1% or more, or from about 0.1% to about 90%, from 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. Exemplary builders include, but are not limited to, alkali metals; ammonium salts and alkanolammonium salts of polyphosphates; alkali metal silicates; alkaline earth metal and alkali metal carbonates; aluminosilicates; polycarboxylate compounds; ether hydroxy polycarboxylates; copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulfonic acid, and carboxymethyloxysuccinic acid; ammonium salts 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 their soluble salts. In some such compositions, the builder forms water-soluble hardness ion complexes (e.g., chelating builders), such as citrates and polyphosphates, such as sodium tripolyphosphate, sodium tripolyphosphate hexahydrate, potassium tripolyphosphate, and mixtures of sodium tripolyphosphate and potassium tripolyphosphate. Exemplary builders are described, for example, in EP 2100949. In some embodiments, the builder includes phosphate builders and non-phosphate builders. In some embodiments, the builder is a phosphate builder. In some embodiments, the builder is a non-phosphate builder. In some embodiments, the builder comprises a mixture of phosphate and non-phosphate builders. Exemplary phosphate builders include, but are not limited to, monophosphates, diphosphates, tripolyphosphates or oligophosphates, including the alkali metal salts of these compounds, including sodium salts. In some embodiments, the builder may be sodium tripolyphosphate (STPP). Additionally, the composition may comprise carbonate and / or citrate. Other suitable non-phosphate builders include polycarboxylic acids and their partially or fully neutralized salts, homopolymers and copolymers of monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts. In some embodiments, the salts of the above compounds include ammonium salts and / or alkali metal salts, i.e., lithium salts, sodium salts and potassium salts, including sodium salts. Suitable polycarboxylic acids include acyclic, cycloaliphatic, heterocyclic and aromatic carboxylic acids, wherein in some embodiments, they may contain at least two carboxyl groups, which in each case are separated from each other and in some cases are separated by no more than two carbon atoms.

[0173] In some embodiments, one or more of the compositions described herein comprise one or more chelating agents. In one embodiment, the composition comprises from about 0.1% to about 15% or about 3% to about 10% chelating agent, by weight of the composition. Exemplary chelating agents include, but are not limited to, for example, copper, iron, manganese and mixtures thereof.

[0174] In some embodiments, one or more of the compositions described herein include one or more deposition aids. Exemplary deposition aids include, but are not limited to, for example, polyethylene glycol; polypropylene glycol; polycarboxylates; soil release polymers such as poly(terephthalic acid); clays such as kaolinite, montmorillonite, attapulgite, illite, bentonite, and halloysite; and mixtures thereof.

[0175] In other embodiments, one or more of the compositions described herein include one or more anti-redeposition agents or nonionic surfactants (which can prevent the redeposition of soil) (see, e.g., EP 2100949). For example, in ADW compositions, nonionic surfactants can be used for surface modification purposes (especially for sheets) to avoid film formation and spotting and to improve gloss. These nonionic surfactants can also be used to prevent the redeposition of soil. In some embodiments, the nonionic surfactant can be an ethoxylated nonionic surfactant, an epoxy-capped poly(alkoxylated) alcohol, and an amine oxide surfactant.

[0176] In some embodiments, one or more of the compositions described herein include one or more dye transfer inhibitors. Exemplary polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyl oxazolidone, polyvinylimidazole, and mixtures thereof. In one embodiment, the composition includes from about 0.0001% to about 10%, about 0.01% to about 5%, or about 0.1% to about 3% of a dye transfer inhibitor, based on the weight of the composition.

[0177] In some embodiments, one or more of the compositions described herein include one or more silicates. Exemplary silicates include, but are not limited to, sodium silicate, for example, disodium silicate, sodium metasilicate, and crystalline sheet silicate. In some embodiments, the silicate is present at a level of from about 1% to about 20% or about 5% to about 15% by weight of the composition.

[0178] In some still further embodiments, one or more of the compositions described herein include one or more dispersants. Exemplary water-soluble organic materials include, but are not limited to, for example, homopolymeric or copolymeric acids or their salts, wherein the polycarboxylic acid contains at least two carboxyl radicals separated from each other by no more than two carbon atoms.

[0179] In some additional embodiments, one or more of the compositions described herein comprise 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 such as calcium salts). In some embodiments, the enzymes used herein are stabilized by a water-soluble source of zinc(II), calcium(II), and / or magnesium(II) ions, 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 vanadyl(IV)) present in the finished composition that provides such ions to the enzyme. Chlorides and sulfates can also be used in some embodiments. Exemplary oligosaccharides and polysaccharides (e.g., dextrin) are described, for example, in WO 07 / 145964. In some embodiments, reversible protease inhibitors can also be used, for example, in boron-containing compounds (e.g., borates, 4-formylphenylboronic acid, and phenylboronic acid derivatives (such as those described in WO 96 / 41859)) and / or peptide aldehydes (such as those further described in WO2009 / 118375 and WO 2013004636).

[0180] As previously described (WO 199813458, WO 2011036153, US20140228274), peptide aldehydes can be used as protease stabilizers in detergent formulations. Examples of peptide aldehyde stabilizers are peptide aldehydes, ketones, or halomethyl ketones, and can be "N-capped", for example, having a ureido, carbamate, or urea moiety, or "doubly N-capped", for example, having a carbonyl, ureido, oxamide, thioureido, dithioxamide, or thioxamide moiety (EP 2358857 B1). The molar ratio of these inhibitors to the protease can be from 0.1:1 to 100:1, for example 0.5:1 - 50:1, 1:1 - 25:1, or 2:1 - 10:1. Other examples of protease stabilizers are benzophenone or benzoic acid aniline derivatives, which may contain a carboxyl group (US 7,968,508 B2). The molar ratio of these stabilizers to the protease is preferably in the range of 1:1 to 1000:1, particularly 1:1 to 500:1, especially preferably from 1:1 to 100:1, and most especially preferably from 1:1 to 20:1.

[0181] In some embodiments, one or more of the compositions described herein comprise one or more bleaching agents, bleach activators, and / or bleach catalysts. In some embodiments, one or more of the compositions described herein comprise one or more inorganic and / or organic bleaching compounds. Exemplary inorganic bleaching agents include, but are not limited to, peroxygenate salts such as perborates, percarbonates, perphosphates, persulfates, and persilicates. In some embodiments, the inorganic peroxygenate salt is an alkali metal salt. In some embodiments, the inorganic peroxygenate salt is a crystalline solid without additional protection, but in some other embodiments, the salt is coated. Bleach activators are typically organic peracid precursors that enhance bleaching during the cleaning process at temperatures of 60 °C and below. Exemplary bleach activators include compounds that give aliphatic peroxycarboxylic acids having from about 1 to about 10 carbon atoms or from about 2 to about 4 carbon atoms, and / or optionally substituted peroxybenzoic acid under perhydrolysis conditions. Exemplary bleach activators are described, for example, in EP 2100949. Exemplary bleach catalysts include, but are not limited to, manganese triazacyclononane and related complexes, and cobalt, copper, manganese, and iron complexes. Additional exemplary bleach catalysts are described, for example, in US 4,246,612; US 5,227,084; US 4,810,410; WO 99 / 06521; and EP2100949.

[0182] In some embodiments, one or more of the compositions described herein comprise one or more catalytic metal complexes. In some embodiments, metal-containing bleach catalysts can be used. In some embodiments, the metal bleach catalyst comprises a catalytic system that includes: a transition metal cation having a defined bleach catalytic activity (e.g., a copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cation), a co-metal cation having little or no bleach catalytic activity (e.g., a zinc or aluminum cation), and a chelate having a defined stability constant for the catalytic and co-metal cations, in particular ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and their water-soluble salts (see, for example, US 4,430,243). In some embodiments, one or more of the compositions described herein are catalyzed by means of a manganese compound. Such compounds and levels of use are described, for example, in US 5,576,282. In additional embodiments, cobalt bleach catalysts can be used and are included in one or more of the compositions described herein. A variety of cobalt bleach catalysts are described, for example, in USPN 5,597,936 and 5,595,967.

[0183] In some additional embodiments, one or more of the compositions described herein comprise a transition metal complex of a mostly polycyclic rigid ligand (MRL). As a practical matter and not by way of limitation, in some embodiments, the compositions and cleaning methods described herein are adjusted to provide an active MRL in the wash liquor of at least on the order of one hundred million parts per, from about 0.005 ppm to about 25 ppm, from about 0.05 ppm to about 10 ppm, or from about 0.1 ppm to about 5 ppm. Exemplary MRLs include, but are not limited to, crosslinked bridged special superrigid ligands such as 5,12 - diethyl - 1,5,8,12 - tetraazabicyclo(6.6.2)hexadecane. Exemplary metal MRLs are described, for example, in WO 2000 / 32601 and US 6,225,464.

[0184] In another embodiment, one or more of the compositions described herein comprise one or more metal care agents. In some embodiments, the composition comprises from about 0.1% to about 5% by weight of the composition of the metal care agent. Exemplary metal care agents include, for example, aluminum, stainless steel, and non - ferrous metals (e.g., silver and copper). Additional exemplary 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.

[0185] In some embodiments, the cleaning composition is a heavy duty liquid (HDL) composition comprising one or more of the subtilisin variants described herein. The HDL liquid laundry detergent may comprise a cleaning surfactant (10% - 40%), the cleaning surfactant comprising an anionic cleaning surfactant selected from the group consisting of 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; and optionally a non - ionic surfactant selected from the group consisting of linear, branched, or random chain, substituted or unsubstituted alkyl alkoxylated alcohols, such as C8 - C 18 alkyl ethoxylated alcohols and / or C6 - C 12 alkyl phenol alkoxylates, optionally wherein the weight ratio of the anionic cleaning surfactant (hydrophilicity index (HIc) from 6.0 to 9) to the non - ionic cleaning surfactant is greater than 1:1. Suitable cleaning surfactants also include cationic cleaning 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 cleaning surfactants (selected from alkanolamine sulfobetaines); amphoteric surfactants; semi - polar non - ionic surfactants; and mixtures thereof.

[0186] In another embodiment, the cleaning composition is a liquid or gel detergent (which is not a unit dose), which can be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70% water by weight, up to about 65% water by weight, up to about 55% water by weight, up to about 45% water by weight, or up to about 35% water by weight. Other types of liquids (including but not limited to alkanols, amines, diols, ethers, and polyols) can be included in the aqueous liquid or gel. The aqueous liquid or gel detergent can contain from 0 to 30% organic solvent. The liquid or gel detergent can be non-aqueous.

[0187] The composition can optionally contain a surfactant-enhancing polymer consisting of: amphiphilic alkoxylated oil cleaning polymers, these amphiphilic alkoxylated oil cleaning polymers being selected from the group consisting of: alkoxylated polymers having branched hydrophilic and hydrophobic properties, such as alkoxylated polyalkyleneimines (in the range of 0.05 wt%-10 wt%); and / or random graft polymers, these random graft polymers typically containing a hydrophilic backbone containing monomers selected from the group consisting of: unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyols (such as glycerol) and mixtures thereof; and one or more hydrophobic side chains, these hydrophobic side chains being selected from the group consisting of: C4-C 25 alkyl groups, polypropylene, polybutene, vinyl esters of saturated C2-C6 monocarboxylic acids, C1-C6 alkyl esters of acrylic acid or methacrylic acid and mixtures thereof.

[0188] The composition may comprise additional polymers such as soil release polymers, which include, for example, anionically terminated polyesters such as SRP1; polymers having a random or block configuration and comprising at least one monomer unit selected from sugars, dicarboxylic acids, polyols, and combinations thereof; polymers based on ethylene terephthalate and copolymers thereof having 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 wt% to 10 wt%, including, for example, carboxylate polymers such as polymers comprising at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid, and any 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; alkylalkoxyalkyl celluloses; carboxyalkyl celluloses; alkylcarboxyalkyl celluloses, examples of which include carboxymethyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, methylcarboxymethyl cellulose; and mixtures thereof); and polymeric carboxylic esters (such as maleate / acrylate random copolymers or polyacrylate homopolymers).

[0189] The composition may further comprise saturated or unsaturated fatty acids, preferably saturated or unsaturated C 12 -C 24 fatty acids (0 - 10 wt%); deposition aids having a random or block configuration (including, for example, polysaccharides, cellulose polymers, poly(diallyldimethylammonium halide) (DADMAC)), and copolymers of DADMAC and vinylpyrrolidone, acrylamide, imidazole, imidazoline halides, and mixtures thereof; cationic guar gum; cationic cellulose such as cationic hydroxyethyl cellulose; cationic starch; cationic polyacrylamide; and mixtures thereof.

[0190] 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 and N-vinylimidazole, polyvinyl oxazolidinone and polyvinylimidazole and / or mixtures thereof; chelating agents, examples of which include ethylenediaminetetraacetic acid (EDTA); diethylenetriaminepenta(methylene phosphonic acid) (DTPMP); hydroxyethane diphosphonic acid (HEDP); ethylenediamine N,N'-disuccinic acid (EDDS); methylglycine diacetic acid (MGDA); diethylenetriaminepentaacetic acid (DTPA); propylenediaminetetraacetic acid (PDTA); 2-hydroxypyridine-N-oxide (HPNO); or methylglycine diacetic acid (MGDA); glutamic acid N,N-diacetic acid (tetrasodium N,N-dicarboxymethylglutamate (GLDA)); nitrilotriacetic acid (NTA); 4,5-dihydroxy-m-benzenedisulfonic acid; citric acid and any of its salts; N-hydroxyethyl ethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP) and derivatives thereof.

[0191] The composition may further comprise a silicone-based or fatty acid-based foam inhibitor; an enzyme stabilizer; a color toner dye, calcium and magnesium cations, a visual signaling component, an antifoaming agent (0.001 wt% to about 4.0 wt%) and / or a structuring agent / thickener (0.01 wt% - 5 wt%), the structuring agent / thickener being selected from the group consisting of diglycerides, triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose-based materials, ultrafine cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof.

[0192] In some embodiments, the cleaning composition is a heavy-duty powder (HDD) composition comprising one or more of the subtilisin variants described herein. The HDD powder laundry detergent may comprise a cleaning surfactant, which includes anionic cleaning surfactants (selected from 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); nonionic cleaning surfactants (selected from linear or branched or random-chain, substituted or unsubstituted C8-C 18 alkyl ethoxylates and / or C6-C 12alkylphenol alkoxylates); cationic cleaning surfactants (selected from alkylpyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds and mixtures thereof); zwitterionic and / or amphoteric cleaning surfactants (selected from alkanolamine sulfobetaines); amphoteric surfactants; semi-polar nonionic surfactants and mixtures thereof; builders (phosphate-free builders such as zeolite builders, examples of which include zeolite A, zeolite X, zeolite P and zeolite MAP in the range of 0 wt% to less than 10 wt%); phosphate builders such as sodium tripolyphosphate in the range of 0 to less than 10 wt%; citric acid, citrate and nitrilotriacetic acid or salts thereof in the range of less than 15 wt%; silicates (sodium silicate or potassium silicate or sodium metasilicate or layered silicate (SKS-6) in the range of 0 wt% to less than 10 wt%); carbonates (sodium carbonate and / or sodium bicarbonate in the range of 0 wt% to less than 10 wt%); and bleaches (photo-bleaches such as sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, xanthene dyes and mixtures thereof); hydrophobic or hydrophilic bleach activators (e.g., dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or salts thereof, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine - TAED, and nonanoyloxybenzenesulfonate - NOBS, nitrile quats, and mixtures thereof); hydrogen peroxide; hydrogen peroxide sources (inorganic hydrogen peroxide salt, such as the mono- or tetra-hydrate sodium salts of perborate, percarbonate, persulfate, perphosphate or persilicate); preformed hydrophilic and / or hydrophobic peracids (selected from percarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acid and salts and mixtures thereof); and / or bleach catalysts (e.g., imine bleach boosters such as imine cations and polyions; imine zwitterions; modified amines; modified amine oxides; N-sulfonylimines; N-phosphonylimines; N-acylimines; thiazole dioxides; perfluoroimines; cyclic glyoxals and mixtures thereof); metal-containing bleach catalysts (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations and auxiliary metal cations (such as zinc or aluminum) and chelates (such as ethylenediaminetetraacetic acid, ethylenediamine tetra(methylenephosphonic acid) and water-soluble salts thereof).

[0193] The composition may further comprise additional detergent ingredients including fragrance microcapsules, starch-encapsulated fragrance modifiers, enzyme stabilizers, colorants, additional polymers (including fabric integrity and cationic polymers), dye-locking ingredients, fabric softeners, brighteners (e.g., C.I. fluorescent brighteners), flocculants, chelating agents, alkoxylated polyamines, fabric deposition aids and / or cyclodextrins.

[0194] In some embodiments, the cleaning composition is an ADW detergent composition comprising one or more subtilisin variants described herein. The ADW detergent composition may comprise two or more nonionic surfactants selected from the group consisting of ethoxylated nonionic surfactants, alcohol alkoxylated surfactants, epoxy-capped poly(alkoxylated) alcohols, and amine oxide surfactants, present in an amount of 0-10% by weight; builders in the range of 5%-60% by weight, including: phosphate builders (monophosphate, diphosphate, tripolyphosphate or oligophosphate), sodium tripolyphosphate - STPP or phosphate-free builders (amino acid-based compounds such as MGDA (methyl-glycine-diacetic acid) and its salts and derivatives, GLDA (glutamic acid-N,N-diacetic acid) and its salts and derivatives, IDS (iminodisuccinic acid) and its salts and derivatives, carboxymethyl inulin and its salts and derivatives and mixtures thereof, nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and β-alanine diacetic acid (β-ADA) and its salts), homopolymers and copolymers of polycarboxylic acids and their partially or fully neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts (in the range of 0.5%-50% by weight); sulfonated / carboxylated polymers (providing dimensional stability to the product), in the range of about 0.1% to about 50% by weight; drying aids in the range of about 0.1% to about 10% by weight (selected from polyesters, especially anionic polyesters optionally together with additional monomers having 3-6 functional groups (especially acid, alcohol or ester functional groups) facilitating polycondensation, polycarbonate-, polyurethane- and / or polyurea-polysiloxane compounds or their reactive cyclic carbonate and urea-type precursor compounds); silicates in the range from about 1% to about 20% by weight (sodium silicate or potassium silicate, such as disodium silicate, sodium metasilicate and crystalline layered silicate); inorganic bleaches (e.g., peroxygenate salts such as perborates, percarbonates, perphosphates, persulfates and persilicates) and organic bleaches (e.g., organic peroxyacids, including diacyl and tetraacyl peroxides, especially diperoxydodecanedioic acid, diperoxytetradecanedioic acid, and diperoxyhexadecanedioic acid); bleach activators - organic peracid precursors, in the range from about 0.1% to about 10% by weight; bleach catalysts (selected from manganese triazacyclononane and related complexes, Co, Cu, Mn and Fe bipyridylamine and related complexes, and pentaaminecobalt(III) acetate and related complexes); metal care agents in the range of about 0.1%-5%.Enzymes (acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, disperse protein, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, mannanase, nuclease, oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, peroxidase, phenol oxidase, phosphatase, phosphodiesterase, phospholipase, phytase, polyesterase, polygalacturonase, additional protease, pullulanase, reductase, rhamnogalacturonase, β-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and mixtures thereof) within the range of 0 mg active enzyme / gram of ADW detergent composition; and an enzyme stabilizer component (selected from oligosaccharides, polysaccharides, and inorganic divalent metal salts).

[0195] Exemplary ADW compositions are provided in Example 2 below or in the table below.

[0196] Exemplary ADW compositions

[0197]

[0198]

[0199] Additional embodiments relate to compositions and methods for treating fabrics (e.g., desizing textiles) using one or more subtilisin variants described herein. Fabric treatment methods are well known in the art (see, e.g., US 6,077,316). For example, the hand and appearance of a fabric can be improved by a method that includes contacting the fabric with a variant described herein in solution. The fabric can be treated with the solution under pressure.

[0200] One or more subtilisin variants described herein can be applied during or after the weaving of textiles, during the desizing stage or in one or more additional fabric processing steps. During the weaving of textiles, the threads are exposed to considerable mechanical strain. Before weaving on a mechanical loom, the warp threads are usually coated with sizing 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 sizing starch or starch derivatives. After weaving, the variants can be used to remove the size coating before further processing the fabric to ensure uniform and wash-fast results. One or more subtilisin variants described herein can be used alone or in combination with other desizing chemicals and / or desizing enzymes as detergent additives (e.g., in aqueous compositions) to desize fabrics, including cotton-containing fabrics. Amylases can also be used in combination with subtilisin variants in compositions and methods for producing a stonewashed appearance on indigo-dyed denim fabrics and garments. For clothing production, the fabric can be cut and sewn into clothes or garments, which are then finished. In particular, for the production of denim, different enzymatic finishing methods have been developed. The finishing process of denim garments usually starts with an enzymatic desizing step, in which the garments are subjected to the action of a proteolytic enzyme to provide 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 the following methods: finishing denim garments (e.g., "bio-stoning methods"), enzymatic desizing and providing softness to the fabric and / or finishing methods.

[0201] The present disclosure also provides methods for cleaning the surface of an article, which include contacting the article with at least one subtilisin variant provided herein (or a composition comprising such a subtilisin variant). In some embodiments, the article may have a protein stain on its surface, for example. In some embodiments, the protein stain can comprise an egg or egg-based stain, such as crème brûlée, baked cheese, BMI, or other protein-containing substances.

[0202] Example 1. A subtilisin variant comprising two or more mutations selected from the group consisting of: X009E, X074D, X085D, X099E, X157D, X176E, X188E, X189E, X211L, X242D, and X256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to a subtilisin having the amino acid sequence of SEQ ID NO:1 or 7.

[0203] Example 2. The subtilisin variant according to Example 1, wherein the variant does not comprise a combination of mutations selected from the following:

[0204] a) Mutated X074D in combination with one or more of X009E, X157D, X176E, X188E and X256E;

[0205] b) Combination X099E - X256E;

[0206] c) Combination X189E - X256E, and

[0207] d) Two or more mutations in X009E, X157D, X176E and X256E,

[0208] wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO:1 or 7.

[0209] Example 3. A subtilisin variant as described in Examples 1 and 2, the subtilisin variant comprising two mutations selected from the group consisting of: X009E - X085D, X009E - X099E, X009E - X188E, X009E - X189E, X009E - X242D, X074D - X085D, X074D - X099E, X074D - X189E, X074D - X242D, X085D - X099E, X085D - X157D, X085D - X176E, X085D - X188E, X085D - X189E, X085D - X242D, X085D - X256E, X099E - X157D, X099E - X176E, X099E - X188E, X099E - X189E, X099E - X242D, X157D - X188E, X157D - X189E, X157D - X242D, X176E - X188E, X176E - X189E, X176E - X242D, X176E - X256E, X188E - X189E, X188E - X242D, X188E - X256E, X189E - X242D, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO:1 or 7.

[0210] Example 4. A subtilisin variant as described in Example 3, wherein the variant comprises two mutations in a combination selected from the group consisting of: S009E-S085D, S009E-S099E, S009E-A188E, S009E-G189E, S009E-N242D, N074D-S085D, N074D-S099E, N074D-G189E, N074D-N242D, S085D-S099E, S085D-G157D, S085D-Q176E, S085D-A188E, S085D-G189E, S085D-N242D, S085D-L256E, S099E-G157D, S099E-Q176E, S099E-A188E, S099E-G189E, S099E-N242D, G157D-A188E, G157D-G189E, G157D-N242D, Q176E-A188E, Q176E-G189E, Q176E-N242D, Q176E-L256E, A188E-G189E, A188E-N242D, A188E-L256E, G189E-N242D, where the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO:1.

[0211] Example 5. A subtilisin variant as described in Example 3, wherein the variant comprises two mutations in a combination selected from the group consisting of: T009E-N085D, T009E-S099E, T009E-T188E, T009E-G189E, T009E-N242D, N074D-N085D, N074D-S099E, N074D-G189E, N074D-N242D, N085D-S099E, N085D-G157D, N085D-Q176E, N085D-T188E, N085D-G189E, N085D-N242D, N085D-Q256E, S099E-G157D, S099E-Q176E, S099E-T188E, S099E-G189E, S099E-N242D, G157D-T188E, G157D-G189E, G157D-N242D, Q176E-T188E, Q176E-G189E, Q176E-N242D, Q176E-Q256E, T188E-G189E, T188E-N242D, T188E-Q256E, G189E-N242D, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to subtilisin having the amino acid sequence of SEQ ID NO:7.

[0212] Example 6. A subtilisin variant as described in any of the preceding examples, wherein the variant has at least a 3-fold improvement in terms of robustness factor relative to the parental subtilisin and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0213] Example 7. A subtilisin variant comprising three mutations selected from the group consisting of: X009E, X074D, X085D, X099E, X157D, X176E, X188E, X189E, X242D and X256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to subtilisin having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0214] Example 8. A subtilisin variant as described in Example 7, wherein the variant does not comprise a combination of mutations selected from:

[0215] a) Three mutations selected from X009E, X074D, X085D, X176E, and X242D;

[0216] b) Mutation X074D in combination with one or more mutations selected from X009E, X157D, X176E, X188E, and X256E; and

[0217] c) Three mutations selected from the group consisting of: X009E, X157D, X176E, X256E

[0218] wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity with a subtilisin having the amino acid sequence of SEQ ID NO:1 or 7.

[0219] Example 9. A subtilisin variant as described in Example 7 or 8, wherein the variant comprises three mutations in a combination selected from the group consisting of: X009E-X085D-X099E, X009E-X085D-X188E, X009E-X085D-X189E, X009E-X085D-X242D, X009E-X099E-X188E, X009E-X099E-X189E, X009E-X099E-X242D, X009E-X188E-X189E, X009E-X188E-X242D, X009E-X189E-X242D, X074D-X085D-X099E, X074D-X085D-X189E, X074D-X085D-X242D, X074D-X099E-X189E, X074D-X099E-X242D, X074D-X189E-X242D, X085D-X099E-X157D, X085D-X099E-X176E, X085D-X099E-X188E, X085D-X099E-X189E, X085D-X099E-X242D, X085D-X157D-X188E, X085D-X157D-X189E, X085D-X157D-X242D, X085D-X176E-X188E, X085D-X176E-X189E, X085D-X176E-X242D, X085D-X188E-X189E, X085D-X188E-X242D, X085D-X188E-X256E, X085D-X189E-X242D, X085D-X242D-X256E, X099E-X157D-X188E, X099E-X157D-X189E, X099E-X157D-X242D, X099E-X176E-X188E, X099E-X176E-X189E, X099E-X176E-X242D, X099E-X188E-X189E, X099E-X188E-X242D, X099E-X189E-X242D, X157D-X188E-X189E, X157D-X188E-X242D, X157D-X189E-X242D, X176E-X188E-X189E, X176E-X188E-X242D, X176E-X189E-X242D, X188E-X189E-X242D, and X188E-X242D-X256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1,wherein said variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO:1.,

[0220] Example 10. The subtilisin variant as described in Example 9, wherein the variant comprises three mutations in a combination selected from the group consisting of: S009E-S085D-S099E, S009E-S085D-A188E, S009E-S085D-G189E, S009E-S085D-N242D, S009E-S099E-A188E, S009E-S099E-G189E, S009E-S099E-N242D, S009E-A188E-G189E, S009E-A188E-N242D, S009E-G189E-N242D, N074D-S085D-S099E, N074D-S085D-G189E, N074D-S085D-N242D, N074D-S099E-G189E, N074D-S099E-N242D, N074D-G189E-N242D, S085D-S099E-G157D, S085D-S099E-Q176E, S085D-S099E-A188E, S085D-S099E-G189E, S085D-S099E-N242D, S085D-G157D-A188E, S085D-G157D-G189E, S085D-G157D-N242D, S085D-Q176E-A188E, S085D-Q176E-G189E, S085D-Q176E-N242D, S085D-A188E-G189E, S085D-A188E-N242D, S085D-A188E-L256E, S085D-G189E-N242D, S085D-N242D-L256E, S099E-G157D-A188E, S099E-G157D-G189E, S099E-G157D-N242D, S099E-Q176E-A188E, S099E-Q176E-G189E, S099E-Q176E-N242D, S099E-A188E-G189E, S099E-A188E-N242D, S099E-G189E-N242D, G157D-A188E-G189E, G157D-A188E-N242D, G157D-A188E-L256E, G157D-G189E-N242D, Q176E-A188E-G189E, Q176E-A188E-N242D, Q176E-G189E-N242D, A188E-G189E-N242D, A188E-N242D-L256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1,wherein said variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO: 1.,

[0221] Example 11. The subtilisin variant as described in Example 9, wherein the variant comprises three mutations in a combination selected from the group consisting of: T009E-N085D-S099E, T009E-N085D-T188E, T009E-N085D-G189E, T009E-N085D-N242D, T009E-S099E-T188E, T009E-S099E-G189E, T009E-S099E-N242D, T009E-T188E-G189E, T009E-T188E-N242D, T009E-G189E-N242D, N074D-N085D-S099E, N074D-N085D-G189E, N074D-N085D-N242D, N074D-S099E-G189E, N074D-S099E-N242D, N074D-G189E-N242D, N085D-S099E-G157D, N085D-S099E-Q176E, N085D-S099E-T188E, N085D-S099E-G189E, N085D-S099E-N242D, N085D-G157D-T188E, N085D-G157D-G189E, N085D-G157D-N242D, N085D-Q176E-T188E, N085D-Q176E-G189E, N085D-Q176E-N242D, N085D-T188E-G189E, N085D-T188E-N242D, N085D-T188E-Q256E, N085D-G189E-N242D, N085D-N242D-Q256E, S099E-G157D-T188E, S099E-G157D-G189E, S099E-G157D-N242D, S099E-Q176E-T188E, S099E-Q176E-G189E, S099E-Q176E-N242D, S099E-T188E-G189E, S099E-T188E-N242D, S099E-G189E-N242D, G157D-T188E-G189E, G157D-T188E-N242D, G157D-T188E-Q256E, G157D-G189E-N242D, Q176E-T188E-G189E, Q176E-T188E-N242D, Q176E-G189E-N242D, T188E-G189E-N242D, T188E-N242D-Q256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1,wherein said variant has at least 60% identity with a subtilisin having the amino acid sequence of SEQ ID NO:7.

[0222] Example 12. A subtilisin variant as described in any of the foregoing examples, wherein said variant has at least a 3-fold improvement in terms of robustness factor relative to the parental subtilisin and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0223] Example 13. A subtilisin variant that comprises X085D and two mutations selected from X009E, X074D, X099E, X157D, X176E, X188E, X189E, X242D and X256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein said variant has at least 60% identity with a subtilisin having the amino acid sequence of SEQ ID NO:1.

[0224] Example 14. The subtilisin variant as described in Example 13, wherein said variant does not comprise a combination of mutations selected from:

[0225] a) two mutations selected from X009E, X074D, X176E and X242D;

[0226] b) the mutation X074D in combination with one of X009E, X157D, X176E, X188E and X256E;

[0227] c) the combination X099E-X256E;

[0228] d) the combination X189E-X256E

[0229] wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein said variant has at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0230] Example 15. A subtilisin variant as described in Example 13 or 14, wherein the variant comprises the mutation X085D and further comprises two mutations selected from the following: X009E-X099E, X009E-X157D, X009E-X176E, X009E-X188E, X009E-X189E, X009E-X242D, X009E-X256E, X074D-X099E, X074D-X189E, X074D-X242D, X099E-X157D, X099E-X176E, X099E-X188E, X099E-X189E, X099E-X242D, X157D-X176E, X157D-X188E, X157D-X189E, X157D-X242D, X157D-X256E, X176E-X188E, X176E-X189E, X176E-X242D, X176E-X256E, X188E-X189E, X188E-X242D, X188E-X256E, X189E-X242D, and X242D-X256E, where the positions are numbered relative to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to subtilisin having the amino acid sequence of SEQ ID NO:1.

[0231] Example 16. A subtilisin variant that comprises X188E and further comprises two mutations selected from X009E, X085D, X099E, X157D, X176E, X189E, X242D, and X256E, where the positions are numbered relative to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to subtilisin having the amino acid sequence of SEQ ID NO:1.

[0232] Example 17. A subtilisin variant as described in Example 16, wherein the variant comprises the mutation X188E and does not comprise a combination of mutations selected from the following:

[0233] a) the combination X099E-X256E;

[0234] b) the combination X189E-X256E,

[0235] where the positions are numbered relative to the amino acid sequence of SEQ ID NO:1 and wherein the variant has at least 60% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0236] Example 18. A subtilisin variant as described in Examples 16 and 17, wherein the variant comprises X188E and further comprises two mutations selected from the following: X009E-S085D, X009E-X099E, X009E-X157D, X009E-X176E, X009E-X189E, X009E-X242D, X009E-X256E, S085D-X099E, S085D-X157D, S085D-X176E, S085D-X189E, S085D-X242D, S085D-X256E, X099E-X157D, X099E-X176E, X099E-X189E, X099E-X242D, X157D-X176E, X157D-X189E, X157D-X242D, X157D-X256E, X176E-X189E, X176E-X242D, X176E-X256E, X189E-X242D and X242D-X256E, where positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO:1.

[0237] Example 19. A subtilisin variant that comprises the substitution X099E and further comprises at least two additional mutations selected from the following: X009E, X085D, X157D, X176E, X188E, X189E, X242D and X256E, where positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO:1.

[0238] Example 20. The subtilisin variant according to Example 19, wherein the variant does not comprise a combination of mutations selected from the following:

[0239] a) the mutation X074D in combination with one of X009E, X157D, X176E, X188E and X256E;

[0240] b) the combination X099E-X256E;

[0241] c) the combination X189E-X256E,

[0242] where positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0243] Example 21. A subtilisin variant as described in Example 19 or 20, wherein the variant comprises X099E and further comprises two mutations selected from: X009E-X085D, X009E-X157D, X009E-X176E, X009E-X188E, X009E-X189E, X009E-X242D, X074D-X085D, X074D-X189E, X074D-X242D, X085D-X157D, X085D-X176E, X085D-X188E, X085D-X189E, X085D-X242D, X157D-X176E, X157D-X188E, X157D-X189E, X157D-X242D, X176E-X188E, X176E-X189E, X176E-X242D, X188E-X189E, X188E-X242D, X189E-X242D, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO:1.

[0244] Example 22. A subtilisin variant comprising mutations from: G157D-A188E-N242D, G157D-Q176E-A188E, G157D-Q176E-N242D, N074D-G189E-N242D, N074D-S085D-G189E, N074D-S085D-N242D, N074D-S085D-S099E, N074D-S099E-G189E, N074D-S099E-N242D, Q176E-A188E-N242D, Q176E-G189E-N242D, S009E-S085D-L256E, S085D-A188E-N242D, S085D-G157D-A188E, S085D-G157D-G189E, S085D-G157D-N242D, S085D-G189E-N242D, S085D-Q176E-G189E, S085D-Q176E-N242D, S085D-S099E-A188E, S085D-S099E-G157D, S085D-S099E-G189E, S085D-S099E-N242D, S085D-S099E-Q176E, S099E-A188E-N242D, S099E-G157D-A188E, S099E-G157D-G189E, S099E-G157D-N242D, S099E-G157D-Q176E, S099E-G189E-N242D, S099E-Q176E-A188E, and S099E-Q176E-N242D, where positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and where the variant has at least 60% identity to subtilisin having the amino acid sequence of SEQ ID NO:1.

[0245] Example 23. A subtilisin variant as described in any of the foregoing examples, wherein the variant has at least a 3-fold improvement in robustness factor relative to the parental subtilisin and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:7.

[0246] Example 24. An enzyme composition comprising one or more subtilisin variants as described in any of the foregoing examples.

[0247] Example 25. The enzyme composition as described in Example 24, wherein the composition is an enzyme granule, an enzyme slurry or a liquid preparation.

[0248] Example 26. The enzyme composition as described in any one of Examples 24 or 25, which further comprises one or more other enzymes selected from the group consisting of: acyltransferase, amylase, α-amylase, β-amylase, α-galactosidase, arabinase, arabinosidase, arylesterase, β-galactosidase, β-glucanase, carrageenase, catalase, chondroitinase, cutinase, dispersin, endo-β-mannanase, exo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipolytic enzyme, lipoxygenase, mannanase, metalloprotease, nuclease, oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenol oxidase, phosphatase, phosphodiesterase, phospholipase, phytase, polyesterase, polygalacturonase, additional protease, pullulanase, reductase, rhamnogalacturonase, cellulase, tannase, transglutaminase, xanthan lyase, xylan acetylesterase, xylanase and xylosidase; and combinations thereof.

[0249] Example 27. The enzyme composition as described in Example 26, wherein the one or more enzymes comprise amylase and its variants, and combinations of the amylase and its variants, and the amylase is selected from the group consisting of: AA707, AA560, AAI10, BspAmy24, SP722 and CspAmy1.

[0250] Example 28. A polynucleotide comprising a nucleic acid sequence encoding a variant as described in any one of Examples 1-23, wherein the polynucleotide is optionally isolated.

[0251] Example 29. The polynucleotide as described in Example 28, wherein the nucleic acid sequence is operably linked to a promoter.

[0252] Example 30. An expression vector or expression cassette comprising the polynucleotide as described in Example 28 or 29.

[0253] Example 31. A recombinant host cell comprising the polynucleotide as described in Example 28 or 29 or the vector or cassette as described in Example 30.

[0254] Example 32. A cleaning composition or a detergent composition comprising at least one subtilisin variant as described in Examples 1-23 and at least one surfactant.

[0255] Example 33. The cleaning composition or detergent composition according to Example 32, wherein the surfactant is selected from the group consisting of: nonionic surfactants, amphoteric surfactants, semi-polar surfactants, anionic surfactants, cationic surfactants, zwitterionic surfactants, and combinations and mixtures thereof.

[0256] Example 34. The cleaning composition or detergent composition according to Example 32 or 33, wherein the surfactant is a nonionic alcohol ethoxylate.

[0257] Example 35. The cleaning composition or detergent composition according to Example 32-34, the cleaning composition or detergent composition further comprising at least one additional polypeptide, wherein the at least one additional polypeptide is an enzyme selected from the group consisting of: acyltransferase, α-amylase, β-amylase, α-galactosidase, arabinosidase, arylesterase, β-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, dispersein, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exo-mannanase, feruloyl esterase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, mannanase, metalloprotease, nuclease (e.g., deoxyribonuclease and ribonuclease), oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, phenol oxidase, phosphatase, phospholipase, phytase, polygalacturonase, polyesterase, additional protease, pullulanase, reductase, rhamnogalacturonase, β-glucanase, tannase, transglutaminase, xanthan lyase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof.

[0258] Example 36. The cleaning composition or detergent composition according to Example 32-35, wherein the composition comprises from about 0.1% to about 60%, from about 1% to about 50%, or from about 5% to about 40% surfactant by weight of the composition.

[0259] Example 37. A cleaning composition or detergent composition as described in Examples 32 - 36, wherein the composition further comprises one or more auxiliary materials selected from the group consisting of: builders, bleaches, bleach activators, bleach catalysts, other enzymes, enzyme stabilizing systems, chelating agents, optical brighteners, soil release polymers, dye transfer agents, dispersants, foam inhibitors, dyes, fragrances, colorants, filler salts, hydrotropes, photoactivators, fluorescent agents, fabric conditioners, hydrolysable surfactants, preservatives, antioxidants, anti - shrinkage agents, anti - wrinkle agents, bactericides, fungicides, color accents, silver care agents, anti - dulling agents and / or anti - corrosion agents, alkaline sources, solubilizers, carriers, processing aids, pigments, and pH control agents.

[0260] Example 38. A cleaning method, the method comprising contacting a surface or article in need of cleaning with a cleaning composition or detergent composition, the cleaning composition or detergent composition comprising at least one subtilisin variant as described in Examples 1 - 23 and at least one surfactant or dispersant polymer; and the method optionally further comprises the step of rinsing the surface or article after contacting the surface or article with the composition.

[0261] Example 39. The method as described in Example 38, wherein the article is tableware or fabric.

[0262] The following examples are provided to demonstrate and illustrate certain preferred embodiments and aspects of the present disclosure and should not be construed as limiting.

[0263] Examples

[0264] Example 1

[0265] Generation of Enzyme Variants

[0266] The wild-type subtilisin of Bacillus lentus (GG36) and its variants were produced as described below. The amino acid sequence of the mature GG36 parental enzyme is shown in SEQ ID NO 1. All GG36 subtilisin variants were expressed using a DNA fragment that sequentially included: a 5'AprE flanking region that contained a variant of the Bacillus subtilis rrnIp2 promoter sequence (SEQ ID NO:2) (the Bacillus subtilis rrnIp2 promoter and engineered variants are more fully described in patent application WO 2020112609); a nucleotide sequence encoding the aprE signal peptide sequence (SEQ ID NO:3); a nucleotide sequence encoding the Bacillus lentus propeptide (SEQ ID NO:4); a sequence corresponding to the gene encoding the mature GG36 subtilisin; the BPN' terminator (SEQ ID NO:5); a 3'AprE flanking sequence that included a kanamycin gene expression cassette (SEQ ID NO:6). This DNA fragment was assembled using standard molecular biotechnology. Competent Bacillus subtilis cells of a suitable strain were transformed with the linear DNA of the expression cassette. A library of GG36 subtilisin variants was generated by the above method. The library contained variants with three amino acid substitutions on the wild-type sequence (SEQ ID NO:1).

[0267] The wild-type subtilisin of Bacillus gibsonii (BG46) and its variants were produced as described below. The amino acid sequence of the mature BG46 parental enzyme is shown in SEQ ID NO 7. All BG46 subtilisin variants were expressed using a DNA fragment that sequentially included: a 5'AprE flanking region that contained a variant of the Bacillus subtilis rrnIp2 promoter sequence (SEQ ID NO:2) (the Bacillus subtilis rrnIp2 promoter and engineered variants are more fully described in patent application No. 62 / 772363 filed on November 28, 2018); a nucleotide sequence encoding the aprE signal peptide sequence (SEQ ID NO:3); a nucleotide sequence encoding the Bacillus lentus propeptide (SEQ ID NO:4); a sequence corresponding to the gene encoding the mature BG46 subtilisin; the BPN' terminator (SEQ ID NO:5); a 3'AprE flanking sequence that included a kanamycin gene expression cassette (SEQ ID NO:6). This DNA fragment was assembled using standard molecular biotechnology. Competent Bacillus subtilis cells of a suitable strain were transformed with the linear DNA of the expression cassette.

[0268] The transformation mixture was plated onto LA plates containing 1.6% skim milk and 5 ppm kanamycin and incubated overnight at 37°C. Single colonies were picked and grown in Luria broth at 37°C under antibiotic selection.

[0269] For protein expression experiments, the transformed cells were grown in a medium (MOPS buffer-based enriched semi-defined medium with urea as the main nitrogen source, glucose as the main carbon source, supplemented with 1% soy peptone for robust cell growth and containing antibiotic selection) in a 96-well microtiter plate (MTP) in an orbital incubator at 32 °C, 250 rpm, and 70% humidity for 3 days. After centrifugation and filtration, the clarified culture supernatant containing the protease of interest was used for the assay.

[0270] Example 2

[0271] Enzyme assay

[0272] Protein concentration determination: Protein concentration quantification was performed using an Agilent Infinity II 1290UHPLC equipped with an Agilent 300SB-C3 RRHD (1.8 μm 2.1x50 mm) column. The column temperature was 60 °C, and the samples were eluted from the column using a gradient of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in acetonitrile. Absorbance was measured at 220 nm, and the peaks were integrated using OpenLab software (Agilent Technologies, USA). The protein concentration of the samples was calculated based on a standard curve of the parental protease.

[0273] Protease activity: The protease activities of the subtilisin parent and its variants were tested by measuring the hydrolysis of the N-suc-AAPF-pNA substrate. For the AAPF assay, the reagent solutions used were: 100 mM Tris pH 8.6, 0.005% -80 and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388). To prepare the working solution, 1 mL of the suc-AAPF-pNA stock solution was added to 100 mL of Tris buffer and mixed. The enzyme samples were added to a microtiter plate (MTP) containing the 1.6 mM suc-AAPF-pNA working solution, and the activity was determined by measuring the absorbance at 405 nm kinetically at room temperature for 3 - 5 min using a SpectraMax microplate reader. The protease activity was expressed as mOD / min.

[0274] Cleaning performance determination:The detergents used for the cleaning performance determination were Persil Small & Mighty non-biological liquid detergent "Persil Non-Bio" (PNB, Unilever) and test detergent A (TDA). PNB was purchased from a UK supermarket on September 26, 2014. The composition of the TDA detergent is shown in Table 1. For the cleaning performance determination, the PNB detergent was diluted to 2.7 g / l in deionized water and 5 mM HEPES (pH 8.2) was added, with a water hardness of 12 gpg (3Ca:1Mg). This detergent is considered boron-free as it contains ≤5 mg / Kg of boron when tested for the element boron content. For the cleaning performance determination, the TDA detergent was diluted to 6.0 g / l in deionized water and 5 mM HEPES (pH 8.2) was added, with a water hardness of 6 gpg (3Ca:1Mg).

[0275]

[0276] The cleaning performance of the test protease variants relative to the parent (wild-type GG36 or BG46) against technical soils C-05 (blood / milk / ink on woven cotton) and C-S-39 (aged whole egg carbon with carbon black on woven cotton) was tested. Both soils were purchased from the Center for Testmaterials BV in Vlaardingen, the Netherlands. The soils were punched into small round cloth samples and distributed into Costar 9017 or Greiner 655101 microtiter plates (MTPs). First, the MTPs containing the micro cloth samples were filled with the detergent. Then, a certain amount of the parent enzyme and the variant were added to a final volume of 200 μl. The determination was carried out by gently shaking at 25 °C for 25 minutes. After the incubation period, 100 - 150 μl of the supernatant was transferred to a fresh MTP and the absorbance of the BMI cloth samples was read at 600 nm or the absorbance of the whole egg cloth samples was read at 405 nm using a SpectraMax microplate reader. The absorbance results were obtained by subtracting the value of the blank control (without enzyme) from each sample value. For each condition and subtilisin variant in Example 2, the cleaning performance index (PI) was calculated by dividing the absorbance of the variant minus the blank by the absorbance of the parent protease at the same concentration. The absorbance value of the parent protease minus the blank at the corresponding concentration of the variant was determined using a standard curve of the parent protease included in the test and generated using a Langmuir fit or a Hill sigmoidal fit, as appropriate.

[0277] General sample setup for stability determination:Test the stability of subtilisin in 10% solutions (v / v) of PNB or TDA detergents. Test GG36 and GG36 variants at 42 °C. Test BG46 and BG46 variants at 37 °C. Set the elevated temperature so that the residual activity of the stressed samples compared to the non-stressed samples can be discerned within a range suitable to distinguish the differences between the variant enzyme and its parental enzyme within a 20-minute incubation time. Mix the enzyme samples with the diluted detergent and immediately measure the protease activity on the AAPF substrate to serve as the non-stressed value. Subsequently, place the samples in a PCR plate, seal it and incubate at high temperature for 20 minutes using a thermal cycler, and then determine the AAPF activity to obtain the stressed value. The percentage of residual activity is calculated by taking the ratio of the stressed activity to the non-stressed activity and multiplying by 100. For these assays, all enzyme samples are assayed in triplicate. The lower limit cut-off value of protein expression applied is 200 ppm, and data with a CV (coefficient of variation) of 20% or less is analyzed.

[0278] Example 3

[0279] Variant subtilisins with increased stability in the presence of detergents

[0280] Table 2 shows the test results of a series of GG36 variants obtained when tested using the method described in Example 2, and these variants have a significant enhancement in stability compared to wild-type GG36. Table 3 shows the test results of a series of BG46 variants obtained when tested using the method described in Example 2, and these variants have a significant enhancement in stability compared to wild-type BG46. All variants exhibited comparable or improved cleaning performance compared to their respective wild-type / parental enzymes.

[0281]

[0282]

[0283]

[0284]

[0285] The parameter called "robustness improvement factor, RIF" is determined for each subtilisin as follows: Multiply the percentage of residual activity value by the cleaning performance index calculated for the C-05 stain and the cleaning performance index calculated for the C-S-39 stain, and divide this number by the product of the corresponding parameters obtained for the wild-type GG36 or BG46 (parental enzyme) tested under the same conditions. As shown in Tables 2 and 3, the RIF values obtained for the variant enzymes reflect the overall ability of the variant enzymes to deliver performance benefits in liquid laundry detergents.

[0286] Although the present disclosure has been described in connection with specific embodiments thereof, it will be apparent that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the present disclosure is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0287] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety into this specification to the extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Additionally, the citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present disclosure. With respect to the use of section headings, they should not be construed as necessarily limiting.

Claims

1. A subtilisin variant comprising two or more mutations selected from the group consisting of: X009E, X074D, X085D, X099E, X157D, X176E, X188E, X189E, X211L, X242D, and X256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to subtilisin having the amino acid sequence of SEQ ID NO:1 or 7.

2. The subtilisin variant according to claim 1, wherein the variant does not comprise a combination of mutations selected from: e) mutation X074D in combination with one or more of X009E, X157D, X176E, X188E, and X256E; f) combination X099E-X256E; g) combination X189E-X256E, and h) two or more mutations of X009E, X157D, X176E, and X256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to subtilisin having the amino acid sequence of SEQ ID NO:1 or 7.

3. The subtilisin variant according to claim 1, comprising two mutations selected from the group consisting of: X009E-X085D, X009E-X099E, X009E-X188E, X009E-X189E, X009E-X242D, X074D-X085D, X074D-X099E, X074D-X189E, X074D-X242D, X085D-X099E, X085D-X157D, X085D-X176E, X085D-X188E, X085D-X189E, X085D-X242D, X085D-X256E, X099E-X157D, X099E-X176E, X099E-X188E, X099E-X189E, X099E-X242D, X157D-X188E, X157D-X189E, X157D-X242D, X176E-X188E, X176E-X189E, X176E-X242D, X176E-X256E, X188E-X189E, X188E-X242D, X188E-X256E, X189E-X242D, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to subtilisin having the amino acid sequence of SEQ ID NO:1 or 7.

4. The subtilisin variant according to claim 3, wherein the variant comprises two mutations in a combination selected from the group consisting of: S009E-S085D, S009E-S099E, S009E-A188E, S009E-G189E, S009E-N242D, N074D-S085D, N074D-S099E, N074D-G189E, N074D-N242D, S085D-S099E, S085D-G157D, S085D-Q176E, S085D-A188E, S085D-G189E, S085D-N242D, S085D-L256E, S099E-G157D, S099E-Q176E, S099E-A188E, S099E-G189E, S099E-N242D, G157D-A188E, G157D-G189E, G157D-N242D, Q176E-A188E, Q176E-G189E, Q176E-N242D, Q176E-L256E, A188E-G189E, A188E-N242D, A188E-L256E, G189E-N242D, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO:

1.

5. The subtilisin variant according to claim 3, wherein the variant comprises two mutations in a combination selected from the group consisting of: T009E-N085D, T009E-S099E, T009E-T188E, T009E-G189E, T009E-N242D, N074D-N085D, N074D-S099E, N074D-G189E, N074D-N242D, N085D-S099E, N085D-G157D, N085D-Q176E, N085D-T188E, N085D-G189E, N085D-N242D, N085D-Q256E, S099E-G157D, S099E-Q176E, S099E-T188E, S099E-G189E, S099E-N242D, G157D-T188E, G157D-G189E, G157D-N242D, Q176E-T188E, Q176E-G189E, Q176E-N242D, Q176E-Q256E, T188E-G189E, T188E-N242D, T188E-Q256E, G189E-N242D, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO:

7.

6. The subtilisin variant according to claim 1, wherein the variant has at least a 3-fold improvement in terms of robustness factor relative to the parental subtilisin and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:

7.

7. A subtilisin variant comprising three mutations selected from the group consisting of: X009E, X074D, X085D, X099E, X157D, X176E, X188E, X189E, X242D and X256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:

7.

8. The subtilisin variant according to claim 7, wherein the variant does not comprise a combination of mutations selected from: d) three mutations selected from X009E, X074D, X085D, X176E and X242D; e) the mutation X074D in combination with one or more mutations selected from X009E, X157D, X176E, X188E and X256E; and f) Three mutations selected from the group consisting of: X009E, X157D, X176E, X256E wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein said variant has at least 60% identity with a subtilisin having the amino acid sequence of SEQ ID NO:1 or 7.

9. The subtilisin variant according to claim 7, wherein the variant comprises a combination of three mutations selected from the group consisting of: X009E-X085D-X099E, X009E-X085D-X188E, X009E-X085D-X189E, X009E-X085D-X242D, X009E-X099E-X188E, X009E-X099E-X189E, X009E-X099E-X242D, X009E-X188E-X189E, X009E-X188E-X242D, X009E-X189E-X242D, X074D-X085D-X099E, X074D-X085D-X189E, X074D-X085D-X242D, X074D-X099E-X189E, X074D-X099E-X242D, X074D-X189E-X242D, X085D-X099E-X157D, X085D-X099E-X176E, X085D-X099E-X188E, X085D-X099E-X189E, X085D-X099E-X242D, X085D-X157D-X188E, X085D-X157D-X189E, X085D-X157D-X242D, X085D-X176E-X188E, X085D-X176E-X189E, X085D-X176E-X242D, X085D-X188E-X189E, X085D-X188E-X242D, X085D-X188E-X256E, X085D-X189E-X242D, X085D-X242D-X256E, X099E-X157D-X188E, X099E-X157D-X189E, X099E-X157D-X242D, X099E-X176E-X188E, X099E-X176E-X189E, X099E-X176E-X242D, X099E-X188E-X189E, X099E-X188E-X242D, X099E-X189E-X242D, X157D-X188E-X189E, X157D-X188E-X242D, X157D-X189E-X242D, X176E-X188E-X189E, X176E-X188E-X242D, X176E-X189E-X242D, X188E-X189E-X242D, and X188E-X242D-X256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1,wherein said variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO: 1., 10. The subtilisin variant according to claim 9, wherein the variant comprises a combination of three mutations selected from the group consisting of: S009E-S085D-S099E, S009E-S085D-A188E, S009E-S085D-G189E, S009E-S085D-N242D, S009E-S099E-A188E, S009E-S099E-G189E, S009E-S099E-N242D, S009E-A188E-G189E, S009E-A188E-N242D, S009E-G189E-N242D, N074D-S085D-S099E, N074D-S085D-G189E, N074D-S085D-N242D, N074D-S099E-G189E, N074D-S099E-N242D, N074D-G189E-N242D, S085D-S099E-G157D, S085D-S099E-Q176E, S085D-S099E-A188E, S085D-S099E-G189E, S085D-S099E-N242D, S085D-G157D-A188E, S085D-G157D-G189E, S085D-G157D-N242D, S085D-Q176E-A188E, S085D-Q176E-G189E, S085D-Q176E-N242D, S085D-A188E-G189E, S085D-A188E-N242D, S085D-A188E-L256E, S085D-G189E-N242D, S085D-N242D-L256E, S099E-G157D-A188E, S099E-G157D-G189E, S099E-G157D-N242D, S099E-Q176E-A188E, S099E-Q176E-G189E, S099E-Q176E-N242D, S099E-A188E-G189E, S099E-A188E-N242D, S099E-G189E-N242D, G157D-A188E-G189E, G157D-A188E-N242D, G157D-A188E-L256E, G157D-G189E-N242D, Q176E-A188E-G189E, Q176E-A188E-N242D, Q176E-G189E-N242D, A188E-G189E-N242D, A188E-N242D-L256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1,wherein said variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO: 1., 11. The subtilisin variant according to claim 9, wherein the variant comprises three mutations in a combination selected from the group consisting of: T009E-N085D-S099E, T009E-N085D-T188E, T009E-N085D-G189E, T009E-N085D-N242D, T009E-S099E-T188E, T009E-S099E-G189E, T009E-S099E-N242D, T009E-T188E-G189E, T009E-T188E-N242D, T009E-G189E-N242D, N074D-N085D-S099E, N074D-N085D-G189E, N074D-N085D-N242D, N074D-S099E-G189E, N074D-S099E-N242D, N074D-G189E-N242D, N085D-S099E-G157D, N085D-S099E-Q176E, N085D-S099E-T188E, N085D-S099E-G189E, N085D-S099E-N242D, N085D-G157D-T188E, N085D-G157D-G189E, N085D-G157D-N242D, N085D-Q176E-T188E, N085D-Q176E-G189E, N085D-Q176E-N242D, N085D-T188E-G189E, N085D-T188E-N242D, N085D-T188E-Q256E, N085D-G189E-N242D, N085D-N242D-Q256E, S099E-G157D-T188E, S099E-G157D-G189E, S099E-G157D-N242D, S099E-Q176E-T188E, S099E-Q176E-G189E, S099E-Q176E-N242D, S099E-T188E-G189E, S099E-T188E-N242D, S099E-G189E-N242D, G157D-T188E-G189E, G157D-T188E-N242D, G157D-T188E-Q256E, G157D-G189E-N242D, Q176E-T188E-G189E, Q176E-T188E-N242D, Q176E-G189E-N242D, T188E-G189E-N242D, T188E-N242D-Q256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO: 1,wherein said variant has at least 60% identity with subtilisin having the amino acid sequence of SEQ ID NO:

7.

12. The subtilisin variant according to claim 1, wherein said variant has at least a 3-fold improvement in terms of robustness factor relative to the parental subtilisin and has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:

7.

13. A subtilisin variant comprising X085D and two mutations selected from X009E, X074D, X099E, X157D, X176E, X188E, X189E, X242D and X256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein said variant has at least 60% identity with a subtilisin having the amino acid sequence of SEQ ID NO:

1.

14. The subtilisin variant according to claim 13, wherein said variant does not comprise a combination of mutations selected from: e) Two mutations selected from X009E, X074D, X176E and X242D; f) The mutation X074D in combination with one of X009E, X157D, X176E, X188E and X256E; g) The combination X099E-X256E; h) The combination X189E-X256E wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein said variant has at least 60% amino acid sequence identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:

7.

15. The subtilisin variant according to claim 13, wherein the variant comprises the mutation X085D and further comprises two mutations selected from the following: X009E-X099E, X009E-X157D, X009E-X176E, X009E-X188E, X009E-X189E, X009E-X242D, X009E-X256E, X074D-X099E, X074D-X189E, X074D-X242D, X099E-X157D, X099E-X176E, X099E-X188E, X099E-X189E, X099E-X242D, X157D-X176E, X157D-X188E, X157D-X189E, X157D-X242D, X157D-X256E, X176E-X188E, X176E-X189E, X176E-X242D, X176E-X256E, X188E-X189E, X188E-X242D, X188E-X256E, X189E-X242D, and X242D-X256E, wherein the positions are numbered corresponding to the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 60% identity to subtilisin having the amino acid sequence of SEQ ID NO:

1.

16. A cleaning composition or a detergent composition, which comprises at least one subtilisin variant according to claim 1 and at least one surfactant.

17. The cleaning composition or the detergent composition according to claim 16, wherein the surfactant is selected from the group consisting of: nonionic surfactants, amphoteric surfactants, semi-polar surfactants, anionic surfactants, cationic surfactants, zwitterionic surfactants, and combinations and mixtures thereof.

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